FEBS Letters 588 (2014) 1983–1988
journal homepage: www.FEBSLetters.org
MicroRNA-152 targets ADAM17 to suppress NSCLC progression Yunshu Su, Yuchang Wang, Hongmin Zhou, Lei Lei, Lijun Xu ⇑ Department of Cardiothoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China
a r t i c l e
i n f o
Article history: Received 19 March 2014 Revised 6 April 2014 Accepted 15 April 2014 Available online 26 April 2014 Edited by Tamas Dalmay Keywords: Non-small cell lung cancer miR-152 ADAM metallopeptidase domain 17 Proliferation Migration Invasion
a b s t r a c t MicroRNAs (miRNAs) are a class of small non-coding RNAs that have been suggested to play an essential role in tumorigenesis. In this study, we show that miR-152 is significantly downregulated in non-small cell lung cancer (NSCLC) tissues and cell lines. Restoration of miR-152 significantly reduces proliferation, colony formation, migration and invasion of NSCLC cells. In addition, ADAM metallopeptidase domain 17 (ADAM17) is identified as a target of miR-152 in NSCLC cells, and miR-152-induced suppression of cell proliferation, colony formation, migration and invasion is partially mediated by silencing of ADAM17 expression. Furthermore, ADAM17 inversely correlates with miR-152 in NSCLC tissues. Collectively, our findings indicate that miR-152 acts as tumor suppressor in NSCLC partially via targeting ADAM17. Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Lung cancer is one of the most life-threatening tumors, with approximately two million new cases diagnosed worldwide annually, and Non-small-cell lung cancer (NSCLC) accounts for 80% of primary lung cancer [1]. Despite the improvements in therapeutic modalities, the 5-year survival rate of NSCLC patients is still around 15% [2]. Thus, it is necessary to explore novel agents for NSCLC treatment. MicroRNAs (miRNAs) are small non-coding RNAs which regulate a variety of cellular processes via binding to the 30 -untranslated region (30 -UTR) of target mRNAs, resulting in the degradation of the mRNAs or inhibition of mRNAs translation [3]. Accumulating evidence shows that miRNAs play critical roles in the regulation of cancer initiation and progression [4,5]. MiRNAs can function as oncogenes or tumor suppressors depending on their specific target genes and different cancers [6–8]. Dysfunction of miRNAs occurred in many cancers and aberrantly altered expression of miRNAs was involved in tumorigenesis. A variety of miRNAs have been found to dysregulated in NSCLC, and many miRNAs regulate NSCLC progression [9,10]. miR-152 functions as a tumor suppressor in some cancers [11,12], and it was also downregulated in NSCLC [13], but the detailed role of miR-152 in NSCLC is unclear.
⇑ Corresponding author. Fax: +86 02783665290.
In this study, we found that miR-152 was significantly downregulated in NSCLC tissues and cell lines, and overexpression of miR-152 substantially suppressed proliferation and motility of NSCLC cells. ADAM metallopeptidase domain 17 (ADAM17) was identified as a target of miR-152 in NSCLC cells, and miR-152induced suppression of proliferation and motility of NSCLC cells was partially mediated by silencing of ADAM17 expression. Furthermore, ADAM17 was inversely correlated with miR-152 in NSCLC tissues. 2. Materials and methods 2.1. NSCLC cancer tissue and cell culture Paired NSCLC and adjacent non-tumor normal lung tissues were obtained from 30 patients with informed consent at our hospital. Tissues were immediately snapfrozen and stored at 80 °C. Four NSCLC cell lines (A549, SK-MES-1, H157, and H520) and a normal lung bronchus epithelial cell line BEAS-2B were obtained from American Type Culture Collection and cultured in DMEM supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA), and incubated in 5% CO2 humid atmosphere at 37 °C. 2.2. Plasmid miR-152 mimic/inhibitor was obtained from RiboBio (Guangzhou, China). pcDNA3-ADAM17 was generated by using primers:
E-mail address: [email protected] (L. Xu). http://dx.doi.org/10.1016/j.febslet.2014.04.022 0014-5793/Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1984
Y. Su et al. / FEBS Letters 588 (2014) 1983–1988
sense 50 -GGGGTACCGAATCTTCCCAGTAGGCG-30 , and antisense 50 TTGCGGCCGCAAAGGAGAAGGGCCAAAC-30 . 30 -UTR of ADAM17 was amplified and cloned into the pGL-3-vector (Promega, Madison, WI, USA). Mutations in the 30 -UTR of ADAM17 with miR-152 target site (Mut) was generated with a fast mutation kit (NEB, Ipswich, Canada). 2.3. RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was isolated with Trizol (Invitrogen, Carlsbad, CA, USA). MicroRNA was isolated with the mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA). Expression of miR-152 was detected using TaqMan MicroRNA Assay primers (ABI, Foster City, CA, USA). U6 or GAPDH was used as an internal control. Experiments were performed on the ABI Prism 7500 HT (ABI, Foster City, CA, USA). The relative expression level was calculated by normalization with the signal for U6 expression using 2DDCt method. 2.4. Cell proliferation assay
