Mol Cell Biochem (2014) 387:171–176 DOI 10.1007/s11010-013-1882-5
Inhibition of miR-92b suppresses nonsmall cell lung cancer cells growth and motility by targeting RECK Lin Lei • Yaping Huang • Wenrong Gong
Received: 7 September 2013 / Accepted: 18 October 2013 / Published online: 27 October 2013 Ó Springer Science+Business Media New York 2013
Abstract microRNAs play critical roles in the progression and metastasis of nonsmall cell lung cancer (NSCLC). miR92b acts as an oncogene in some malignancies; however, its role in NSCLC remains poorly understood. Here, we found that miR-92b was significantly increased in human NSCLC tissues and cell lines. Inhibition of miR-92b remarkably suppressed cell proliferation, migration, and invasion of NSCLC cells. Reversion-inducing-cysteine-rich protein with kazal motifs (RECK) was identified to be a target of miR-92b. Expression of miR-92b was negatively correlated with RECK in NSCLC tissues. Collectively, miR-92b might promote NSCLC cell growth and motility partially by inhibiting RECK. Keywords miR-92 Nonsmall cell lung cancer Growth Migration Invasion
Introduction Lung cancer is one of the most common causes of cancerrelated mortality worldwide, and metastasis is the main cause of death in lung cancer. Nonsmall cell lung cancer
L. Lei Department of Oncology, Xiangyang Central Hospital, Xiangyang 441021, China Y. Huang (&) Department of Respiratory Medicine, Xiangyang Central Hospital, Xiangyang 441021, China e-mail: [email protected] W. Gong Institute of Oncology, Medical College of Hubei University of Arts and Science, Xiangyang 441053, China
(NSCLC) accounts for over 80 % of all lung cancer cases [1]. The majority of patients are diagnosed at an advanced stage, and the prognosis of NSCLC remains very poor, with a 5-year survival of 11 %, despite there have been some advances in the treatment of NSCLC in recent decades [2]. Therefore, it is of great significance to understand the molecular mechanisms involved in NSCLC carcinogenesis, identify diagnostic, and prognostic biomarkers, and develop effective treatment for NSCLC. microRNAs (miRNAs) are a class of small (approximately 20–25 nucleotides), noncoding RNAs which negatively regulate gene expression posttranscriptionally. miRNAs suppress protein translation or trigger target mRNAs degradation through binding to a complementary sequence within the 30 -untranslated regions of target mRNAs [3, 4]. An individual miRNA can modulate the expression of multiple genes containing target binding sites for interaction with miRNAs, and an individual mRNA can be regulated by multiple miRNAs [5]. Recent studies have revealed that miRNAs were involved in a variety of biological processes, including cell proliferation, development, differentiation, and metabolism [6]. Thus, the aberrant alteration of miRNA expression might be involved in carcinogenesis and disease progression. Emerging evidence shows that aberrantly expressed miRNAs might serve as oncogenes or tumor-suppressor genes in NSCLC [7–9]. miR-92b has been found to be upregulated and act as oncogenes in several tumors, including ovarian epithelial carcinoma, colon cancer, neuroblastoma [10–12]. Recently, miR-92b has been reported to be elevated in NSCLC [13, 14]; however, the detailed role of miR-92b in NSCLC remains poorly understood. Herein, we studied the effects of miR-92b on cell proliferation, colony formation, migration, and invasion in NSCLC cells. Reversion-inducing-cysteine-rich protein
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with kazal motifs (RECK) was found to be a target of miR92b. Our results suggested that miR-92b might promote NSCLC cell growth and motility partially by inhibiting RECK.
Mol Cell Biochem (2014) 387:171–176
(Promega, Madison, WI, USA). Mutation in the miR-92bbinding sites of RECK mRNA was performed using a fast mutation kit (NEB, Ipswich, Canada). MTT cell proliferation assay
Materials and methods Tissue samples, cell lines, and transfection A total of 33 NSCLC tissues and matched nontumor normal tissues were surgically obtained in our department. This work was approved by the Ethics Committee of Medical College of Hubei University of Arts and Science, and informed consent was taken from all subjects. Four NSCLC cell lines, A549, SPC-A1, H1299, and H1650, and normal bronchial epithelial cell line 16HBE were obtained from the Chinese Academy of Sciences (Shanghai, China). Cells were grown in DMEM media (Invitrogen, Carlsbad, CA, USA) containing 10 % FBS at 37 °C with 5 % CO2. Cell transfection was performed using Lipofectamine 2000 in Opti-MEM medium (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol, and 100 nM mimic or inhibitor was used.
