Journal of Human Hypertension (2014) 28, 610–616 & 2014 Macmillan Publishers Limited All rights reserved 0950-9240/14 www.nature.com/jhh
ORIGINAL ARTICLE
MiR-101 regulates apoptosis of trophoblast HTR-8/SVneo cells by targeting endoplasmic reticulum (ER) protein 44 during preeclampsia Y Zou1,3, Z Jiang1,3, X Yu2,3, Y Zhang1, M Sun2, W Wang1, Z Ge1, W De2 and L Sun1 To investigate a possible association between miR-101 and apoptosis of human trophoblast cells mediated by endoplasmic reticulum protein 44 (ERp44) in preeclampsia (PE), we explored the expression of miR-101 in PE placentas (n ¼ 30) compared with normotensive pregnant placentas (n ¼ 30) and the correlation between miR-101 and ERp44 was also analyzed. Furthermore, both the apoptotic rate of trophoblast cells and the ER stress-induced apoptotic proteins were assayed when the HTR-8/SVneo cells were treated with miR-101 mimics or inhibitors in vitro. We found a lower expression of miR-101 and an inverse correlation between miR101 and ERp44 protein in PE placentas. Upregulation of miR-101 expression could inhibit trophoblast HTR-8/SVneo cell apoptosis and repress ER stress-induced apoptotic proteins by targeting ERp44 in vitro, whereas inhibition of miR-101 could induce HTR-8/ SVneo cell apoptosis. Our findings indicated that overexpression of miR-101 could downregulate ERp44 and suppress apoptosis in trophoblast cells during PE. Therefore, loss of miR-101 expression could contribute to ER stress-induced trophoblast cell apoptosis by targeting ERp44. Journal of Human Hypertension (2014) 28, 610–616; doi:10.1038/jhh.2014.35; published online 8 May 2014
INTRODUCTION Preeclampsia (PE), as one of the most common pathologic complications of pregnancy, afflicts B3–5%1 of pregnancies and is a leading contributor to perinatal morbidity and pregnancyassociated mortality, especially in developing countries. The clinical characteristics of this disease include: a new onset of hypertension (blood pressure X140/90 mm Hg in previously normotensive women after the 20th week of pregnancy) and proteinuria (X300 mg per 24-h urine collection) during the second stage of pregnancy. The delivery of the baby and placenta is the only curative treatment to reverse the syndromes.2 Because of the adverse outcome of this disease, more and more research on it has been continuously carried out; however, the specific pathogenesis of this disorder remains to be elucidated. So far, there have been many mechanisms associated with the pathogenesis of PE, such as inflammatory cytokines,3–5 endothelial dysfunction,6 imbalance between proangiogenic and antiangiogenic factors,7,8 oxidative stress9,10 and finally genetic and dietary factors.11 Recent evidence indicates placental endoplasmic reticulum stress (ERS) involved in pathophysiological characteristics of PE.12,13 In endoplasmic reticulum, molecular chaperone proteins have crucial roles in the process of synthesizing and packing proteins in the early stages of ERS.14 Severe or prolonged ERS induces activation of unique pathways that lead to cell death through apoptosis.15 Therefore, the generation of placental ERS is a key intermediary event in the pathology of PE.16 However, the abnormal details of the molecular mechanism of ERS still remain to be studied.
Our previous studies identified several dysregulated expressions of endoplasmic reticulum molecular chaperone proteins in PE placenta by two-dimensional polyacrylamide gel electrophoresis.17 Four proteins (protein disulfide isomerase precursor, endoplasmic reticulum resident protein, dihydrolipoyl dehydrogenase and TIM21-like protein) were found to be significantly upregulated in PE and three were downregulated (disulfide isomerase ER-60, peroxiredoxin 2 and D3, 5-D2, 4-dienoyl-CoA isomerase). Among those upregulated proteins, levels of endoplasmic reticulum protein 44 (ERp44) are about sixfold in PE placenta compared with normal controls. This indicates that ERp44 may have an important role in the pathogenesis of PE; however, there is no report about the regulatory role of ERp44 in PE. Recent studies have revealed that micro RNAs (miRNAs) could inhibit protein expression through base pairing with sequences located predominantly in the 30 untranslated regions (30 -UTRs) of mRNA targets.18 The functions of miRNAs often become pronounced under conditions of stress,19 so we postulate that ERp44 might be modulated by miRNA in ERS. MiRNAs are a class of highly conserved endogenous small noncoding RNA molecules (19–22 nt), which have important posttranscriptional regulatory roles of targeting mRNAs for cleavage or translational repression.20,21 Recent data indicate that miRNAs have a fundamental role in many biologic and pathologic processes, which include cell proliferation,22 invasion,23 apoptosis,24,25 cell development,26 carcinogenesis27 and cardiovascular disease.28 There have been many reports suggesting that many miRNAs have differential expression in PE, including miR-101.29 In this study, we found a decreased expression
1 Department of Obstetrics and Gynecology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China and 2Department of Biochemistry and Molecular Biology, Nanjing Medical University, Nanjing, China. Correspondence: Professor L Sun, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing 210000, China. E-mail: sunlizhou@njmu. edu. cn 3 These authors contributed equally to this work and should be regarded as joint first authors. Received 4 November 2013; revised 2 March 2014; accepted 26 March 2014; published online 8 May 2014
MiR-101 regulates apoptosis of HTR-8/SVneo cells Y Zou et al
611 of miR-101(-3p) in 30 severe human PE placentas by quantitative PCR on comparing with 30 normal pregnant ones and detecting 23 miRNAs in all. By using bioinformatics analysis (softwares: Target Scan (Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA, USA), PicTar (Max Del Brunet Center, Berlin, Germany), miTarget (http://cbit.snu.ac.kr/~miTarget), we found that there are miR-101-binding sites in the 30 -UTR of ERp44 mRNA (CAUGACA: GUACUGU). In addition, this was proved by the luciferase reporter assay in our study. Then, overexpression of miR-101 inhibited trophoblast HTR-8/SVneo cell apoptosis and repressed ER stress-induced apoptotic proteins by targeting ERp44 in vitro, whereas inhibition of miR-101 induced cell apoptosis. Thus, miR-101 might contribute to ER stress-induced trophoblast cell apoptosis by targeting ERp44. MATERIALS AND METHODS Sample collection Placental tissues were obtained from primipara women aged 20–36 years who underwent cesarean deliveries between 2011 and 2012 in the Department of Gynecology and Obstetrics of the People’s Hospital of Jiangsu Province, China. All tissues were collected immediately after placental delivery from the uterus and washed with sterile phosphatebuffered saline before being snap-frozen in liquid nitrogen for transportation to the lab. Tissues were temporarily stored at 80 1C for RNA and protein extraction. All of our experiments were approved by the Ethics Board of the First Affiliated Hospital of Nanjing Medical University, and informed consents have been obtained from all the pregnant women as well.
Cell culture and treatment Human embryonic kidney HEK 293T cells (used for luciferase assay) were maintained in Dulbecco’s modified Eagle medium, supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U ml 1 penicillin and 100 mg ml 1 streptomycin in standard culture conditions (37 1C in 5% humidified CO2 incubator). After placing into 48-well plates, HEK 293T cells were cotransfected with pCDNA-miR-101 or pCDNA (as negative control), pRLTK (2 ng; Promega, Shanghai, China) and luciferase reporter plasmids (50 ng) containing wild type or mutant type of ERp44 30 -UTR using Lipofectamine 2000 (Invitrogen, Shanghai, China). An immortalized first-trimester extravillous trophoblast cells (EVT) cell line HTR-8/SVneo30 (kindly provided by Dr Charles Graham, Queen’s University, Canada), which is derived from a short-lived primary EVT cell line, was used in the present study. With the behavior of trophoblast cells, many studies31,32 have been based on this cell line to simulate the behavior of trophoblast cells in pregnancy. The human trophoblast-derived cell line was maintained in 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U ml 1 penicillin and 100 mg ml 1 streptomycin in standard culture conditions (37 1C in 5% humidified CO2 incubator). Cells were sowed into six-well plates for 12 h before being transiently transfected with miRNA duplexes at 100 nM final concentrations (hsa-mir-101 miRIDIAN Mimics MI0000739) and its inhibitor (MIMAT0000099). After transfection for 48 h, cells were collected to detect the transfection efficiency.
RNA extraction and real-time reverse transcription-PCR Total RNA was extracted from B0.1 g placental tissues or cultured cells with Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) and 1bromo-3-chloropropane and isopropanol.33,34 Then, a Reverse Transcription Kit (Takara) was used for the synthesis of complementary DNA by adding 1 mg of total RNA to the RT Reaction Mix. For quantitative PCR, the amplification of complementary DNA was performed by adding Power SYBR Green (Takara) to a total volume of 20 ml reaction mix. According to the manufacturer’s instructions, the reverse transcription was performed at 37 1C for 15 min and at 85 1C for 5 s. To normalize the results for the quantitative PCR, U6 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expressions were used. The sequence of the primer was as follows: miR-101: reverse transcribed primer: 50 -CGCATGAATACGCCAAACAACTTCAGTTAT-30 ; forward: 50 -CTCGCATGAATACGCCAAACAA-30 ; reverse: 50 -CCAGCGGTTACAGTACTGTGATA-30 . U6: reverse transcribed primer: 50 -AACGCTTCACGAATTTGCGT-30 ; forward: 50 -CTCGCTTCGGCAGCACA-30 ; reverse: 50 -AACGCTTCACGAATTTGCGT-30 . GAPDH: forward: 50 -GACTCATGACCACAGTCCATGC-30 ; reverse: 50 -AGAGGCAGGGATGATGTTCTG-30 . An ABI 7500 (Applied Biosystems, Paisley, UK) was used to carry out quantitative PCR and data collections.