2.7. Western blotting Cultured cells were lysed in RIPA buffer with 1% PMSF. Protein was loaded and seperated by SDS–PAGE gel and transferred onto PVDF membrane. The blots were probed with primary antibodies at 4 °C overnight and subsequently incubated with HRPconjugated secondary antibodies. Signals were visualized using ECL Substrates (Pierce, Rockford, IL, USA). GAPDH was used as an endogenous control. 2.8. Luciferase assays About 1 105 HEK293 cells per well were cultured in 24-well plates. Cells were co-transfected with 10 ng pRL-TK Renilla luciferase reporter, 50 ng pGL-3 firefly luciferase reporter, and 100 nM miR-152 mimics or control mimics using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Cell lysates were prepared 48 h after transfection using Passive Lysis Buffer, and luciferase activity was examined using the Dual-Luciferase Reporter Assay (Promega, Madison, WI, USA). Results were normalized to the Renilla luciferase.
Transfected cells were seeded into 96-well plates with a density of 4000 cells/well, and cultured for different time. 10 lL of MTT was added into each well, and incubated for 4 h. Then the supernatant was discarded, and 200 lL of DMSO was added to each well. Optical density (OD) was detected at the wavelength of 490 nm. Data were derived from three independent experiments.
Data are presented as mean ± SD. Statistical differences were determined by ANOVA or Student t test. P < 0.05 was considered significant.
2.5. Colony formation assay
3. Results
2000 transfected cells were seeded into 12-well plate. Approximately 10 days later, the clones were washed with PBS and stained with 0.5% crystal violet for 15 min. Finally the clones were taken pictures and counted under a microscope (Olympus, Tokyo, Japan).
3.1. miR-152 was downregulated in NSCLC tissues and cell lines
2.6. Migration and invasion assays Migration and invasion were examined using a transwell chamber (Millipore, Billerica, MA, USA). For the migration assay, 1 105 transfected cells were plated into the upper chamber, and cultured in DMEM, while DMEM with 10% FBS was added to the lower chamber. After 24 h incubation at 37 °C, cells remaining on the upper surface of membrane was removed, and membrane was stained with 20% methanol and 0.5% crystal violet. Cell images were obtained using an inverted microscope (Olympus, Tokyo, Japan). For invasion assays, the upper chamber was precoated with Matrigel (BD, San Jose, CA, USA).
2.9. Statistical analysis
Expression of miR-152 in 30 paired NSCLC tissues was examined by qRT-PCR. We discovered that miR-152 was significantly downregulated in NSCLC tissues compared with the adjacent normal tissues (Fig. 1A). Furthermore, miR-152 was also downregulated in four NSCLC cell lines compared with the normal lung bronchus epithelial cell line BEAS-2B (Fig. 1B). 3.2. miR-152 suppressed NSCLC cell proliferation and colony formation MTT and colony formation were used to determine the effects of miR-152 on A549 and H520 cells. The expression of miR-152 mimics was identified by qRT-PCR (Fig. 2A). Overexpression of miR-152 significantly suppressed cell proliferation of A549 and H520 cells compared with the scramble control (Fig. 2B). In consistent with this result, miR-152 overexpression substantially suppressed colony formation of A549 and H520 cells (Fig. 2C).
Fig. 1. miR-152 was downregulated in NSCLC tissues and cell lines. (A) miR-152 was significantly downregulated in NSCLC tissues compared with the corresponding nontumor normal tissues. (B) miR-152 was significantly decreased in four NSCLC cell lines compared with the lung bronchus epithelial cell line BEAS-2B. ⁄P < 0.05, ⁄⁄P < 0.01 compared with the control group.
Y. Su et al. / FEBS Letters 588 (2014) 1983–1988
1985
Fig. 2. miR-152 suppressed NSCLC cell proliferation and colony formation. (A) Expression of miR-152 in A549 or H520 cells transfected with miR-152 or the scramble control. (B) MTT assay of A549 or H520 cells transfected with miR-152 or the scramble control. (C) Colony formation of A549 or H520 cells transfected with miR-152 or the scramble control. Data were drawn from three independent experiments. ⁄P < 0.05, ⁄⁄P < 0.01 compared with the control group.