Cell viability was measured by using the MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Briefly, cells were incubated with 0.5 mg/ml MTT at 37 °C for 4 h. Then, cell culture supernatants were removed, and DMSO was added. The absorbance values at 490 nm was detected under a microplate reader (Pharmacia Biotech, Uppsala, Sweden). Colony formation assay Five hundred treated cells were placed into a fresh 6-well plates and maintained in DMEM containing 10 % FBS for 2 weeks. Colonies were then fixed and stained with 0.5 % crystal violet for 20 min. Visible colonies were photographed and quantified with the aid of Olympus INT-2 inverted microscope (Tokyo, Japan). Cell migration and invasion assays
RNA extraction and quantitative real time PCR (qRT-PCR) Total RNAs were isolated from tissues and cells by Trizol reagent (Invitrogen, Carlsbad, CA, USA), and reversely transcripted by Takara RNA PCR kit (Takara, Tokyo, Japan). qRT-PCR was performed using a SYBR Green Ò Premix Ex Taq (Takara, Tokyo, Japan) on ABI Stepone plus (ABI, Foster City, CA, USA). U6 small nuclear RNA and GAPDH were used as internal controls. Primers for U6 and miR-92b were purchased from GeneCopoeia (Carlsbad, CA, USA). The primers for RECK were 50 CCTCAGTGAGCACAGTTCAGA-30 and 50 -GCAGCACACACACTGCTGTA-30 .
For the migration assays, 5 9 104 cells were seeded into the upper chamber of 8-lm transwells (BD Bioscience, San Jose, CA, USA). For the invasion assays, 1 9 105 cells were placed into the upper chamber of 8-lm transwells precoated with matrigel (BD Bioscience, San Jose, CA, USA). Cells were cultured without serum in the upper chamber, and medium containing 10 % FBS was added into the lower chamber as chemoattractant. After 24 h incubation at 37 °C, cells on the upper surface of the filter were carefully wiped out. Then the lower surface of the filter was fixed in 90 % alcohol and followed by 0.1 % crystal violet staining. Four random fields per chamber were counted by using an inverted microscope (Olympus, Japan).
Plasmids
Luciferase reporter assays
miR-92b mimic/inhibitor and the controls were purchased from RiboBio (Guangzhou, China). pcDNA3-RECK was generated by using the following primers: 50 -GGGGTACC GGGTCCGAGCATCCCGC-30 and 50 -TTGCGGCCGCC ACGAATGGAAAGCACTGGG-30 . The PCR fragment was inserted into pcDNA3.0 within KpnI and NotI restriction sites (Invitrogen, Carlsbad, CA, USA). The 30 UTR of RECK mRNA was amplified using the following primers: sense, 50 -CCCTCGAGCCAAAATGTTGCTGAA-30 and antisense, 50 -TTGCGGCCGCGCTTTCCAGTATATTGC-30 . The PCR fragment was subcloned into psiCHECK2 vector within XhoI and NotI restriction sites
HEK293 cells cultured in 24-well plates were cotransfected with 100 nM of miR-92b mimic or control mimic and WT or Mut 30 -UTR of RECK by Lipofectamine 2000. 48 h after transfection, cells were harvested, and the relative luciferase activity was measured by a Dual-Luciferase Reporter Assay System (Promega, Wisconsin, WI, USA) according to the manufacturer’s instructions.
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Western blotting Treated cells were lysed with lysis buffer containing 50 mmol/l Tris–HCl (pH 7.4), 150 mmol/l NaCl, 1 mmol/l
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Fig. 1 miR-92b was elevated in NSCLC tissues and cell lines. a miR-92b was significantly elevated in NSCLC tissues compared with that in the corresponding nontumoral normal tissues (NC). b miR-92b was significantly elevated in four NSCLC cell lines
compared with that in normal bronchial epithelial cell line 16HBE. U6 was used as an internal control. *P \ 0.05, **P \ 0.01 compared with the control group
EDTA, 1 mmol/l phenylmethylsulfonyl fluoride, 1 % NP-40; and a proteinase inhibitor mixture (Roche, Indianapolis, IN, USA), and separated by 10 % SDS-PAGE gel. Proteins were electrophoretically transferred to PVDF membranes (Millipore, Billerica, MA, USA), and probed with primary antibodies against RECK (Abcam, Cambridge, UK) or GAPDH (Millipore, Billerica, MA, USA), followed by incubation with HRP-linked secondary antibodies (Cell Signaling Technology, Danvers, MA, USA) and detected by ECL.