Hoechst staining assay HTR-8/SVneo cells planted into six-well plates were transiently transfected with has-mir-101 and its inhibitor for 48 h, and then Hoechst 33258 (Beyotime) was added into the culture medium to induce nuclear morphology changes. Finally, the percentage of Hoechst-positive nuclei per optical field (at least 50 fields) was counted under fluorescence microscopy by using a filter for Hoechst33258.
Flow cytometry Cells transiently transfected with has-mir-101 and inhibitor for 48 h were collected using trypsin without EDTA, washed with phosphate-buffered saline, resuspended in 1 ml binding buffer and stained with fluorescein isothiocyanate-annexin V and propidium iodide at room temperature for 15 min in the dark according to the manufacturer’s recommendations. The analysis of the cells was carried out by a flow cytometry analysis (FCM, FAC Scan; BD Biosciences, Becton, Dickinson and Company, Franklin Lakes, NJ, USA) equipped with a CellQuest software (BD Biosciences).
Luciferase assay HEK 293T cells were placed in 48-well plates and cotransfected with pCDNA-miR-101 or pCDNA (200 ng), pRL-TK (2 ng; Promega) and Luciferase reporter plasmids (50 ng) containing wild type or mutant type of ERp44 30 -UTR using Lipofectamine 2000 (Invitrogen). The firefly and Renilla luciferase activities were measured by the Dual-Luciferase Reporter Assay System (Promega) after transfection for 48 h.
Treatment of HTR-8/SVneo cells with sodium 4-phenylbutyrate Western blotting analysis Cells (HTR-8/SVneo) transfected with hsa-mir-101 and inhibitor were lysed for protein extraction by using mammalian reagent RIPA (Beyotime, Shanghai, China) supplemented with protease inhibitors cocktail (Roche, Penzberg, Germany) and phenylmethylsulfonyl-fluoride (Roche). The Bradford method was used for the determination of protein concentration in each sample. Samples containing 50–100 mg of protein extractions were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Next, the separated proteins were transferred to 0.22 mm nitrocellulose (Sigma, St Louis, MO, USA) and incubated with specific antibodies (ERp44, caspase-12, chop, ATF-6, PERK Caspase-3, BCL-2, 1:1000, all purchased from Cell Signaling Technology (Boston, MA, USA); ATF-4, 1:500, Bioworld, Shanghai, China; and IRE 1, 1:500, SAB Biotech, Nanjing, China). The secondary antibody was horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1:1000; Beijing ZhongShan Biotechnology Co., Beijing, China). Quantity One software (Bio-Rad, Hercules, CA, USA) was used to quantify the intensity of the autoradiograms protein bands, and the b-actin antibody (1:1000, Santa Cruz, CA, USA) was used as control. & 2014 Macmillan Publishers Limited
HTR-8/SVneo cells were seeded into six-well culture plates overnight and exposed to 20 nm of sodium 4-phenylbutyrate (PBA, Sigma) for 24 h. Next, the cells were collected for protein extraction and assays of ERS signaling cascade proteins.
Statistical analysis All data were expressed as mean±s.d. and all statistical analysis was performed using SPSS statistical software package (SPSS Inc., Chicago, IL, USA). Po0.05 was considered statistically significant.
RESULTS Clinical characteristics The clinical data were obtained from all the patients who were sampled. Placental tissues were classified into two groups: PE (PE, n ¼ 30) and normal pregnant (N, n ¼ 30). PE was diagnosed by the standard criteria, whereas normal pregnancy was defined as not Journal of Human Hypertension (2014) 610 – 616
MiR-101 regulates apoptosis of HTR-8/SVneo cells Y Zou et al
612 having PE or any other complications (including maternal history of hypertension and/or renal disease, maternal infection, smoking, alcoholism, chemical dependency and fetal congenital anomalies). There were no significant differences between the two groups in maternal age, gestational age (week) and C-reaction protein levels (P40.05), although the blood pressure and proteinuria were significantly higher in preeclamptic women (Po0.01) as compared with women with normal pregnancies (Table 1). The expression level of miR-101 is downregulated in human placental tissues By detecting 30 PE compared with normal pregnant placental tissues using quantitative reverse transcription-PCR analysis, we found that the expression level of miR-101 in PE was 25% lower than that of normal pregnancy (Figure 1a). Table 1, Po0.01, shows the clinical characteristics of 30 normal and preeclamptic pregnancies. According to our previous studies, the expression level of ERp44 in human PE placental tissues was six times upregulated compared with normal placenta. The correlation analysis showed that there was a negative correlation between miR-101 and Erp44 (Figure 1b). Thus, the negative correlation between miR-101 and ERp44 indicated that miR-101 may have a part in PE through ERp44. MiR-101 contributes to the expression of ERp44 by interacting with its 30 -UTR To further explore the relationship between miR-101 and ERp44, we conducted a bioinformatics screen to identify that there are seven
Table 1. Clinical characteristics of normal and preeclamptic pregnancies a
Variable
PE (n ¼ 30)
N (n ¼ 30)
P-value Control vs PE
Maternal age Proteinuria (g per day) Gestational age (week) Systolic blood pressure, mm Hg Diastolic blood pressure, mm Hg Body weight of infant (g) C-reaction protein Maternal smoking (number)
30.2±5.7 40.3 36.5±3.7 169±20.1
30.6±3.5 o0.3 39.1±1.2 112±6.8
40.05 o0.01 40.05 o0.01
115±12.8
77±7.1
o0.01
2582±740 8.1±3.1 2
3322±413 5.9±2.9 1
o0.05 40.05 40.05
All results are presented as mean±s.d. aObtained by one-way analysis of variance using SPSS 13.0 software (SPSS Inc.).
continuous complementary base pairings between the mRNA sequence of ERp44 and miR-101. Next, luciferase assays were used to verify the target relationship between miR-101 and ERp44. We found that miR-101 decreased 30% of the activity of the luciferase reporter containing the wild-type 30 -UTR of ERp44 mRNA (Figure 2a, Po0.01). However, when the predicted binding site of miR-101 with the 30 -UTR of ERp44 was mutated, luciferase activity did not drop sharply in the 30 -UTRs with mutant-binding sites. Thus, the data proved that miR-101 may serve as a target of ERp44 in regulating the apoptosis of trophoblast cells when ERS occurs. Exogenous downregulation or overexpression of miR-101 in HTR-8/SVneo cells and its contribution to ERp44 expression To explore the role of miR-101 in ERp44, we conducted the upregulation as well as downregulation of miR-101; miR-101 mimics or miR-101 inhibitors (constructed by Invitrogen) were transiently transfected into HTR-8/SVneo cells. After transfection for 48 h, cells were collected to detect the expression of miR-101 to make sure of the success of overexpression and downregulation. Quantitative reverse transcription-PCR assays revealed that expression of miR101 transfected with miR-101 mimics was 28-fold higher than that in normal control and 0.45-fold in miR-101 inhibitors (Figure 2b, Po0.01). To assess the intermediary molecular mechanism by which miR-101 acts as an inhibitor of trophoblast cell apoptosis and the ERS pathway, we confirmed the expression level of ERp44 proteins after miR-101 was overexpressed or inhibited in HTR-8/ SVneo cells by using western blotting (WB). There was a significant increase in ERp44 protein levels in cells transfected with siRNA targeting miR-101, whereas a decrease occurred in cells treated with miR-101 mimics (Figures 2c and d, Po0.01). Modulation of miR-101 on cell apoptosis in vitro The decrease in miR-101 in PE placentas prompted us to detect the possible biological significance of miR-101 in trophoblast cell apoptosis. We conducted Hoechst staining assay and FCM to detect the apoptotic cells and WB assays to identify apoptotic proteins in cells treated with miR-101. When HTR-8/SVneo cells were transfected with si-RNAs targeting miR-101, a significant increase in apoptosis was observed (Figures 3c and d, Po0.01) as compared with those infected with miR-101 mimics using both FCM (Figure 3a, Po0.01) and Hoechst staining assay (Figure 3b, Po0.01). These results indicated that higher miR-101 could inhibit HTR-8/SVneo cell apoptosis in vitro. The apoptotic protein caspase3 was significantly increased in cells transfected with siRNA targeting miR-101, whereas it was decreased in anti-apoptotic protein Bcl-2 (Figure 3f, Po0.01). On the other hand, WB analysis also indicated that the expression level of caspase-3 protein displayed a decrease in cells treated with miR-101 mimics
Figure 1. (a and b) Relative expression of miR-101 was lower in PE placental tissues as compared with the normal pregnancies, as determined by quantitative PCR analysis (a) and the negative correlation between miR-101 and ERp44 (b). Values are mean±s.d.; **Po0.01. Journal of Human Hypertension (2014) 610 – 616
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Figure 2. Target relationship between miR-101 and ERp44 and the overexpression of miR-01 could partially block the expression level of ERp44 protein. (a) The luciferase reporter plasmid containing wild-type or mutant ERp44 30 -UTR was cotransfected into HEK-293T cells with pCDNA-miR-101 or pCDNA-miR-NC. Luciferase activity was determined by the dual-luciferase assay and shown as the relative firefly activity normalized to Renilla activity. (b) Relative expression of miR-101 after HTR-8/SVneo cells were transfected with miR-101 mimics or anti-miR101. (c and d) The expression of ERp44 protein was analyzed by WB when cells were treated with miR-101 or anti-miR-101 and b-actin was used as control. Values are mean±s.d.; **Po0.01. NS, not significant.