3.3. miR-152 suppressed NSCLC cell migration and invasion We further investigated whether miR-152 could also inhibit migration and invasion of NSCLC cells. Overexpression of miR152 dramatically suppressed tumor cell migration in A549 cells compared with the scramble control (Fig. 3A). Similarly, transwell assays with Matrigel demonstrated that miR-152 markedly decreased the invasive capacity of A549 cells (Fig. 3B).
3.5. miR-152 suppressed NSCLC progression partially by targeting ADAM17 We further investigated that whether miR-152 suppressed NSCLC progression by targeting ADAM17. MTT assay, colony formation, migration and invasion (Fig. 5A–D) showed that supplement of ADAM17 by pcDNA3-ADAM17 significantly attenuated the tumor suppressive effects of miR-152. The effect of ADAM17 plasmid was confirmed by Western blotting (Fig. 5E).
3.4. ADAM17 was a target of miR-152 in NSCLC cells To identify the target of miR-152 in NSCLC, TargetScan 6.2 was used. ADAM17, which was increased in NSCLC, was predicted to be a potential target of miR-152 (Fig. 4A). Luciferase activity assay found that miR-152 significantly inhibited the WT but not Mut luciferase activity in HEK293 cells (Fig. 4B). Moreover, overexpression of miR-152 significantly inhibited ADAM17 expression (Fig. 4C).
3.6. miR-152 was inversely correlated with ADAM17 expression in NSCLC tissues Expression of ADAM17 in 30 NSCLC tissues was examined by qRT-PCR. ADAM17 mRNA was remarkably increased in NSCLC tissues (Fig. 6A). Furthermore, ADAM17 mRNA level was inversely correlated with miR-152 level in NSCLC tissues (Fig. 6B).
1986
Y. Su et al. / FEBS Letters 588 (2014) 1983–1988
Fig. 3. miR-152 suppressed NSCLC cell migration and invasion. (A) Migration assay of A549 cells transfected with miR-152 or the scramble control. (B) Invasion assay of A549 cells transfected with miR-152 or the scramble control. Data were drawn from three independent experiments. ⁄P < 0.05, ⁄⁄P < 0.01 compared with the control group.
Fig. 4. ADAM17 was a target of miR-152 in NSCLC cells. (A) The potential miR-152 binding sites of ADAM17 30 -UTR and the mutant. (B) HEK293 cells were co-transfected with miR-152 or the scramble control with WT or Mut ADAM17 30 -UTR. Luciferase activity was assayed. (C) Protein level of ADAM17 was detected by Western blotting in A549 cells transfected with miR-152 or the scramble control. GAPDH was used as an internal control. Experiments were performed in triplicate. ⁄⁄P < 0.01 compared with the control group.
4. Discussion In this study, we provide important evidence in support of miR152 functioning as a tumor suppressor in NSCLC. In addition, we identified ADAM17 as a direct target of miR-152 in NSCLC cells. Restoring the expression of ADAM17 could abolish miR-152mediated suppression of NSCLC progression. Moreover, ADAM17 was inversely correlated with miR-152 in NSCLC tissues. miR-152 is a member of the miR-148/152 family which includes miR-148a/b and miR-152. miR-152 was an tumor suppressive miRNA and decreased in various types of cancer. Aberrant expression of miR-152 was thought to contribute to the malignant phenotype of several tumors. Huang et al. showed that miR-152 was decreased in hepatitis B virus-related hepatocellular carcinoma (HCC), and forced expression of miR-152 in liver cells suppressed the expression of DNA methyltransferase 1 (DNMT1), leading to decrease in global DNA methylation [14]. These data were consistent with the study of pancreatic cancer cells by Azizi et al. [15]. Zhu et al. reported that miR-152 expression was decreased in primary prostate cancer (PC), and low miR-152 expression is correlated with advanced pathological T-stages. They further identified that TGFa, which was increased in PC, was a target of miR-152 [16]. Zheng et al. found that miR-152 and miR-15b
suppressed glioma cell invasion and angiogenesis by targeting NRP-2 and MMP-3 [11]. Our data expanded the tumor suppressive role of miR-152 in NSCLC. Forced expression of miR-152 suppressed A549 cells proliferation, colony formation, migration and invasion. ADAM17 is one kind of the superfamily of Zn-dependent metalloproteinases, and regulates the cleavage of ectodomain of several transmembrane proteins [17]. ADAM17 sheds many essential cell surface molecules, including cytokines and adhesion molecules [18]. Accumulating evidence suggests that ADAM17 was increased in a variety of tumors, including gastric cancer, HCC, colon cancer, breast cancer, and NSCLC [18–21]. The elevated ADAM17 was involved in proliferation, chemoresistance, and malignant phenotypes. Thus, ADAM17 has been considered as a potential therapeutic target in several tumors [22]. Zheng et al. showed that suppression of ADAM17 abolished hypoxia-mediated brain tumor cell invasiveness [23]. Lv et al. found that downregulation of ADAM17 expression suppressed the migration and invasion of NSCLC cells [21]. In the present study, we identified that ADAM17 was a target of miR-152 in NSCLC cells, and supplement of ADAM17 remarkably attenuated the tumor suppressive effects of miR-152 on NSCLC cells. In addition, ADAM17 was inversely correlated with miR-152 in NSCLC tissues.