(antimiR-92b) or control inhibitor (antimiR-NC). Overexpression of miR-92 inhibitor significantly inhibited the growth of A549 cells (Fig. 2a). Moreover, overexpression of miR-92 inhibitor remarkably suppressed colony formation of A549 (Fig. 2b). The effect of miR-92 inhibitor was validated by qRT-PCR (Fig. 2c).
Statistical analysis
To investigate the effect of miR-92b on the motility of NSCLC cells, antimiR-92b or antimiR-NC was transfected into A549 cells and in vitro migration and invasion assays were performed. Inhibition of miR-92b significantly suppressed the in vitro migration and invasion abilities of NSCLC cells (Fig. 3a, b).
All data are presented as mean ± standard deviation and analyzed by using SPSS 12.0. Two-tail Student’s t test and one-way analysis of variance (ANOVA) were performed to determine the statistical significance of differences. P \ 0.05 was considered statistically significant.
Inhibition of miR-92b suppressed NSCLC cell migration and invasion
RECK was a direct target of miR-92b in NSCLC cells Results miR-92b was elevated in NSCLC tissues and cell lines Expression of miR-92b in 33 NSCLC patient tissues and matched adjacent nontumoral normal tissues was determined by qRT-PCR. miR-92b was significantly elevated in NSCLC patient tissues compared with matched adjacent nontumor normal tissues (Fig. 1a). In addition, expression of miR-92b in four NSCLC cell lines, A549, SPC-A1, H1299, and H1650 was remarkably increased compared with that in 16HBE cells (Fig. 1b). Inhibition of miR-92b suppressed NSCLC cell growth in vitro To examine the role of miR-92b in NSCLC cell growth, A549 cells were transfected with miR-92b inhibitor
To explore the function of miR-92b in NSCLC, we used TargetScan 6.2 to search the potential target gene of miR92b. RECK was predicted to be a target of miR-92b (Fig. 4a). Luciferase activity assay showed that miR-92b significantly suppressed the luciferase activity of the WT 30 -UTR but not that of Mut 30 -UTR of RECK in HEK293 cells (Fig. 4b). Furthermore, Overexpression of miR-92b significantly suppressed RECK mRNA and protein levels, while inhibition of miR-92b significantly increased RECK mRNA and protein levels (Fig. 4c, d). miR-92b was negatively correlated with RECK in NSCLC tissues Expression of RECK mRNA in 33 NSCLC and the corresponding nontumor tissues was measured. Results showed that RECK mRNA was remarkably decreased in NSCLC tissues compared with the corresponding nontumor tissues
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Fig. 2 Inhibition of miR-92b suppressed NSCLC cell growth in vitro. a At 24, 48, 36, 72, or 96 h after transfection, MTT assay was performed to examine A549 cell proliferation. AntimiR-92b transfection significantly inhibited A549 cell proliferation. b AntimiR-92b transfection significantly inhibited A549 cells colony
formation. c The expression of miR-92 was significantly decreased after miR-92 inhibitor transfection. Data were drawn from three independent experiments. *P \ 0.05, **P \ 0.01 compared with the control group
Fig. 3 Inhibition of miR-92b suppressed NSCLC cell migration and invasion. a In vitro migration assay of A549 cells transfected with antimiR-92b or antimiR-NC. Inhibition of miR-92b significantly suppressed A549 cell migration. b In vitro invasion assay of A549
cells transfected with antimiR-92b or antimiR-NC. Inhibition of miR92b significantly suppressed A549 cell invasion. Data were drawn from three independent experiments. *P \ 0.05, **P \ 0.01 compared with the control group
(Fig. 5a), and the protein level of RECK was also significantly decreased in NSCLC tissues compared with the corresponding nontumor tissues (Fig. 5b). Moreover, RECK was inversely correlated with miR-92b level in NSCLC tissues (Fig. 5c).