(Figure 3e, Po0.01), whereas the expression level of Bcl-2 protein presented reverse results (Figure 3e, Po0.01). Effects of miR-101 expression on the ERS signaling pathway A series of proteins such as PERK and ATF-4, ATF-6, CHOP, caspase12 and IRE1p, which are involved in the ERS pathways, were tested after HTR-8/SVneo cells were treated with miR-101 mimics and inhibitor. WB assays showed that PERK, ATF-6, CHOP and caspase12 showed a significant decrease in cells treated with miR-101 mimics (Figures 4a and b, Po0.01) and an increase in cells transfected with miR-101 inhibitor (Figures 4c and d, Po0.01) compared with anti-miR-NC. These data indicated that miR-101 could prevent the progression of ERS by inhibiting some participants in this way. Reverse activation of Anti-miR-101 to ERS under the inhibition effect of PBA To further investigate the possible biological significance of antimiR-101 to ERS, HTR-8/SVneo cells were treated with anti-miR-101 under the effect of PBA (inhibition of ERS). A decrease in ERSassociated proteins was detected when cells treated with both anti-miR-101 and PBA were compared with those treated with anti-miR-101 alone, whereas an increase in these proteins was detected when cells treated with PBA alone were compared with none of both treatments (Figures 4e and f, Po0.05). DISCUSSION It was evidenced that PE is associated with alterations in placental miRNA expression.35 Many miRNAs have been found to be involved & 2014 Macmillan Publishers Limited
in PE, such as miR-210,36 miR-155,37 miR-18238 and so on. Zhu et al.29 also detected a series of downregulated miRNAs in severe preeclamptic placentas compared with normal pregnant placentas, and miR-101 is among these. In the present study, we verified that miR-101 was markedly decreased in PE placentas than in normal pregnancy, suggesting that the low expression of miR-101 was involved in PE pathogenesis. Accordingly, recent studies have revealed that miRNAs often deeply influence the responses of organisms to physiologic and pathophysiologic stress.39 In addition, genetic knockout of specific miRNAs renders the organisms incapable of coping with many stresses.40 For example, mice deficient in miR-208 cannot deal with cardiac overload, and inactivation of miR-8 renders zebra fish incapable of responding to osmotic stress.41 So miRNAs may be closely related to the pathogenesis of PE by regulating the stress response. In recent years, many studies have focused on the association of miRNA with cell apoptosis through interaction with their target genes.42 PE is also characterized by an increase in apoptosis of trophoblast cells in placenta.43,44 To explore the role of miR-101 in modulating trophoblast cell apoptosis in PE, we conducted FCM analysis. The results suggested that low expression of miR-101 promoted trophoblast cell apoptosis in PE. MiRNAs are primarily referred to bind to the 30 -UTR of targets to regulate expression levels of multiple proteins.18 Accordingly, a negative relationship between miR-101 and ERp44 and luciferase assay in our study indicates that the apoptosis role of miR-101 in PE may be mediated by ERp44. ERp44 contains a thioredoxin domain with a CRFS motif and can be induced by agents that cause the accumulation of unfolded proteins in the ER during ER stress.45 There are several specific signaling pathways triggered by ER stress, including ER-associated Journal of Human Hypertension (2014) 610 – 616
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Figure 3. Cell apoptosis assays by FCM, Hoechst staining and WB. (a and b) HTR-8/SVneo cells treated with miR-101 mimics showed a significant decrease in apoptotic rate as compared with that of the miR-NC as demonstrated by FCM (a) and Hoechst staining (b). (c and d) Cells treated with siRNA that targeted miR-101 showed a significantly higher rate of apoptosis by FCM (c) and Hoechst staining (d). (e) WB analysis of apoptotic protein in cells that overexpressed miR-101 displayed a decrease in caspase-3 and an increase in Bcl-2. (f ) Cells treated with anti-miR-101 showed an increase in the expression of caspase-3 and a reduction in the expression of Bcl-2. Values are mean±s.d.; **Po0.01. A full colour version of this figure is available at the Journal of Human Hypertension journal online.
Journal of Human Hypertension (2014) 610 – 616
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Figure 4. Effects of miR-101 expression on the ERS signaling pathway. (a and b) HTR-8/SVneo cells treated with miR-101 mimics showed an inhibition of ERS-induced proteins compared with cells treated with miR-NC by WB. (c and d) Cells treated with anti-miR-101 showed a significantly higher activation of the ERS signaling cascade proteins by WB. (e and f ) HTR-8/SVneo cells treated with anti-miR-101 presented an obvious activation of ERS and this promotion could be partly reversed by the use of PBA. Values are mean±s.d.
protein degradation and unfolded protein response.46 Accumulation of unfolded proteins is closely related with the imbalanced homeostasis in ER, which then promotes apoptosis by activating PERK, ATF-6, CHOP, caspase-12 and so on. The unfolded protein response comprises three conserved signaling cascades including inositol-requiring protein1 (IRE1), PKR-like ER kinase (PERK) and activating transcription factor 6 (ATF-6); CHOP and caspase-12 were important executors inducing apoptosis in this pathway. In this study, we found that decreased miR-101 activated a series of ER cascade proteins PERK, ATF-6, CHOP and so on in trophoblast cells, suggesting that miR-101 has a role in inducing imbalanced homeostasis in ER of trophoblast cells, probably through targeting chaperone protein ERp44, and this needs further study. On the other hand, IP3R was reported to modulate cell apoptosis through regulating of Ca2 þ in ER.47 Although IP3R could be considered the ‘most valuable player’ of cellular Ca2 þ signaling,47 ERp44 could be considered long-term coaches for Ca2 þ signaling pathways.48 ERp44 could modulate IP3R activity and thus inhibit elevation of calcium, which probably contributes to an anti-apoptotic activity of cells in ER.49 Thus, this may offset part of its role on apoptosis promotion. Also, the specific regulation mode involved in this process remains to be studied deeply. Furthermore, many studies have already evidenced that one miRNA could modulate multiple genes, although one gene could also be regulated by multiple miRNAs.50 Therefore, miR-101 might regulate other target genes that are involved in cell apoptosis in PE and further studies are needed to clarify this point. In conclusion, our study showed that miR-101 was significantly decreased in PE placentas and was involved in regulating some apoptotic proteins during ERS. MiR-101 and its target ERp44 might be associated with the pathogenesis of PE and further insights into the functional and clinical implications of miR-101 may contribute to the early diagnosis and treatment of PE. However, the specific mechanism involved in ER stress-induced apoptosis and in the miR-101 targeting of ERp44 in regulating trophoblast cell apoptosis in PE remains to be further studied. & 2014 Macmillan Publishers Limited
What is known about this topic? PE placentas presented a lower expression level of miR-101. The differential expression of miR-101 could regulate trophoblast cell apoptosis. The regulation of miR-101 on trophoblast cells is associated with ERS by targeting ERp44. What this study adds? This study showed that miR-101 was significantly decreased in preeclampsia placentas and suppressed apoptosis in trophoblast cells during PE. Our findings indicated that the overexpression of miR-101 could downregulate ERp44 and was involved in regulating some apoptotic proteins during ERS. The functional and clinical implications of miR-101 may contribute to the early diagnosis and treatment of PE.
CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS This study was supported by The National Natural Science Foundation of China, Grant Nos 81070511 and 81270710.
REFERENCES 1 Powe CE, Levine RJ, Karumanchi SA. Preeclampsia, a disease of the maternal endothelium: the role of antiangiogenic factors and implications for later cardiovascular disease. Circulation 2011; 123(24): 2856–2869. 2 Askelund KJ, Chamley LW. Trophoblast deportation part I: review of the evidence demonstrating trophoblast shedding and deportation during human pregnancy. Placenta 2011; 32(10): 716–723. 3 Sacks GP, Studena K, Sargent K, Redman CW. Normal pregnancy and preeclampsia both produce inflammatory changes in peripheral blood leukocytes akin to those of sepsis. Am J Obstet Gynecol 1998; 179(1): 80–86.