Y. Su et al. / FEBS Letters 588 (2014) 1983–1988
1987
Fig. 5. miR-152 suppressed NSCLC progression partially by targeting ADAM17. (A) A549 cells were transfected with miR-152 with/without ADAM17 overexpression plasmid, and MTT assay was performed. (B) Colony formation assay. (C) Migration assay. (D) Invasion assay. (E) Protein level of ADAM17. Experiments were performed in triplicate. ⁄ P < 0.05, ⁄⁄P < 0.01 compared with control; #P < 0.05, compared with miR-152 transfected group.
Fig. 6. miR-152 was negatively correlated with ADAM17 in NSCLC tissues. (A) The expression of ADAM17 in 30 pairs of NSCLC tissues was measured by qRT-PCR. (B) ADAM17 mRNA level was inversely correlated with miR-152 level in NSCLC tissues (Spearman’s correlation analysis, r = 0.377, P = 0.04). ⁄P < 0.05 compared with control.
In conclusion, we demonstrate in the present study that miR152 acts as a tumor suppressor in NSCLC cells, which are largely mediated through inhibiting of ADAM17 expression. These findings provide novel insight into the mechanisms of NSCLC.
Conflict of interest The authors declare that they have no conflicts of interest concerning this article.
References [1] Siegel, R., Naishadham, D. and Jemal, A. (2012) Cancer statistics, 2012. CA Cancer J. Clin. 62, 10–29. [2] Nakamura, H., Taniguchi, Y., Miwa, K., Adachi, Y., Fujioka, S., Haruki, T., Takagi, Y. and Yurugi, Y. (2011) Comparison of the surgical outcomes of thoracoscopic lobectomy, segmentectomy, and wedge resection for clinical stage I non-small cell lung cancer. Thorac. Cardiovasc. Surg. 59, 137–141. [3] Aryal, B., Rotllan, N. and Fernandez-Hernando, C. (2014) Noncoding RNAs and atherosclerosis. Curr. Atheroscler. Rep. 16, 407. [4] Xin, C., Zhang, H. and Liu, Z. (2014) MiR-154 suppresses colorectal cancer cell growth and motility by targeting TLR2. Mol. Cell. Biochem. 387, 271–277.