data suggest that miR-92b might be involved in the development of NSCLC. Studies have revealed that miR-92b was associated with neuroblastoma tumorigenesis by targeting the tumor suppressor DICKKOPF-3 (DKK3) [12], the G1/S progression in human embryonic stem cells by targeting p57, a gene whose product suppressed G1 to S-phase progression [15], intestinal epithelial differentiation of Caco2-BBE cells by targeting PepT1, an epithelial di/tripeptide membrane transporter [16], and proliferation and invasion of glioblastomas cells by targeting Nemo-like kinase [17]. Here we expanded the function of miR-92b in the development of NSCLC. Furthermore, we identified that RECK, a tumor suppressor gene, was a target of miR-92b. RECK was firstly identified to induce reversion in ras-activated fibroblasts, which suppressed matrix metalloproteinases (MMPs) including MMP-2, MMP-9, and MMP14, which were known to be critical for cancer progression [18]. Numerous
Discussion Recently miRNAs have been widely explored in various types of tumors; the knowledge of aberrantly expressed miRNAs and their detailed function in NSCLC remains poorly understood. Here we found that expression of miR92b was significantly increased in NSCLC tissues and cell lines compared with normal controls. We also found that inhibition of miR-92b could remarkably suppress cell proliferation and colony formation. In addition, inhibition of miR-92b could dramatically decrease in vitro cell migration and invasion abilities of A549 cells. Thus, our
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Fig. 4 RECK was a direct target of miR-92b in NSCLC cells. a The potential miR-92 binding sites of RECK 30 -UTR and the mutant. b HEK293 cells were cotransfected with miR-92b or miR-NC with WT or Mut RECK 30 -UTR. Relative luciferase activity was assayed. c Expression of RECK mRNA was detected by qRT-PCR in A549 cells transfected with miR-92b/miR-NC, or antimiR-92b/antimiR-
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NC. d Protein level was detected by Western blotting in A549 cells transfected with miR-92b/miR-NC, or antimiR-92b/antimiR-NC. GAPDH was used as an internal control. Data were drawn from three independent experiments. *P \ 0.05, **P \ 0.01 compared with the control group
Fig. 5 miR-92b was negatively correlated with RECK in NSCLC tissues. a RECK mRNA level was detected by qRT-PCR, and it was significantly decreased in NSCLC tissues. b RECK protein level was detected by Western blotting, and it was significantly decreased in NSCLC tissues. c RECK mRNA level was inversely correlated with miR-92b level in NSCLC tissues (Spearman’s correlation analysis, r = -0.431; P = 0.012). *P \ 0.05 **P \ 0.01 compared with the control group
tumors have been linked to RECK down-regulation, and decreased expression of RECK was associated with poor survival [19]. Moreover, RECK plays an important roles in
embryogenesis, vasculogenesis, and tumorigenesis [20]. RECK aberrant expression was related with the prognosis of several cancers, including breast, gastric, and pancreatic
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tumors [20–22]. RECK could be regulated by several miRNAs [18, 23, 24]. In our study, we found that miR-92b negatively regulated RECK expression, and RECK was inversely correlated with miR-92b level in NSCLC tissues. In conclusion, miR-92b was overexpressed in NSCLC, and aberrantly expressed miR-92b could influence various biological processes of NSCLC cells, including proliferation, migration, and invasion, partially by targeting RECK expression. miR-92b might serve as a biomarker for NSCLC, and an inhibitory strategy against miR-92b might be of great help for treatment of NSCLC. Acknowledgments This work was supported by Key Natural Science Foundation of Hubei Province (No. 2011CDA065) Conflict of interest
We declare that we have no conflict of interest.
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12. Haug BH, Henriksen JR, Buechner J, Geerts D, Tomte E, Kogner P, Martinsson T, Flaegstad T, Sveinbjornsson B, Einvik C (2011) MYCN-regulated miRNA-92 inhibits secretion of the tumor suppressor DICKKOPF-3 (DKK3) in neuroblastoma. Carcinogenesis 32:1005–1012 13. Vosa U, Vooder T, Kolde R, Fischer K, Valk K, Tonisson N, Roosipuu R, Vilo J, Metspalu A, Annilo T (2011) Identification of miR-374a as a prognostic marker for survival in patients with earlystage nonsmall cell lung cancer. Genes Chromosomes Cancer 50: 812–822 14. Wang R, Wang ZX, Yang JS, Pan X, De W, Chen LB (2011) microRNA-451 functions as a tumor suppressor in human nonsmall cell lung cancer by targeting ras-related protein 14 (RAB14). Oncogene 30:2644–2658 15. Sengupta S, Nie J, Wagner RJ, Yang C, Stewart R, Thomson JA (2009) microRNA-92b controls the G1/S checkpoint gene p57 in human embryonic stem cells. Stem Cells 27:1524–1528 16. Dalmasso G, Nguyen HT, Yan Y, Laroui H, Charania MA, Obertone TS, Sitaraman SV, Merlin D (2011) microRNA-92b regulates expression of the oligopeptide transporter PepT1 in intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 300:G52–G59 17. Wang K, Wang X, Zou J, Zhang A, Wan Y, Pu P, Song Z, Qian C, Chen Y, Yang S, Wang Y (2013) miR-92b controls glioma proliferation and invasion through regulating Wnt/beta-catenin