Journal of Human Hypertension (2014) 610 – 616
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616 4 Saftlas AF, Beydoun H, Triche E. Immunogenetic determinants of preeclampsia and related pregnancy disorders: a systematic review. Obstet Gynecol 2005; 106(1): 162–172. 5 Conrad KP, Benyo DF. Placental cytokines and the pathogenesis of preeclampsia. Am J Reprod Immunol 1997; 37(3): 240–249. 6 Granger JP, Alexander BT, Llinas MT, Bennett WA, Khalil RA. Pathophysiology of hypertension during preeclampsia linking placental ischemia with endothelial dysfunction. Hypertension 2001; 38(3 Pt 2): 718–722. 7 Rana S, Karumanchi SA, Levine RJ, Venkatesha S, Rauh-Hain JA, Tamez H et al. Sequential changes in antiangiogenic factors in early pregnancy and risk of developing preeclampsia. Hypertension 2007; 50(1): 137–142. 8 Lam C, Lim KH, Karumanchi SA. Circulating angiogenic factors in the pathogenesis and prediction of preeclampsia. Hypertension 2005; 46(5): 1077–1085. 9 Noris M, Todeschini M, Cassis P, Pasta F, Cappellini A, Bonazzola S et al. L-arginine depletion in preeclampsia orients nitric oxide synthase toward oxidant species. Hypertension 2004; 43(3): 614–622. 10 Walsh SW. Maternal-placental interactions of oxidative stress and antioxidants in preeclampsia. Semin Reprod Endocrinol 1998; 16(1): 93–104. 11 Bartha JL, Romero-Carmona R, Torrejon-Cardoso R, Comino-Delgado R. Insulin, insulin-like growth factor-1, and insulin resistance in women with pregnancyinduced hypertension. Am J Obstet Gynecol 2002; 187(3): 735–740. 12 Jain A, Olovsson M, Burton GJ, Yung HW. Endothelin-1 induces endoplasmic reticulum stress by activating the PLC-IP(3) pathway: implications for placental pathophysiology in preeclampsia. Am J Pathol 2012; 180(6): 2309–2320. 13 Burton GJ, Yung HW. Endoplasmic reticulum stress in the pathogenesis of earlyonset pre-eclampsia. Pregnancy Hypertens 2011; 1(1-2): 72–78. 14 Yung HW, Calabrese S, Hynx D, Hemmings BA, Cetin I, Charnock-Jones DS et al. Evidence of placental translation inhibition and endoplasmic reticulum stress in the etiology of human intrauterine growth restriction. Am J Pathol 2008; 173(2): 451–462. 15 Lin WC, Chuang YC, Chang YS, Lai MD, Teng YN, Su IJ et al. Endoplasmic reticulum stress stimulates p53 expression through NF-kappaB activation. PLoS ONE 2012; 7(7): e39120. 16 Burton GJ, Jauniaux E. Placental oxidative stress: from miscarriage to preeclampsia. J Soc Gynecol Investig 2004; 11(6): 342–352. 17 Sun LZ, Yang NN, De W, Xiao YS. Proteomic analysis of proteins differentially expressed in preeclamptic trophoblasts. Gynecol Obstet Invest 2007; 64(1): 17–23. 18 Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004; 116(2): 281–297. 19 Small EM, Olson EN. Pervasive roles of microRNAs in cardiovascular biology. Nature 2011; 469(7330): 336–342. 20 Fineberg SK, Kosik KS, Davidson BL. MicroRNAs potentiate neural development. Neuron 2009; 64(3): 303–309. 21 Weidhaas J. Using microRNAs to understand cancer biology. Lancet Oncol 2010; 11(2): 106–107. 22 Forbes K, Farrokhnia F, Aplin JD, Westwood M. Dicer-dependent miRNAs provide an endogenous restraint on cytotrophoblast proliferation. Placenta 2012; 33(7): 581–585. 23 Kota J, Chivukula RR, O’Donnell KA, Wentzel EA, Montgomery CL, Hwang HW et al. Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model. Cell 2009; 137(6): 1005–1017. 24 Gartner JJ, Sethupathy P, Hatzigeorgiou AG, Fraser NW. Anti-apoptotic function of a microRNA encoded by the HSV-1 latency-associated transcript. Nature 2008; 451(7178): 600. 25 Geeleher P, Huang SR, Gamazon ER, Golden A, Seoighe C. The regulatory effect of miRNAs is a heritable genetic trait in humans. BMC Genomics 2012; 13: 383. 26 Follert P, Cremer H, Beclin C. MicroRNAs in brain development and function: a matter of flexibility and stability. Front Mol Neurosci 2014; 7: 5. 27 Bouyssou JM, Manier S, Huynh D, Issa S, Roccaro AM, Ghobrial IM. Regulation of microRNAs in Cancer Metastasis. Biochim Biophys Acta 2014; 1845(2): 255–265. 28 Fernandez-Hernando C, Baldan A. MicroRNAs and cardiovascular disease. Curr Genet Med Rep 2013; 1(1): 30–38.
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29 Zhu XM, Han T, Sargent IL, Yin GW, Yao YQ. Differential expression profile of microRNAs in human placentas from preeclamptic pregnancies vs normal pregnancies. Am J Obstet Gynecol 2009; 200(6): 661. 30 Kitroser E, Pomeranz M, Epstein SG, Fishman A, Drucker L, Sadeh-Mestechkin D et al. The involvement of eukaryotic translation initiation factor 4E in extravillous trophoblast cell function. Placenta 2012; 33(9): 717–724. 31 Liu S, Li Q, Na Q, Liu C. Endothelin-1 stimulates human trophoblast cell migration through Cdc42 activation. Placenta 2012; 33(9): 712–716. 32 Graham CH, Hawley TS, Hawley RG, MacDougall JR, Kerbel RS, Khoo N et al. Establishment and characterization of first trimester human trophoblast cells with extended lifespan. Exp Cell Res 1993; 206(2): 204–211. 33 Trobaugh DW, Gardner CL, Sun C, Haddow AD, Wang E, Chapnik E et al. RNA viruses can hijack vertebrate microRNAs to suppress innate immunity. Nature 2014; 506(7487): 245–248. 34 Su L, Liu R, Cheng W, Zhu M, Li X, Zhao S et al. Expression patterns of microRNAs in porcine endometrium and their potential roles in embryo implantation and Placentation. PLoS ONE 2014; 9(2): e87867. 35 Pineles BL, Romero R, Montenegro D, Tarca AL, Han YM, Kim YM et al. Distinct subsets of microRNAs are expressed differentially in the human placentas of patients with preeclampsia. Am J Obstet Gynecol 2007; 196(3): 261. 36 Muralimanoharan S, Maloyan A, Mele J, Guo C, Myatt LG, Myatt L. MIR-210 modulates mitochondrial respiration in placenta with preeclampsia. Placenta 2012; 33(10): 816–823. 37 Dai Y, Qiu Z, Diao Z, Shen L, Xue P, Sun H et al. MicroRNA-155 inhibits proliferation and migration of human extravillous trophoblast derived HTR-8/SVneo cells via down-regulating cyclin D1. Placenta 2012; 33(10): 824–829. 38 Zhang Y, Diao Z, Su L, Sun H, Li R, Cui H et al. MicroRNA-155 contributes to preeclampsia by down-regulating CYR61. Am J Obstet Gynecol 2010; 202(5): 461–466. 39 Leung AK, Sharp PA. MicroRNA functions in stress responses. Mol Cell 2010; 40(2): 205–215. 40 van Rooij E, Sutherland LB, Qi X, Richardson JA, Hill J, Olson EN. Control of stressdependent cardiac growth and gene expression by a microRNA. Science 2007; 316(5824): 575–579. 41 Flynt AS, Thatcher EJ, Burkewitz K, Li N, Liu Y, Patton JG. miR-8 microRNAs regulate the response to osmotic stress in zebrafish embryos. J Cell Biol 2009; 185(1): 115–127. 42 Brennecke J, Hipfner DR, Stark A, Russell RB, Cohen SM. bantam encodes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila. Cell 2003; 113(1): 25–36. 43 Whitley GS, Dash PR, Ayling LJ, Prefumo F, Thilaganathan B, Cartwright JE. Increased apoptosis in first trimester extravillous trophoblasts from pregnancies at higher risk of developing preeclampsia. Am J Pathol 2007; 170(6): 1903–1909. 44 Longtine MS, Chen B, Odibo AO, Zhong Y, Nelson DM. Villous trophoblast apoptosis is elevated and restricted to cytotrophoblasts in pregnancies complicated by preeclampsia, IUGR, or preeclampsia with IUGR. Placenta 2012; 33(5): 352–359. 45 Anelli T, Alessio M, Mezghrani A, Simmen T, Talamo F, Bachi A et al. ERp44, a novel endoplasmic reticulum folding assistant of the thioredoxin family. EMBO J 2002; 21(4): 835–844. 46 Moenner M, Pluquet O, Bouchecareilh M, Chevet E. Integrated endoplasmic reticulum stress responses in cancer. Cancer Res 2007; 67(22): 10631–10634. 47 Hamada K, Miyata T, Mayanagi K, Hirota J, Mikoshiba K. Two-state conformational changes in inositol 1,4,5-trisphosphate receptor regulated by calcium. J Biol Chem 2002; 277(24): 21115–21118. 48 Choe CU, Ehrlich BE. The inositol 1,4,5-trisphosphate receptor (IP3R) and its regulators: sometimes good and sometimes bad teamwork. Sci STKE 2006; 2006(363): e15. 49 Marks AR. Intracellular calcium-release channels: regulators of cell life and death. Am J Physiol 1997; 272(2 Pt 2): H597–H605. 50 Peter ME. Targeting of mRNAs by multiple miRNAs: the next step. Oncogene 2010; 29(15): 2161–2164.