1988
Y. Su et al. / FEBS Letters 588 (2014) 1983–1988
[5] Kolesnikoff, N., Attema, J.L., Roslan, S., Bert, A.G., Schwarz, Q.P., Gregory, P.A. and Goodall, G.J. (2014) Specificity protein 1 (Sp1) maintains basal epithelial expression of the miR-200 family: implications for epithelial–mesenchymal transition. J. Biol. Chem. 289, 11194–11205, http://dx.doi.org/10.1074/ jbc.M113.529172. [6] Yang, H., Li, Q., Zhao, W., Yuan, D., Zhao, H. and Zhou, Y. (2014) MiR-329 suppresses the growth and motility of neuroblastoma by targeting KDM1A. FEBS Lett. 588, 192–197. [7] Ke, Y., Zhao, W., Xiong, J. and Cao, R. (2013) Downregulation of miR-16 promotes growth and motility by targeting HDGF in non-small cell lung cancer cells. FEBS Lett. 587, 3153–3157. [8] Wang, X., Yu, H., Lu, X., Zhang, P., Wang, M. and Hu, Y. (2014) MiR-22 suppresses the proliferation and invasion of gastric cancer cells by inhibiting CD151. Biochem. Biophys. Res. Commun. 445, 175–179. [9] Lei, L., Huang, Y. and Gong, W. (2013) MiR-205 promotes the growth, metastasis and chemoresistance of NSCLC cells by targeting PTEN. Oncol. Rep. 30, 2897–2902. [10] Wan, L., Zhang, L., Fan, K. and Wang, J. (2014) MiR-27b targets LIMK1 to inhibit growth and invasion of NSCLC cells. Mol. Cell. Biochem. 390, 85–91, http:// dx.doi.org/10.1007/s11010-013-1959-1. [11] Zheng, X., Chopp, M., Lu, Y., Buller, B. and Jiang, F. (2013) MiR-15b and miR152 reduce glioma cell invasion and angiogenesis via NRP-2 and MMP-3. Cancer Lett. 329, 146–154. [12] Tsuruta, T. et al. (2011) MiR-152 is a tumor suppressor microRNA that is silenced by DNA hypermethylation in endometrial cancer. Cancer Res. 71, 6450–6462. [13] Sanfiorenzo, C., Ilie, M.I., Belaid, A., Barlesi, F., Mouroux, J., Marquette, C.H., Brest, P. and Hofman, P. (2013) Two panels of plasma microRNAs as noninvasive biomarkers for prediction of recurrence in resectable NSCLC. PLoS One 8, e54596.
[14] Huang, J., Wang, Y., Guo, Y. and Sun, S. (2010) Down-regulated microRNA-152 induces aberrant DNA methylation in hepatitis B virus-related hepatocellular carcinoma by targeting DNA methyltransferase 1. Hepatology 52, 60–70. [15] Azizi, M., Teimoori-Toolabi, L., Arzanani, M.K., Azadmanesh, K., Fard-Esfahani, P. and Zeinali, S. (2014) MicroRNA-148b and microRNA-152 reactivate tumor suppressor genes through suppression of DNA methyltransferase-1 gene in pancreatic cancer cell lines. Cancer Biol. Ther. 15. [16] Zhu, C. et al. (2013) MiR-152 controls migration and invasive potential by targeting TGFalpha in prostate cancer cell lines. Prostate 73, 1082–1089. [17] Primakoff, P. and Myles, D.G. (2000) The ADAM gene family: surface proteins with adhesion and protease activity. Trends Genet. 16, 83–87. [18] Ding, X., Yang, L.Y., Huang, G.W., Wang, W. and Lu, W.Q. (2004) ADAM17 mRNA expression and pathological features of hepatocellular carcinoma. World J. Gastroenterol. 10, 2735–2739. [19] Yoshimura, T., Tomita, T., Dixon, M.F., Axon, A.T., Robinson, P.A. and Crabtree, J.E. (2002) ADAMs (a disintegrin and metalloproteinase) messenger RNA expression in Helicobacter pylori-infected, normal, and neoplastic gastric mucosa. J. Infect. Dis. 185, 332–340. [20] Blanchot-Jossic, F., Jarry, A., Masson, D., Bach-Ngohou, K., Paineau, J., Denis, M.G., Laboisse, C.L. and Mosnier, J.F. (2005) Up-regulated expression of ADAM17 in human colon carcinoma: co-expression with EGFR in neoplastic and endothelial cells. J. Pathol. 207, 156–163. [21] Lv, X., Li, Y., Qian, M., Ma, C., Jing, H., Wen, Z. and Qian, D. (2014) ADAM17 silencing suppresses the migration and invasion of non-small cell lung cancer. Mol. Med. Rep. 9, 1935–1940, http://dx.doi.org/10.3892/mmr.2014.2029. [22] Zheng, X., Jiang, F., Katakowski, M., Lu, Y. and Chopp, M. (2012) ADAM17 promotes glioma cell malignant phenotype. Mol. Carcinog. 51, 150–164. [23] Zheng, X. et al. (2007) Inhibition of ADAM17 reduces hypoxia-induced brain tumor cell invasiveness. Cancer Sci. 98, 674–684.