signaling via Nemo-like kinase. Neuro Oncol 15:578–588 18. Li N, Tang B, Zhu ED, Li BS, Zhuang Y, Yu S, Lu DS, Zou QM, Xiao B, Mao XH (2012) Increased miR-222 in H. pylori-associated gastric cancer correlated with tumor progression by promoting cancer cell proliferation and targeting RECK. FEBS Lett 586:722–728 19. Clark JC, Thomas DM, Choong PF, Dass CR (2007) RECK—a newly discovered inhibitor of metastasis with prognostic significance in multiple forms of cancer. Cancer Metastasis Rev 26: 675–683 20. Lu XX, Zhang SM, Fang Y, Wang ZT, Xie JJ, Zhan Q, Deng XX, Chen H, Jin JB, Peng CH, Li HW, Shen BY (2013) Clinical significance of RECK promoter methylation in pancreatic ductal adenocarcinoma. Tumor Biol. doi:10.1007/s13277-013-0903-z 21. Zhang Y, Cheng S, Zhang G, Ma W, Liu Y, Zhao R, Zhang Q, Pang D (2012) Low expression of RECK indicates a shorter survival for patients with invasive breast cancer. Cancer Sci 103:1084–1089 22. Du YY, Dai DQ, Yang Z (2010) Role of RECK methylation in gastric cancer and its clinical significance. World J Gastroenterol 16:904–908 23. Wang N, Zhang CQ, He JH, Duan XF, Wang YY, Ji X, Zang WQ, Li M, Ma YY, Wang T, Zhao GQ (2013) miR-21 downregulation suppresses cell growth, invasion and induces cell apoptosis by targeting FASL, TIMP3, and RECK genes in esophageal carcinoma. Dig Dis Sci 58:1863–1870 24. Xin C, Buhe B, Hongting L, Chuanmin Y, Xiwei H, Hong Z, Lulu H, Qian D, Renjie W (2013) microRNA-15a promotes neuroblastoma migration by targeting reversion-inducing cysteine-rich protein with Kazal motifs (RECK) and regulating matrix metalloproteinase-9 expression. FEBS J 280:855–866
Inhibition of miR-92b suppresses nonsmall cell lung cancer cells growth and motility by targeting RECK Lin Lei • Yaping Huang • Wenrong Gong
Received: 7 September 2013 / Accepted: 18 October 2013 / Published online: 27 October 2013 Ó Springer Science+Business Media New York 2013
Abstract microRNAs play critical roles in the progression and metastasis of nonsmall cell lung cancer (NSCLC). miR92b acts as an oncogene in some malignancies; however, its role in NSCLC remains poorly understood. Here, we found that miR-92b was significantly increased in human NSCLC tissues and cell lines. Inhibition of miR-92b remarkably suppressed cell proliferation, migration, and invasion of NSCLC cells. Reversion-inducing-cysteine-rich protein with kazal motifs (RECK) was identified to be a target of miR-92b. Expression of miR-92b was negatively correlated with RECK in NSCLC tissues. Collectively, miR-92b might promote NSCLC cell growth and motility partially by inhibiting RECK. Keywords miR-92 Nonsmall cell lung cancer Growth Migration Invasion
Introduction Lung cancer is one of the most common causes of cancerrelated mortality worldwide, and metastasis is the main cause of death in lung cancer. Nonsmall cell lung cancer
L. Lei Department of Oncology, Xiangyang Central Hospital, Xiangyang 441021, China Y. Huang (&) Department of Respiratory Medicine, Xiangyang Central Hospital, Xiangyang 441021, China e-mail: [email protected] W. Gong Institute of Oncology, Medical College of Hubei University of Arts and Science, Xiangyang 441053, China
(NSCLC) accounts for over 80 % of all lung cancer cases [1]. The majority of patients are diagnosed at an advanced stage, and the prognosis of NSCLC remains very poor, with a 5-year survival of 11 %, despite there have been some advances in the treatment of NSCLC in recent decades [2]. Therefore, it is of great significance to understand the molecular mechanisms involved in NSCLC carcinogenesis, identify diagnostic, and prognostic biomarkers, and develop effective treatment for NSCLC. microRNAs (miRNAs) are a class of small (approximately 20–25 nucleotides), noncoding RNAs which negatively regulate gene expression posttranscriptionally. miRNAs suppress protein translation or trigger target mRNAs degradation through binding to a complementary sequence within the 30 -untranslated regions of target mRNAs [3, 4]. An individual miRNA can modulate the expression of multiple genes containing target binding sites for interaction with miRNAs, and an individual mRNA can be regulated by multiple miRNAs [5]. Recent studies have revealed that miRNAs were involved in a variety of biological processes, including cell proliferation, development, differentiation, and metabolism [6]. Thus, the aberrant alteration of miRNA expression might be involved in carcinogenesis and disease progression. Emerging evidence shows that aberrantly expressed miRNAs might serve as oncogenes or tumor-suppressor genes in NSCLC [7–9]. miR-92b has been found to be upregulated and act as oncogenes in several tumors, including ovarian epithelial carcinoma, colon cancer, neuroblastoma [10–12]. Recently, miR-92b has been reported to be elevated in NSCLC [13, 14]; however, the detailed role of miR-92b in NSCLC remains poorly understood. Herein, we studied the effects of miR-92b on cell proliferation, colony formation, migration, and invasion in NSCLC cells. Reversion-inducing-cysteine-rich protein
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with kazal motifs (RECK) was found to be a target of miR92b. Our results suggested that miR-92b might promote NSCLC cell growth and motility partially by inhibiting RECK.