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ORIGINAL ARTICLE
MiR-101 regulates apoptosis of trophoblast HTR-8/SVneo cells by targeting endoplasmic reticulum (ER) protein 44 during preeclampsia Y Zou1,3, Z Jiang1,3, X Yu2,3, Y Zhang1, M Sun2, W Wang1, Z Ge1, W De2 and L Sun1 To investigate a possible association between miR-101 and apoptosis of human trophoblast cells mediated by endoplasmic reticulum protein 44 (ERp44) in preeclampsia (PE), we explored the expression of miR-101 in PE placentas (n ¼ 30) compared with normotensive pregnant placentas (n ¼ 30) and the correlation between miR-101 and ERp44 was also analyzed. Furthermore, both the apoptotic rate of trophoblast cells and the ER stress-induced apoptotic proteins were assayed when the HTR-8/SVneo cells were treated with miR-101 mimics or inhibitors in vitro. We found a lower expression of miR-101 and an inverse correlation between miR101 and ERp44 protein in PE placentas. Upregulation of miR-101 expression could inhibit trophoblast HTR-8/SVneo cell apoptosis and repress ER stress-induced apoptotic proteins by targeting ERp44 in vitro, whereas inhibition of miR-101 could induce HTR-8/ SVneo cell apoptosis. Our findings indicated that overexpression of miR-101 could downregulate ERp44 and suppress apoptosis in trophoblast cells during PE. Therefore, loss of miR-101 expression could contribute to ER stress-induced trophoblast cell apoptosis by targeting ERp44. Journal of Human Hypertension (2014) 28, 610–616; doi:10.1038/jhh.2014.35; published online 8 May 2014
INTRODUCTION Preeclampsia (PE), as one of the most common pathologic complications of pregnancy, afflicts B3–5%1 of pregnancies and is a leading contributor to perinatal morbidity and pregnancyassociated mortality, especially in developing countries. The clinical characteristics of this disease include: a new onset of hypertension (blood pressure X140/90 mm Hg in previously normotensive women after the 20th week of pregnancy) and proteinuria (X300 mg per 24-h urine collection) during the second stage of pregnancy. The delivery of the baby and placenta is the only curative treatment to reverse the syndromes.2 Because of the adverse outcome of this disease, more and more research on it has been continuously carried out; however, the specific pathogenesis of this disorder remains to be elucidated. So far, there have been many mechanisms associated with the pathogenesis of PE, such as inflammatory cytokines,3–5 endothelial dysfunction,6 imbalance between proangiogenic and antiangiogenic factors,7,8 oxidative stress9,10 and finally genetic and dietary factors.11 Recent evidence indicates placental endoplasmic reticulum stress (ERS) involved in pathophysiological characteristics of PE.12,13 In endoplasmic reticulum, molecular chaperone proteins have crucial roles in the process of synthesizing and packing proteins in the early stages of ERS.14 Severe or prolonged ERS induces activation of unique pathways that lead to cell death through apoptosis.15 Therefore, the generation of placental ERS is a key intermediary event in the pathology of PE.16 However, the abnormal details of the molecular mechanism of ERS still remain to be studied.
Our previous studies identified several dysregulated expressions of endoplasmic reticulum molecular chaperone proteins in PE placenta by two-dimensional polyacrylamide gel electrophoresis.17 Four proteins (protein disulfide isomerase precursor, endoplasmic reticulum resident protein, dihydrolipoyl dehydrogenase and TIM21-like protein) were found to be significantly upregulated in PE and three were downregulated (disulfide isomerase ER-60, peroxiredoxin 2 and D3, 5-D2, 4-dienoyl-CoA isomerase). Among those upregulated proteins, levels of endoplasmic reticulum protein 44 (ERp44) are about sixfold in PE placenta compared with normal controls. This indicates that ERp44 may have an important role in the pathogenesis of PE; however, there is no report about the regulatory role of ERp44 in PE. Recent studies have revealed that micro RNAs (miRNAs) could inhibit protein expression through base pairing with sequences located predominantly in the 30 untranslated regions (30 -UTRs) of mRNA targets.18 The functions of miRNAs often become pronounced under conditions of stress,19 so we postulate that ERp44 might be modulated by miRNA in ERS. MiRNAs are a class of highly conserved endogenous small noncoding RNA molecules (19–22 nt), which have important posttranscriptional regulatory roles of targeting mRNAs for cleavage or translational repression.20,21 Recent data indicate that miRNAs have a fundamental role in many biologic and pathologic processes, which include cell proliferation,22 invasion,23 apoptosis,24,25 cell development,26 carcinogenesis27 and cardiovascular disease.28 There have been many reports suggesting that many miRNAs have differential expression in PE, including miR-101.29 In this study, we found a decreased expression
1 Department of Obstetrics and Gynecology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China and 2Department of Biochemistry and Molecular Biology, Nanjing Medical University, Nanjing, China. Correspondence: Professor L Sun, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing 210000, China. E-mail: sunlizhou@njmu. edu. cn 3 These authors contributed equally to this work and should be regarded as joint first authors. Received 4 November 2013; revised 2 March 2014; accepted 26 March 2014; published online 8 May 2014
MiR-101 regulates apoptosis of HTR-8/SVneo cells Y Zou et al
611 of miR-101(-3p) in 30 severe human PE placentas by quantitative PCR on comparing with 30 normal pregnant ones and detecting 23 miRNAs in all. By using bioinformatics analysis (softwares: Target Scan (Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA, USA), PicTar (Max Del Brunet Center, Berlin, Germany), miTarget (http://cbit.snu.ac.kr/~miTarget), we found that there are miR-101-binding sites in the 30 -UTR of ERp44 mRNA (CAUGACA: GUACUGU). In addition, this was proved by the luciferase reporter assay in our study. Then, overexpression of miR-101 inhibited trophoblast HTR-8/SVneo cell apoptosis and repressed ER stress-induced apoptotic proteins by targeting ERp44 in vitro, whereas inhibition of miR-101 induced cell apoptosis. Thus, miR-101 might contribute to ER stress-induced trophoblast cell apoptosis by targeting ERp44. MATERIALS AND METHODS Sample collection Placental tissues were obtained from primipara women aged 20–36 years who underwent cesarean deliveries between 2011 and 2012 in the Department of Gynecology and Obstetrics of the People’s Hospital of Jiangsu Province, China. All tissues were collected immediately after placental delivery from the uterus and washed with sterile phosphatebuffered saline before being snap-frozen in liquid nitrogen for transportation to the lab. Tissues were temporarily stored at 80 1C for RNA and protein extraction. All of our experiments were approved by the Ethics Board of the First Affiliated Hospital of Nanjing Medical University, and informed consents have been obtained from all the pregnant women as well.
Cell culture and treatment Human embryonic kidney HEK 293T cells (used for luciferase assay) were maintained in Dulbecco’s modified Eagle medium, supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U ml 1 penicillin and 100 mg ml 1 streptomycin in standard culture conditions (37 1C in 5% humidified CO2 incubator). After placing into 48-well plates, HEK 293T cells were cotransfected with pCDNA-miR-101 or pCDNA (as negative control), pRLTK (2 ng; Promega, Shanghai, China) and luciferase reporter plasmids (50 ng) containing wild type or mutant type of ERp44 30 -UTR using Lipofectamine 2000 (Invitrogen, Shanghai, China). An immortalized first-trimester extravillous trophoblast cells (EVT) cell line HTR-8/SVneo30 (kindly provided by Dr Charles Graham, Queen’s University, Canada), which is derived from a short-lived primary EVT cell line, was used in the present study. With the behavior of trophoblast cells, many studies31,32 have been based on this cell line to simulate the behavior of trophoblast cells in pregnancy. The human trophoblast-derived cell line was maintained in 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U ml 1 penicillin and 100 mg ml 1 streptomycin in standard culture conditions (37 1C in 5% humidified CO2 incubator). Cells were sowed into six-well plates for 12 h before being transiently transfected with miRNA duplexes at 100 nM final concentrations (hsa-mir-101 miRIDIAN Mimics MI0000739) and its inhibitor (MIMAT0000099). After transfection for 48 h, cells were collected to detect the transfection efficiency.
RNA extraction and real-time reverse transcription-PCR Total RNA was extracted from B0.1 g placental tissues or cultured cells with Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) and 1bromo-3-chloropropane and isopropanol.33,34 Then, a Reverse Transcription Kit (Takara) was used for the synthesis of complementary DNA by adding 1 mg of total RNA to the RT Reaction Mix. For quantitative PCR, the amplification of complementary DNA was performed by adding Power SYBR Green (Takara) to a total volume of 20 ml reaction mix. According to the manufacturer’s instructions, the reverse transcription was performed at 37 1C for 15 min and at 85 1C for 5 s. To normalize the results for the quantitative PCR, U6 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expressions were used. The sequence of the primer was as follows: miR-101: reverse transcribed primer: 50 -CGCATGAATACGCCAAACAACTTCAGTTAT-30 ; forward: 50 -CTCGCATGAATACGCCAAACAA-30 ; reverse: 50 -CCAGCGGTTACAGTACTGTGATA-30 . U6: reverse transcribed primer: 50 -AACGCTTCACGAATTTGCGT-30 ; forward: 50 -CTCGCTTCGGCAGCACA-30 ; reverse: 50 -AACGCTTCACGAATTTGCGT-30 . GAPDH: forward: 50 -GACTCATGACCACAGTCCATGC-30 ; reverse: 50 -AGAGGCAGGGATGATGTTCTG-30 . An ABI 7500 (Applied Biosystems, Paisley, UK) was used to carry out quantitative PCR and data collections.