journal homepage: www.FEBSLetters.org
MicroRNA-152 targets ADAM17 to suppress NSCLC progression Yunshu Su, Yuchang Wang, Hongmin Zhou, Lei Lei, Lijun Xu ⇑ Department of Cardiothoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China
a r t i c l e
i n f o
Article history: Received 19 March 2014 Revised 6 April 2014 Accepted 15 April 2014 Available online 26 April 2014 Edited by Tamas Dalmay Keywords: Non-small cell lung cancer miR-152 ADAM metallopeptidase domain 17 Proliferation Migration Invasion
a b s t r a c t MicroRNAs (miRNAs) are a class of small non-coding RNAs that have been suggested to play an essential role in tumorigenesis. In this study, we show that miR-152 is significantly downregulated in non-small cell lung cancer (NSCLC) tissues and cell lines. Restoration of miR-152 significantly reduces proliferation, colony formation, migration and invasion of NSCLC cells. In addition, ADAM metallopeptidase domain 17 (ADAM17) is identified as a target of miR-152 in NSCLC cells, and miR-152-induced suppression of cell proliferation, colony formation, migration and invasion is partially mediated by silencing of ADAM17 expression. Furthermore, ADAM17 inversely correlates with miR-152 in NSCLC tissues. Collectively, our findings indicate that miR-152 acts as tumor suppressor in NSCLC partially via targeting ADAM17. Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Lung cancer is one of the most life-threatening tumors, with approximately two million new cases diagnosed worldwide annually, and Non-small-cell lung cancer (NSCLC) accounts for 80% of primary lung cancer [1]. Despite the improvements in therapeutic modalities, the 5-year survival rate of NSCLC patients is still around 15% [2]. Thus, it is necessary to explore novel agents for NSCLC treatment. MicroRNAs (miRNAs) are small non-coding RNAs which regulate a variety of cellular processes via binding to the 30 -untranslated region (30 -UTR) of target mRNAs, resulting in the degradation of the mRNAs or inhibition of mRNAs translation [3]. Accumulating evidence shows that miRNAs play critical roles in the regulation of cancer initiation and progression [4,5]. MiRNAs can function as oncogenes or tumor suppressors depending on their specific target genes and different cancers [6–8]. Dysfunction of miRNAs occurred in many cancers and aberrantly altered expression of miRNAs was involved in tumorigenesis. A variety of miRNAs have been found to dysregulated in NSCLC, and many miRNAs regulate NSCLC progression [9,10]. miR-152 functions as a tumor suppressor in some cancers [11,12], and it was also downregulated in NSCLC [13], but the detailed role of miR-152 in NSCLC is unclear.
⇑ Corresponding author. Fax: +86 02783665290.
In this study, we found that miR-152 was significantly downregulated in NSCLC tissues and cell lines, and overexpression of miR-152 substantially suppressed proliferation and motility of NSCLC cells. ADAM metallopeptidase domain 17 (ADAM17) was identified as a target of miR-152 in NSCLC cells, and miR-152induced suppression of proliferation and motility of NSCLC cells was partially mediated by silencing of ADAM17 expression. Furthermore, ADAM17 was inversely correlated with miR-152 in NSCLC tissues. 2. Materials and methods 2.1. NSCLC cancer tissue and cell culture Paired NSCLC and adjacent non-tumor normal lung tissues were obtained from 30 patients with informed consent at our hospital. Tissues were immediately snapfrozen and stored at 80 °C. Four NSCLC cell lines (A549, SK-MES-1, H157, and H520) and a normal lung bronchus epithelial cell line BEAS-2B were obtained from American Type Culture Collection and cultured in DMEM supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA), and incubated in 5% CO2 humid atmosphere at 37 °C. 2.2. Plasmid miR-152 mimic/inhibitor was obtained from RiboBio (Guangzhou, China). pcDNA3-ADAM17 was generated by using primers:
E-mail address: [email protected] (L. Xu). http://dx.doi.org/10.1016/j.febslet.2014.04.022 0014-5793/Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1984
Y. Su et al. / FEBS Letters 588 (2014) 1983–1988
sense 50 -GGGGTACCGAATCTTCCCAGTAGGCG-30 , and antisense 50 TTGCGGCCGCAAAGGAGAAGGGCCAAAC-30 . 30 -UTR of ADAM17 was amplified and cloned into the pGL-3-vector (Promega, Madison, WI, USA). Mutations in the 30 -UTR of ADAM17 with miR-152 target site (Mut) was generated with a fast mutation kit (NEB, Ipswich, Canada). 2.3. RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was isolated with Trizol (Invitrogen, Carlsbad, CA, USA). MicroRNA was isolated with the mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA). Expression of miR-152 was detected using TaqMan MicroRNA Assay primers (ABI, Foster City, CA, USA). U6 or GAPDH was used as an internal control. Experiments were performed on the ABI Prism 7500 HT (ABI, Foster City, CA, USA). The relative expression level was calculated by normalization with the signal for U6 expression using 2DDCt method. 2.4. Cell proliferation assay
2.7. Western blotting Cultured cells were lysed in RIPA buffer with 1% PMSF. Protein was loaded and seperated by SDS–PAGE gel and transferred onto PVDF membrane. The blots were probed with primary antibodies at 4 °C overnight and subsequently incubated with HRPconjugated secondary antibodies. Signals were visualized using ECL Substrates (Pierce, Rockford, IL, USA). GAPDH was used as an endogenous control. 2.8. Luciferase assays About 1 105 HEK293 cells per well were cultured in 24-well plates. Cells were co-transfected with 10 ng pRL-TK Renilla luciferase reporter, 50 ng pGL-3 firefly luciferase reporter, and 100 nM miR-152 mimics or control mimics using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Cell lysates were prepared 48 h after transfection using Passive Lysis Buffer, and luciferase activity was examined using the Dual-Luciferase Reporter Assay (Promega, Madison, WI, USA). Results were normalized to the Renilla luciferase.