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(Promega, Madison, WI, USA). Mutation in the miR-92bbinding sites of RECK mRNA was performed using a fast mutation kit (NEB, Ipswich, Canada). MTT cell proliferation assay
Materials and methods Tissue samples, cell lines, and transfection A total of 33 NSCLC tissues and matched nontumor normal tissues were surgically obtained in our department. This work was approved by the Ethics Committee of Medical College of Hubei University of Arts and Science, and informed consent was taken from all subjects. Four NSCLC cell lines, A549, SPC-A1, H1299, and H1650, and normal bronchial epithelial cell line 16HBE were obtained from the Chinese Academy of Sciences (Shanghai, China). Cells were grown in DMEM media (Invitrogen, Carlsbad, CA, USA) containing 10 % FBS at 37 °C with 5 % CO2. Cell transfection was performed using Lipofectamine 2000 in Opti-MEM medium (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol, and 100 nM mimic or inhibitor was used.
Cell viability was measured by using the MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Briefly, cells were incubated with 0.5 mg/ml MTT at 37 °C for 4 h. Then, cell culture supernatants were removed, and DMSO was added. The absorbance values at 490 nm was detected under a microplate reader (Pharmacia Biotech, Uppsala, Sweden). Colony formation assay Five hundred treated cells were placed into a fresh 6-well plates and maintained in DMEM containing 10 % FBS for 2 weeks. Colonies were then fixed and stained with 0.5 % crystal violet for 20 min. Visible colonies were photographed and quantified with the aid of Olympus INT-2 inverted microscope (Tokyo, Japan). Cell migration and invasion assays
RNA extraction and quantitative real time PCR (qRT-PCR) Total RNAs were isolated from tissues and cells by Trizol reagent (Invitrogen, Carlsbad, CA, USA), and reversely transcripted by Takara RNA PCR kit (Takara, Tokyo, Japan). qRT-PCR was performed using a SYBR Green Ò Premix Ex Taq (Takara, Tokyo, Japan) on ABI Stepone plus (ABI, Foster City, CA, USA). U6 small nuclear RNA and GAPDH were used as internal controls. Primers for U6 and miR-92b were purchased from GeneCopoeia (Carlsbad, CA, USA). The primers for RECK were 50 CCTCAGTGAGCACAGTTCAGA-30 and 50 -GCAGCACACACACTGCTGTA-30 .
For the migration assays, 5 9 104 cells were seeded into the upper chamber of 8-lm transwells (BD Bioscience, San Jose, CA, USA). For the invasion assays, 1 9 105 cells were placed into the upper chamber of 8-lm transwells precoated with matrigel (BD Bioscience, San Jose, CA, USA). Cells were cultured without serum in the upper chamber, and medium containing 10 % FBS was added into the lower chamber as chemoattractant. After 24 h incubation at 37 °C, cells on the upper surface of the filter were carefully wiped out. Then the lower surface of the filter was fixed in 90 % alcohol and followed by 0.1 % crystal violet staining. Four random fields per chamber were counted by using an inverted microscope (Olympus, Japan).
Plasmids
Luciferase reporter assays
miR-92b mimic/inhibitor and the controls were purchased from RiboBio (Guangzhou, China). pcDNA3-RECK was generated by using the following primers: 50 -GGGGTACC GGGTCCGAGCATCCCGC-30 and 50 -TTGCGGCCGCC ACGAATGGAAAGCACTGGG-30 . The PCR fragment was inserted into pcDNA3.0 within KpnI and NotI restriction sites (Invitrogen, Carlsbad, CA, USA). The 30 UTR of RECK mRNA was amplified using the following primers: sense, 50 -CCCTCGAGCCAAAATGTTGCTGAA-30 and antisense, 50 -TTGCGGCCGCGCTTTCCAGTATATTGC-30 . The PCR fragment was subcloned into psiCHECK2 vector within XhoI and NotI restriction sites
HEK293 cells cultured in 24-well plates were cotransfected with 100 nM of miR-92b mimic or control mimic and WT or Mut 30 -UTR of RECK by Lipofectamine 2000. 48 h after transfection, cells were harvested, and the relative luciferase activity was measured by a Dual-Luciferase Reporter Assay System (Promega, Wisconsin, WI, USA) according to the manufacturer’s instructions.