Hoechst staining assay HTR-8/SVneo cells planted into six-well plates were transiently transfected with has-mir-101 and its inhibitor for 48 h, and then Hoechst 33258 (Beyotime) was added into the culture medium to induce nuclear morphology changes. Finally, the percentage of Hoechst-positive nuclei per optical field (at least 50 fields) was counted under fluorescence microscopy by using a filter for Hoechst33258.
Flow cytometry Cells transiently transfected with has-mir-101 and inhibitor for 48 h were collected using trypsin without EDTA, washed with phosphate-buffered saline, resuspended in 1 ml binding buffer and stained with fluorescein isothiocyanate-annexin V and propidium iodide at room temperature for 15 min in the dark according to the manufacturer’s recommendations. The analysis of the cells was carried out by a flow cytometry analysis (FCM, FAC Scan; BD Biosciences, Becton, Dickinson and Company, Franklin Lakes, NJ, USA) equipped with a CellQuest software (BD Biosciences).
Luciferase assay HEK 293T cells were placed in 48-well plates and cotransfected with pCDNA-miR-101 or pCDNA (200 ng), pRL-TK (2 ng; Promega) and Luciferase reporter plasmids (50 ng) containing wild type or mutant type of ERp44 30 -UTR using Lipofectamine 2000 (Invitrogen). The firefly and Renilla luciferase activities were measured by the Dual-Luciferase Reporter Assay System (Promega) after transfection for 48 h.
Treatment of HTR-8/SVneo cells with sodium 4-phenylbutyrate Western blotting analysis Cells (HTR-8/SVneo) transfected with hsa-mir-101 and inhibitor were lysed for protein extraction by using mammalian reagent RIPA (Beyotime, Shanghai, China) supplemented with protease inhibitors cocktail (Roche, Penzberg, Germany) and phenylmethylsulfonyl-fluoride (Roche). The Bradford method was used for the determination of protein concentration in each sample. Samples containing 50–100 mg of protein extractions were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Next, the separated proteins were transferred to 0.22 mm nitrocellulose (Sigma, St Louis, MO, USA) and incubated with specific antibodies (ERp44, caspase-12, chop, ATF-6, PERK Caspase-3, BCL-2, 1:1000, all purchased from Cell Signaling Technology (Boston, MA, USA); ATF-4, 1:500, Bioworld, Shanghai, China; and IRE 1, 1:500, SAB Biotech, Nanjing, China). The secondary antibody was horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1:1000; Beijing ZhongShan Biotechnology Co., Beijing, China). Quantity One software (Bio-Rad, Hercules, CA, USA) was used to quantify the intensity of the autoradiograms protein bands, and the b-actin antibody (1:1000, Santa Cruz, CA, USA) was used as control. & 2014 Macmillan Publishers Limited
HTR-8/SVneo cells were seeded into six-well culture plates overnight and exposed to 20 nm of sodium 4-phenylbutyrate (PBA, Sigma) for 24 h. Next, the cells were collected for protein extraction and assays of ERS signaling cascade proteins.
Statistical analysis All data were expressed as mean±s.d. and all statistical analysis was performed using SPSS statistical software package (SPSS Inc., Chicago, IL, USA). Po0.05 was considered statistically significant.
RESULTS Clinical characteristics The clinical data were obtained from all the patients who were sampled. Placental tissues were classified into two groups: PE (PE, n ¼ 30) and normal pregnant (N, n ¼ 30). PE was diagnosed by the standard criteria, whereas normal pregnancy was defined as not Journal of Human Hypertension (2014) 610 – 616
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612 having PE or any other complications (including maternal history of hypertension and/or renal disease, maternal infection, smoking, alcoholism, chemical dependency and fetal congenital anomalies). There were no significant differences between the two groups in maternal age, gestational age (week) and C-reaction protein levels (P40.05), although the blood pressure and proteinuria were significantly higher in preeclamptic women (Po0.01) as compared with women with normal pregnancies (Table 1). The expression level of miR-101 is downregulated in human placental tissues By detecting 30 PE compared with normal pregnant placental tissues using quantitative reverse transcription-PCR analysis, we found that the expression level of miR-101 in PE was 25% lower than that of normal pregnancy (Figure 1a). Table 1, Po0.01, shows the clinical characteristics of 30 normal and preeclamptic pregnancies. According to our previous studies, the expression level of ERp44 in human PE placental tissues was six times upregulated compared with normal placenta. The correlation analysis showed that there was a negative correlation between miR-101 and Erp44 (Figure 1b). Thus, the negative correlation between miR-101 and ERp44 indicated that miR-101 may have a part in PE through ERp44. MiR-101 contributes to the expression of ERp44 by interacting with its 30 -UTR To further explore the relationship between miR-101 and ERp44, we conducted a bioinformatics screen to identify that there are seven
Table 1. Clinical characteristics of normal and preeclamptic pregnancies a
Variable
PE (n ¼ 30)
N (n ¼ 30)
P-value Control vs PE
Maternal age Proteinuria (g per day) Gestational age (week) Systolic blood pressure, mm Hg Diastolic blood pressure, mm Hg Body weight of infant (g) C-reaction protein Maternal smoking (number)
30.2±5.7 40.3 36.5±3.7 169±20.1
30.6±3.5 o0.3 39.1±1.2 112±6.8
40.05 o0.01 40.05 o0.01
115±12.8
77±7.1
o0.01
2582±740 8.1±3.1 2
3322±413 5.9±2.9 1
o0.05 40.05 40.05
All results are presented as mean±s.d. aObtained by one-way analysis of variance using SPSS 13.0 software (SPSS Inc.).
continuous complementary base pairings between the mRNA sequence of ERp44 and miR-101. Next, luciferase assays were used to verify the target relationship between miR-101 and ERp44. We found that miR-101 decreased 30% of the activity of the luciferase reporter containing the wild-type 30 -UTR of ERp44 mRNA (Figure 2a, Po0.01). However, when the predicted binding site of miR-101 with the 30 -UTR of ERp44 was mutated, luciferase activity did not drop sharply in the 30 -UTRs with mutant-binding sites. Thus, the data proved that miR-101 may serve as a target of ERp44 in regulating the apoptosis of trophoblast cells when ERS occurs. Exogenous downregulation or overexpression of miR-101 in HTR-8/SVneo cells and its contribution to ERp44 expression To explore the role of miR-101 in ERp44, we conducted the upregulation as well as downregulation of miR-101; miR-101 mimics or miR-101 inhibitors (constructed by Invitrogen) were transiently transfected into HTR-8/SVneo cells. After transfection for 48 h, cells were collected to detect the expression of miR-101 to make sure of the success of overexpression and downregulation. Quantitative reverse transcription-PCR assays revealed that expression of miR101 transfected with miR-101 mimics was 28-fold higher than that in normal control and 0.45-fold in miR-101 inhibitors (Figure 2b, Po0.01). To assess the intermediary molecular mechanism by which miR-101 acts as an inhibitor of trophoblast cell apoptosis and the ERS pathway, we confirmed the expression level of ERp44 proteins after miR-101 was overexpressed or inhibited in HTR-8/ SVneo cells by using western blotting (WB). There was a significant increase in ERp44 protein levels in cells transfected with siRNA targeting miR-101, whereas a decrease occurred in cells treated with miR-101 mimics (Figures 2c and d, Po0.01). Modulation of miR-101 on cell apoptosis in vitro The decrease in miR-101 in PE placentas prompted us to detect the possible biological significance of miR-101 in trophoblast cell apoptosis. We conducted Hoechst staining assay and FCM to detect the apoptotic cells and WB assays to identify apoptotic proteins in cells treated with miR-101. When HTR-8/SVneo cells were transfected with si-RNAs targeting miR-101, a significant increase in apoptosis was observed (Figures 3c and d, Po0.01) as compared with those infected with miR-101 mimics using both FCM (Figure 3a, Po0.01) and Hoechst staining assay (Figure 3b, Po0.01). These results indicated that higher miR-101 could inhibit HTR-8/SVneo cell apoptosis in vitro. The apoptotic protein caspase3 was significantly increased in cells transfected with siRNA targeting miR-101, whereas it was decreased in anti-apoptotic protein Bcl-2 (Figure 3f, Po0.01). On the other hand, WB analysis also indicated that the expression level of caspase-3 protein displayed a decrease in cells treated with miR-101 mimics
Figure 1. (a and b) Relative expression of miR-101 was lower in PE placental tissues as compared with the normal pregnancies, as determined by quantitative PCR analysis (a) and the negative correlation between miR-101 and ERp44 (b). Values are mean±s.d.; **Po0.01. Journal of Human Hypertension (2014) 610 – 616
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Figure 2. Target relationship between miR-101 and ERp44 and the overexpression of miR-01 could partially block the expression level of ERp44 protein. (a) The luciferase reporter plasmid containing wild-type or mutant ERp44 30 -UTR was cotransfected into HEK-293T cells with pCDNA-miR-101 or pCDNA-miR-NC. Luciferase activity was determined by the dual-luciferase assay and shown as the relative firefly activity normalized to Renilla activity. (b) Relative expression of miR-101 after HTR-8/SVneo cells were transfected with miR-101 mimics or anti-miR101. (c and d) The expression of ERp44 protein was analyzed by WB when cells were treated with miR-101 or anti-miR-101 and b-actin was used as control. Values are mean±s.d.; **Po0.01. NS, not significant.