Transfected cells were seeded into 96-well plates with a density of 4000 cells/well, and cultured for different time. 10 lL of MTT was added into each well, and incubated for 4 h. Then the supernatant was discarded, and 200 lL of DMSO was added to each well. Optical density (OD) was detected at the wavelength of 490 nm. Data were derived from three independent experiments.
Data are presented as mean ± SD. Statistical differences were determined by ANOVA or Student t test. P < 0.05 was considered significant.
2.5. Colony formation assay
3. Results
2000 transfected cells were seeded into 12-well plate. Approximately 10 days later, the clones were washed with PBS and stained with 0.5% crystal violet for 15 min. Finally the clones were taken pictures and counted under a microscope (Olympus, Tokyo, Japan).
3.1. miR-152 was downregulated in NSCLC tissues and cell lines
2.6. Migration and invasion assays Migration and invasion were examined using a transwell chamber (Millipore, Billerica, MA, USA). For the migration assay, 1 105 transfected cells were plated into the upper chamber, and cultured in DMEM, while DMEM with 10% FBS was added to the lower chamber. After 24 h incubation at 37 °C, cells remaining on the upper surface of membrane was removed, and membrane was stained with 20% methanol and 0.5% crystal violet. Cell images were obtained using an inverted microscope (Olympus, Tokyo, Japan). For invasion assays, the upper chamber was precoated with Matrigel (BD, San Jose, CA, USA).
2.9. Statistical analysis
Expression of miR-152 in 30 paired NSCLC tissues was examined by qRT-PCR. We discovered that miR-152 was significantly downregulated in NSCLC tissues compared with the adjacent normal tissues (Fig. 1A). Furthermore, miR-152 was also downregulated in four NSCLC cell lines compared with the normal lung bronchus epithelial cell line BEAS-2B (Fig. 1B). 3.2. miR-152 suppressed NSCLC cell proliferation and colony formation MTT and colony formation were used to determine the effects of miR-152 on A549 and H520 cells. The expression of miR-152 mimics was identified by qRT-PCR (Fig. 2A). Overexpression of miR-152 significantly suppressed cell proliferation of A549 and H520 cells compared with the scramble control (Fig. 2B). In consistent with this result, miR-152 overexpression substantially suppressed colony formation of A549 and H520 cells (Fig. 2C).
Fig. 1. miR-152 was downregulated in NSCLC tissues and cell lines. (A) miR-152 was significantly downregulated in NSCLC tissues compared with the corresponding nontumor normal tissues. (B) miR-152 was significantly decreased in four NSCLC cell lines compared with the lung bronchus epithelial cell line BEAS-2B. ⁄P < 0.05, ⁄⁄P < 0.01 compared with the control group.
Y. Su et al. / FEBS Letters 588 (2014) 1983–1988
1985
Fig. 2. miR-152 suppressed NSCLC cell proliferation and colony formation. (A) Expression of miR-152 in A549 or H520 cells transfected with miR-152 or the scramble control. (B) MTT assay of A549 or H520 cells transfected with miR-152 or the scramble control. (C) Colony formation of A549 or H520 cells transfected with miR-152 or the scramble control. Data were drawn from three independent experiments. ⁄P < 0.05, ⁄⁄P < 0.01 compared with the control group.
3.3. miR-152 suppressed NSCLC cell migration and invasion We further investigated whether miR-152 could also inhibit migration and invasion of NSCLC cells. Overexpression of miR152 dramatically suppressed tumor cell migration in A549 cells compared with the scramble control (Fig. 3A). Similarly, transwell assays with Matrigel demonstrated that miR-152 markedly decreased the invasive capacity of A549 cells (Fig. 3B).