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Western blotting Treated cells were lysed with lysis buffer containing 50 mmol/l Tris–HCl (pH 7.4), 150 mmol/l NaCl, 1 mmol/l
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Fig. 1 miR-92b was elevated in NSCLC tissues and cell lines. a miR-92b was significantly elevated in NSCLC tissues compared with that in the corresponding nontumoral normal tissues (NC). b miR-92b was significantly elevated in four NSCLC cell lines
compared with that in normal bronchial epithelial cell line 16HBE. U6 was used as an internal control. *P \ 0.05, **P \ 0.01 compared with the control group
EDTA, 1 mmol/l phenylmethylsulfonyl fluoride, 1 % NP-40; and a proteinase inhibitor mixture (Roche, Indianapolis, IN, USA), and separated by 10 % SDS-PAGE gel. Proteins were electrophoretically transferred to PVDF membranes (Millipore, Billerica, MA, USA), and probed with primary antibodies against RECK (Abcam, Cambridge, UK) or GAPDH (Millipore, Billerica, MA, USA), followed by incubation with HRP-linked secondary antibodies (Cell Signaling Technology, Danvers, MA, USA) and detected by ECL.
(antimiR-92b) or control inhibitor (antimiR-NC). Overexpression of miR-92 inhibitor significantly inhibited the growth of A549 cells (Fig. 2a). Moreover, overexpression of miR-92 inhibitor remarkably suppressed colony formation of A549 (Fig. 2b). The effect of miR-92 inhibitor was validated by qRT-PCR (Fig. 2c).
Statistical analysis
To investigate the effect of miR-92b on the motility of NSCLC cells, antimiR-92b or antimiR-NC was transfected into A549 cells and in vitro migration and invasion assays were performed. Inhibition of miR-92b significantly suppressed the in vitro migration and invasion abilities of NSCLC cells (Fig. 3a, b).
All data are presented as mean ± standard deviation and analyzed by using SPSS 12.0. Two-tail Student’s t test and one-way analysis of variance (ANOVA) were performed to determine the statistical significance of differences. P \ 0.05 was considered statistically significant.
Inhibition of miR-92b suppressed NSCLC cell migration and invasion
RECK was a direct target of miR-92b in NSCLC cells Results miR-92b was elevated in NSCLC tissues and cell lines Expression of miR-92b in 33 NSCLC patient tissues and matched adjacent nontumoral normal tissues was determined by qRT-PCR. miR-92b was significantly elevated in NSCLC patient tissues compared with matched adjacent nontumor normal tissues (Fig. 1a). In addition, expression of miR-92b in four NSCLC cell lines, A549, SPC-A1, H1299, and H1650 was remarkably increased compared with that in 16HBE cells (Fig. 1b). Inhibition of miR-92b suppressed NSCLC cell growth in vitro To examine the role of miR-92b in NSCLC cell growth, A549 cells were transfected with miR-92b inhibitor
To explore the function of miR-92b in NSCLC, we used TargetScan 6.2 to search the potential target gene of miR92b. RECK was predicted to be a target of miR-92b (Fig. 4a). Luciferase activity assay showed that miR-92b significantly suppressed the luciferase activity of the WT 30 -UTR but not that of Mut 30 -UTR of RECK in HEK293 cells (Fig. 4b). Furthermore, Overexpression of miR-92b significantly suppressed RECK mRNA and protein levels, while inhibition of miR-92b significantly increased RECK mRNA and protein levels (Fig. 4c, d). miR-92b was negatively correlated with RECK in NSCLC tissues Expression of RECK mRNA in 33 NSCLC and the corresponding nontumor tissues was measured. Results showed that RECK mRNA was remarkably decreased in NSCLC tissues compared with the corresponding nontumor tissues
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Fig. 2 Inhibition of miR-92b suppressed NSCLC cell growth in vitro. a At 24, 48, 36, 72, or 96 h after transfection, MTT assay was performed to examine A549 cell proliferation. AntimiR-92b transfection significantly inhibited A549 cell proliferation. b AntimiR-92b transfection significantly inhibited A549 cells colony
formation. c The expression of miR-92 was significantly decreased after miR-92 inhibitor transfection. Data were drawn from three independent experiments. *P \ 0.05, **P \ 0.01 compared with the control group
Fig. 3 Inhibition of miR-92b suppressed NSCLC cell migration and invasion. a In vitro migration assay of A549 cells transfected with antimiR-92b or antimiR-NC. Inhibition of miR-92b significantly suppressed A549 cell migration. b In vitro invasion assay of A549
cells transfected with antimiR-92b or antimiR-NC. Inhibition of miR92b significantly suppressed A549 cell invasion. Data were drawn from three independent experiments. *P \ 0.05, **P \ 0.01 compared with the control group
(Fig. 5a), and the protein level of RECK was also significantly decreased in NSCLC tissues compared with the corresponding nontumor tissues (Fig. 5b). Moreover, RECK was inversely correlated with miR-92b level in NSCLC tissues (Fig. 5c).