(Figure 3e, Po0.01), whereas the expression level of Bcl-2 protein presented reverse results (Figure 3e, Po0.01). Effects of miR-101 expression on the ERS signaling pathway A series of proteins such as PERK and ATF-4, ATF-6, CHOP, caspase12 and IRE1p, which are involved in the ERS pathways, were tested after HTR-8/SVneo cells were treated with miR-101 mimics and inhibitor. WB assays showed that PERK, ATF-6, CHOP and caspase12 showed a significant decrease in cells treated with miR-101 mimics (Figures 4a and b, Po0.01) and an increase in cells transfected with miR-101 inhibitor (Figures 4c and d, Po0.01) compared with anti-miR-NC. These data indicated that miR-101 could prevent the progression of ERS by inhibiting some participants in this way. Reverse activation of Anti-miR-101 to ERS under the inhibition effect of PBA To further investigate the possible biological significance of antimiR-101 to ERS, HTR-8/SVneo cells were treated with anti-miR-101 under the effect of PBA (inhibition of ERS). A decrease in ERSassociated proteins was detected when cells treated with both anti-miR-101 and PBA were compared with those treated with anti-miR-101 alone, whereas an increase in these proteins was detected when cells treated with PBA alone were compared with none of both treatments (Figures 4e and f, Po0.05). DISCUSSION It was evidenced that PE is associated with alterations in placental miRNA expression.35 Many miRNAs have been found to be involved & 2014 Macmillan Publishers Limited
in PE, such as miR-210,36 miR-155,37 miR-18238 and so on. Zhu et al.29 also detected a series of downregulated miRNAs in severe preeclamptic placentas compared with normal pregnant placentas, and miR-101 is among these. In the present study, we verified that miR-101 was markedly decreased in PE placentas than in normal pregnancy, suggesting that the low expression of miR-101 was involved in PE pathogenesis. Accordingly, recent studies have revealed that miRNAs often deeply influence the responses of organisms to physiologic and pathophysiologic stress.39 In addition, genetic knockout of specific miRNAs renders the organisms incapable of coping with many stresses.40 For example, mice deficient in miR-208 cannot deal with cardiac overload, and inactivation of miR-8 renders zebra fish incapable of responding to osmotic stress.41 So miRNAs may be closely related to the pathogenesis of PE by regulating the stress response. In recent years, many studies have focused on the association of miRNA with cell apoptosis through interaction with their target genes.42 PE is also characterized by an increase in apoptosis of trophoblast cells in placenta.43,44 To explore the role of miR-101 in modulating trophoblast cell apoptosis in PE, we conducted FCM analysis. The results suggested that low expression of miR-101 promoted trophoblast cell apoptosis in PE. MiRNAs are primarily referred to bind to the 30 -UTR of targets to regulate expression levels of multiple proteins.18 Accordingly, a negative relationship between miR-101 and ERp44 and luciferase assay in our study indicates that the apoptosis role of miR-101 in PE may be mediated by ERp44. ERp44 contains a thioredoxin domain with a CRFS motif and can be induced by agents that cause the accumulation of unfolded proteins in the ER during ER stress.45 There are several specific signaling pathways triggered by ER stress, including ER-associated Journal of Human Hypertension (2014) 610 – 616
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Figure 3. Cell apoptosis assays by FCM, Hoechst staining and WB. (a and b) HTR-8/SVneo cells treated with miR-101 mimics showed a significant decrease in apoptotic rate as compared with that of the miR-NC as demonstrated by FCM (a) and Hoechst staining (b). (c and d) Cells treated with siRNA that targeted miR-101 showed a significantly higher rate of apoptosis by FCM (c) and Hoechst staining (d). (e) WB analysis of apoptotic protein in cells that overexpressed miR-101 displayed a decrease in caspase-3 and an increase in Bcl-2. (f ) Cells treated with anti-miR-101 showed an increase in the expression of caspase-3 and a reduction in the expression of Bcl-2. Values are mean±s.d.; **Po0.01. A full colour version of this figure is available at the Journal of Human Hypertension journal online.
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Figure 4. Effects of miR-101 expression on the ERS signaling pathway. (a and b) HTR-8/SVneo cells treated with miR-101 mimics showed an inhibition of ERS-induced proteins compared with cells treated with miR-NC by WB. (c and d) Cells treated with anti-miR-101 showed a significantly higher activation of the ERS signaling cascade proteins by WB. (e and f ) HTR-8/SVneo cells treated with anti-miR-101 presented an obvious activation of ERS and this promotion could be partly reversed by the use of PBA. Values are mean±s.d.
protein degradation and unfolded protein response.46 Accumulation of unfolded proteins is closely related with the imbalanced homeostasis in ER, which then promotes apoptosis by activating PERK, ATF-6, CHOP, caspase-12 and so on. The unfolded protein response comprises three conserved signaling cascades including inositol-requiring protein1 (IRE1), PKR-like ER kinase (PERK) and activating transcription factor 6 (ATF-6); CHOP and caspase-12 were important executors inducing apoptosis in this pathway. In this study, we found that decreased miR-101 activated a series of ER cascade proteins PERK, ATF-6, CHOP and so on in trophoblast cells, suggesting that miR-101 has a role in inducing imbalanced homeostasis in ER of trophoblast cells, probably through targeting chaperone protein ERp44, and this needs further study. On the other hand, IP3R was reported to modulate cell apoptosis through regulating of Ca2 þ in ER.47 Although IP3R could be considered the ‘most valuable player’ of cellular Ca2 þ signaling,47 ERp44 could be considered long-term coaches for Ca2 þ signaling pathways.48 ERp44 could modulate IP3R activity and thus inhibit elevation of calcium, which probably contributes to an anti-apoptotic activity of cells in ER.49 Thus, this may offset part of its role on apoptosis promotion. Also, the specific regulation mode involved in this process remains to be studied deeply. Furthermore, many studies have already evidenced that one miRNA could modulate multiple genes, although one gene could also be regulated by multiple miRNAs.50 Therefore, miR-101 might regulate other target genes that are involved in cell apoptosis in PE and further studies are needed to clarify this point. In conclusion, our study showed that miR-101 was significantly decreased in PE placentas and was involved in regulating some apoptotic proteins during ERS. MiR-101 and its target ERp44 might be associated with the pathogenesis of PE and further insights into the functional and clinical implications of miR-101 may contribute to the early diagnosis and treatment of PE. However, the specific mechanism involved in ER stress-induced apoptosis and in the miR-101 targeting of ERp44 in regulating trophoblast cell apoptosis in PE remains to be further studied. & 2014 Macmillan Publishers Limited
What is known about this topic? PE placentas presented a lower expression level of miR-101. The differential expression of miR-101 could regulate trophoblast cell apoptosis. The regulation of miR-101 on trophoblast cells is associated with ERS by targeting ERp44. What this study adds? This study showed that miR-101 was significantly decreased in preeclampsia placentas and suppressed apoptosis in trophoblast cells during PE. Our findings indicated that the overexpression of miR-101 could downregulate ERp44 and was involved in regulating some apoptotic proteins during ERS. The functional and clinical implications of miR-101 may contribute to the early diagnosis and treatment of PE.
CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS This study was supported by The National Natural Science Foundation of China, Grant Nos 81070511 and 81270710.
REFERENCES 1 Powe CE, Levine RJ, Karumanchi SA. Preeclampsia, a disease of the maternal endothelium: the role of antiangiogenic factors and implications for later cardiovascular disease. Circulation 2011; 123(24): 2856–2869. 2 Askelund KJ, Chamley LW. Trophoblast deportation part I: review of the evidence demonstrating trophoblast shedding and deportation during human pregnancy. Placenta 2011; 32(10): 716–723. 3 Sacks GP, Studena K, Sargent K, Redman CW. Normal pregnancy and preeclampsia both produce inflammatory changes in peripheral blood leukocytes akin to those of sepsis. Am J Obstet Gynecol 1998; 179(1): 80–86.