data suggest that miR-92b might be involved in the development of NSCLC. Studies have revealed that miR-92b was associated with neuroblastoma tumorigenesis by targeting the tumor suppressor DICKKOPF-3 (DKK3) [12], the G1/S progression in human embryonic stem cells by targeting p57, a gene whose product suppressed G1 to S-phase progression [15], intestinal epithelial differentiation of Caco2-BBE cells by targeting PepT1, an epithelial di/tripeptide membrane transporter [16], and proliferation and invasion of glioblastomas cells by targeting Nemo-like kinase [17]. Here we expanded the function of miR-92b in the development of NSCLC. Furthermore, we identified that RECK, a tumor suppressor gene, was a target of miR-92b. RECK was firstly identified to induce reversion in ras-activated fibroblasts, which suppressed matrix metalloproteinases (MMPs) including MMP-2, MMP-9, and MMP14, which were known to be critical for cancer progression [18]. Numerous
Discussion Recently miRNAs have been widely explored in various types of tumors; the knowledge of aberrantly expressed miRNAs and their detailed function in NSCLC remains poorly understood. Here we found that expression of miR92b was significantly increased in NSCLC tissues and cell lines compared with normal controls. We also found that inhibition of miR-92b could remarkably suppress cell proliferation and colony formation. In addition, inhibition of miR-92b could dramatically decrease in vitro cell migration and invasion abilities of A549 cells. Thus, our
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Fig. 4 RECK was a direct target of miR-92b in NSCLC cells. a The potential miR-92 binding sites of RECK 30 -UTR and the mutant. b HEK293 cells were cotransfected with miR-92b or miR-NC with WT or Mut RECK 30 -UTR. Relative luciferase activity was assayed. c Expression of RECK mRNA was detected by qRT-PCR in A549 cells transfected with miR-92b/miR-NC, or antimiR-92b/antimiR-
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NC. d Protein level was detected by Western blotting in A549 cells transfected with miR-92b/miR-NC, or antimiR-92b/antimiR-NC. GAPDH was used as an internal control. Data were drawn from three independent experiments. *P \ 0.05, **P \ 0.01 compared with the control group
Fig. 5 miR-92b was negatively correlated with RECK in NSCLC tissues. a RECK mRNA level was detected by qRT-PCR, and it was significantly decreased in NSCLC tissues. b RECK protein level was detected by Western blotting, and it was significantly decreased in NSCLC tissues. c RECK mRNA level was inversely correlated with miR-92b level in NSCLC tissues (Spearman’s correlation analysis, r = -0.431; P = 0.012). *P \ 0.05 **P \ 0.01 compared with the control group
tumors have been linked to RECK down-regulation, and decreased expression of RECK was associated with poor survival [19]. Moreover, RECK plays an important roles in
embryogenesis, vasculogenesis, and tumorigenesis [20]. RECK aberrant expression was related with the prognosis of several cancers, including breast, gastric, and pancreatic
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tumors [20–22]. RECK could be regulated by several miRNAs [18, 23, 24]. In our study, we found that miR-92b negatively regulated RECK expression, and RECK was inversely correlated with miR-92b level in NSCLC tissues. In conclusion, miR-92b was overexpressed in NSCLC, and aberrantly expressed miR-92b could influence various biological processes of NSCLC cells, including proliferation, migration, and invasion, partially by targeting RECK expression. miR-92b might serve as a biomarker for NSCLC, and an inhibitory strategy against miR-92b might be of great help for treatment of NSCLC. Acknowledgments This work was supported by Key Natural Science Foundation of Hubei Province (No. 2011CDA065) Conflict of interest
We declare that we have no conflict of interest.
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