Journal of Human Hypertension (2014) 610 – 616
MiR-101 regulates apoptosis of HTR-8/SVneo cells Y Zou et al
616 4 Saftlas AF, Beydoun H, Triche E. Immunogenetic determinants of preeclampsia and related pregnancy disorders: a systematic review. Obstet Gynecol 2005; 106(1): 162–172. 5 Conrad KP, Benyo DF. Placental cytokines and the pathogenesis of preeclampsia. Am J Reprod Immunol 1997; 37(3): 240–249. 6 Granger JP, Alexander BT, Llinas MT, Bennett WA, Khalil RA. Pathophysiology of hypertension during preeclampsia linking placental ischemia with endothelial dysfunction. Hypertension 2001; 38(3 Pt 2): 718–722. 7 Rana S, Karumanchi SA, Levine RJ, Venkatesha S, Rauh-Hain JA, Tamez H et al. Sequential changes in antiangiogenic factors in early pregnancy and risk of developing preeclampsia. Hypertension 2007; 50(1): 137–142. 8 Lam C, Lim KH, Karumanchi SA. Circulating angiogenic factors in the pathogenesis and prediction of preeclampsia. Hypertension 2005; 46(5): 1077–1085. 9 Noris M, Todeschini M, Cassis P, Pasta F, Cappellini A, Bonazzola S et al. L-arginine depletion in preeclampsia orients nitric oxide synthase toward oxidant species. Hypertension 2004; 43(3): 614–622. 10 Walsh SW. Maternal-placental interactions of oxidative stress and antioxidants in preeclampsia. Semin Reprod Endocrinol 1998; 16(1): 93–104. 11 Bartha JL, Romero-Carmona R, Torrejon-Cardoso R, Comino-Delgado R. Insulin, insulin-like growth factor-1, and insulin resistance in women with pregnancyinduced hypertension. Am J Obstet Gynecol 2002; 187(3): 735–740. 12 Jain A, Olovsson M, Burton GJ, Yung HW. Endothelin-1 induces endoplasmic reticulum stress by activating the PLC-IP(3) pathway: implications for placental pathophysiology in preeclampsia. Am J Pathol 2012; 180(6): 2309–2320. 13 Burton GJ, Yung HW. Endoplasmic reticulum stress in the pathogenesis of earlyonset pre-eclampsia. Pregnancy Hypertens 2011; 1(1-2): 72–78. 14 Yung HW, Calabrese S, Hynx D, Hemmings BA, Cetin I, Charnock-Jones DS et al. Evidence of placental translation inhibition and endoplasmic reticulum stress in the etiology of human intrauterine growth restriction. Am J Pathol 2008; 173(2): 451–462. 15 Lin WC, Chuang YC, Chang YS, Lai MD, Teng YN, Su IJ et al. Endoplasmic reticulum stress stimulates p53 expression through NF-kappaB activation. PLoS ONE 2012; 7(7): e39120. 16 Burton GJ, Jauniaux E. Placental oxidative stress: from miscarriage to preeclampsia. J Soc Gynecol Investig 2004; 11(6): 342–352. 17 Sun LZ, Yang NN, De W, Xiao YS. Proteomic analysis of proteins differentially expressed in preeclamptic trophoblasts. Gynecol Obstet Invest 2007; 64(1): 17–23. 18 Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004; 116(2): 281–297. 19 Small EM, Olson EN. Pervasive roles of microRNAs in cardiovascular biology. Nature 2011; 469(7330): 336–342. 20 Fineberg SK, Kosik KS, Davidson BL. MicroRNAs potentiate neural development. Neuron 2009; 64(3): 303–309. 21 Weidhaas J. Using microRNAs to understand cancer biology. Lancet Oncol 2010; 11(2): 106–107. 22 Forbes K, Farrokhnia F, Aplin JD, Westwood M. Dicer-dependent miRNAs provide an endogenous restraint on cytotrophoblast proliferation. Placenta 2012; 33(7): 581–585. 23 Kota J, Chivukula RR, O’Donnell KA, Wentzel EA, Montgomery CL, Hwang HW et al. Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model. Cell 2009; 137(6): 1005–1017. 24 Gartner JJ, Sethupathy P, Hatzigeorgiou AG, Fraser NW. Anti-apoptotic function of a microRNA encoded by the HSV-1 latency-associated transcript. Nature 2008; 451(7178): 600. 25 Geeleher P, Huang SR, Gamazon ER, Golden A, Seoighe C. The regulatory effect of miRNAs is a heritable genetic trait in humans. BMC Genomics 2012; 13: 383. 26 Follert P, Cremer H, Beclin C. MicroRNAs in brain development and function: a matter of flexibility and stability. Front Mol Neurosci 2014; 7: 5. 27 Bouyssou JM, Manier S, Huynh D, Issa S, Roccaro AM, Ghobrial IM. Regulation of microRNAs in Cancer Metastasis. Biochim Biophys Acta 2014; 1845(2): 255–265. 28 Fernandez-Hernando C, Baldan A. MicroRNAs and cardiovascular disease. Curr Genet Med Rep 2013; 1(1): 30–38.
Journal of Human Hypertension (2014) 610 – 616
29 Zhu XM, Han T, Sargent IL, Yin GW, Yao YQ. Differential expression profile of microRNAs in human placentas from preeclamptic pregnancies vs normal pregnancies. Am J Obstet Gynecol 2009; 200(6): 661. 30 Kitroser E, Pomeranz M, Epstein SG, Fishman A, Drucker L, Sadeh-Mestechkin D et al. The involvement of eukaryotic translation initiation factor 4E in extravillous trophoblast cell function. Placenta 2012; 33(9): 717–724. 31 Liu S, Li Q, Na Q, Liu C. Endothelin-1 stimulates human trophoblast cell migration through Cdc42 activation. Placenta 2012; 33(9): 712–716. 32 Graham CH, Hawley TS, Hawley RG, MacDougall JR, Kerbel RS, Khoo N et al. Establishment and characterization of first trimester human trophoblast cells with extended lifespan. Exp Cell Res 1993; 206(2): 204–211. 33 Trobaugh DW, Gardner CL, Sun C, Haddow AD, Wang E, Chapnik E et al. RNA viruses can hijack vertebrate microRNAs to suppress innate immunity. Nature 2014; 506(7487): 245–248. 34 Su L, Liu R, Cheng W, Zhu M, Li X, Zhao S et al. Expression patterns of microRNAs in porcine endometrium and their potential roles in embryo implantation and Placentation. PLoS ONE 2014; 9(2): e87867. 35 Pineles BL, Romero R, Montenegro D, Tarca AL, Han YM, Kim YM et al. Distinct subsets of microRNAs are expressed differentially in the human placentas of patients with preeclampsia. Am J Obstet Gynecol 2007; 196(3): 261. 36 Muralimanoharan S, Maloyan A, Mele J, Guo C, Myatt LG, Myatt L. MIR-210 modulates mitochondrial respiration in placenta with preeclampsia. Placenta 2012; 33(10): 816–823. 37 Dai Y, Qiu Z, Diao Z, Shen L, Xue P, Sun H et al. MicroRNA-155 inhibits proliferation and migration of human extravillous trophoblast derived HTR-8/SVneo cells via down-regulating cyclin D1. Placenta 2012; 33(10): 824–829. 38 Zhang Y, Diao Z, Su L, Sun H, Li R, Cui H et al. MicroRNA-155 contributes to preeclampsia by down-regulating CYR61. Am J Obstet Gynecol 2010; 202(5): 461–466. 39 Leung AK, Sharp PA. MicroRNA functions in stress responses. Mol Cell 2010; 40(2): 205–215. 40 van Rooij E, Sutherland LB, Qi X, Richardson JA, Hill J, Olson EN. Control of stressdependent cardiac growth and gene expression by a microRNA. Science 2007; 316(5824): 575–579. 41 Flynt AS, Thatcher EJ, Burkewitz K, Li N, Liu Y, Patton JG. miR-8 microRNAs regulate the response to osmotic stress in zebrafish embryos. J Cell Biol 2009; 185(1): 115–127. 42 Brennecke J, Hipfner DR, Stark A, Russell RB, Cohen SM. bantam encodes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila. Cell 2003; 113(1): 25–36. 43 Whitley GS, Dash PR, Ayling LJ, Prefumo F, Thilaganathan B, Cartwright JE. Increased apoptosis in first trimester extravillous trophoblasts from pregnancies at higher risk of developing preeclampsia. Am J Pathol 2007; 170(6): 1903–1909. 44 Longtine MS, Chen B, Odibo AO, Zhong Y, Nelson DM. Villous trophoblast apoptosis is elevated and restricted to cytotrophoblasts in pregnancies complicated by preeclampsia, IUGR, or preeclampsia with IUGR. Placenta 2012; 33(5): 352–359. 45 Anelli T, Alessio M, Mezghrani A, Simmen T, Talamo F, Bachi A et al. ERp44, a novel endoplasmic reticulum folding assistant of the thioredoxin family. EMBO J 2002; 21(4): 835–844. 46 Moenner M, Pluquet O, Bouchecareilh M, Chevet E. Integrated endoplasmic reticulum stress responses in cancer. Cancer Res 2007; 67(22): 10631–10634. 47 Hamada K, Miyata T, Mayanagi K, Hirota J, Mikoshiba K. Two-state conformational changes in inositol 1,4,5-trisphosphate receptor regulated by calcium. J Biol Chem 2002; 277(24): 21115–21118. 48 Choe CU, Ehrlich BE. The inositol 1,4,5-trisphosphate receptor (IP3R) and its regulators: sometimes good and sometimes bad teamwork. Sci STKE 2006; 2006(363): e15. 49 Marks AR. Intracellular calcium-release channels: regulators of cell life and death. Am J Physiol 1997; 272(2 Pt 2): H597–H605. 50 Peter ME. Targeting of mRNAs by multiple miRNAs: the next step. Oncogene 2010; 29(15): 2161–2164.
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