3.5. miR-152 suppressed NSCLC progression partially by targeting ADAM17 We further investigated that whether miR-152 suppressed NSCLC progression by targeting ADAM17. MTT assay, colony formation, migration and invasion (Fig. 5A–D) showed that supplement of ADAM17 by pcDNA3-ADAM17 significantly attenuated the tumor suppressive effects of miR-152. The effect of ADAM17 plasmid was confirmed by Western blotting (Fig. 5E).
3.4. ADAM17 was a target of miR-152 in NSCLC cells To identify the target of miR-152 in NSCLC, TargetScan 6.2 was used. ADAM17, which was increased in NSCLC, was predicted to be a potential target of miR-152 (Fig. 4A). Luciferase activity assay found that miR-152 significantly inhibited the WT but not Mut luciferase activity in HEK293 cells (Fig. 4B). Moreover, overexpression of miR-152 significantly inhibited ADAM17 expression (Fig. 4C).
3.6. miR-152 was inversely correlated with ADAM17 expression in NSCLC tissues Expression of ADAM17 in 30 NSCLC tissues was examined by qRT-PCR. ADAM17 mRNA was remarkably increased in NSCLC tissues (Fig. 6A). Furthermore, ADAM17 mRNA level was inversely correlated with miR-152 level in NSCLC tissues (Fig. 6B).
1986
Y. Su et al. / FEBS Letters 588 (2014) 1983–1988
Fig. 3. miR-152 suppressed NSCLC cell migration and invasion. (A) Migration assay of A549 cells transfected with miR-152 or the scramble control. (B) Invasion assay of A549 cells transfected with miR-152 or the scramble control. Data were drawn from three independent experiments. ⁄P < 0.05, ⁄⁄P < 0.01 compared with the control group.
Fig. 4. ADAM17 was a target of miR-152 in NSCLC cells. (A) The potential miR-152 binding sites of ADAM17 30 -UTR and the mutant. (B) HEK293 cells were co-transfected with miR-152 or the scramble control with WT or Mut ADAM17 30 -UTR. Luciferase activity was assayed. (C) Protein level of ADAM17 was detected by Western blotting in A549 cells transfected with miR-152 or the scramble control. GAPDH was used as an internal control. Experiments were performed in triplicate. ⁄⁄P < 0.01 compared with the control group.
4. Discussion In this study, we provide important evidence in support of miR152 functioning as a tumor suppressor in NSCLC. In addition, we identified ADAM17 as a direct target of miR-152 in NSCLC cells. Restoring the expression of ADAM17 could abolish miR-152mediated suppression of NSCLC progression. Moreover, ADAM17 was inversely correlated with miR-152 in NSCLC tissues. miR-152 is a member of the miR-148/152 family which includes miR-148a/b and miR-152. miR-152 was an tumor suppressive miRNA and decreased in various types of cancer. Aberrant expression of miR-152 was thought to contribute to the malignant phenotype of several tumors. Huang et al. showed that miR-152 was decreased in hepatitis B virus-related hepatocellular carcinoma (HCC), and forced expression of miR-152 in liver cells suppressed the expression of DNA methyltransferase 1 (DNMT1), leading to decrease in global DNA methylation [14]. These data were consistent with the study of pancreatic cancer cells by Azizi et al. [15]. Zhu et al. reported that miR-152 expression was decreased in primary prostate cancer (PC), and low miR-152 expression is correlated with advanced pathological T-stages. They further identified that TGFa, which was increased in PC, was a target of miR-152 [16]. Zheng et al. found that miR-152 and miR-15b
suppressed glioma cell invasion and angiogenesis by targeting NRP-2 and MMP-3 [11]. Our data expanded the tumor suppressive role of miR-152 in NSCLC. Forced expression of miR-152 suppressed A549 cells proliferation, colony formation, migration and invasion. ADAM17 is one kind of the superfamily of Zn-dependent metalloproteinases, and regulates the cleavage of ectodomain of several transmembrane proteins [17]. ADAM17 sheds many essential cell surface molecules, including cytokines and adhesion molecules [18]. Accumulating evidence suggests that ADAM17 was increased in a variety of tumors, including gastric cancer, HCC, colon cancer, breast cancer, and NSCLC [18–21]. The elevated ADAM17 was involved in proliferation, chemoresistance, and malignant phenotypes. Thus, ADAM17 has been considered as a potential therapeutic target in several tumors [22]. Zheng et al. showed that suppression of ADAM17 abolished hypoxia-mediated brain tumor cell invasiveness [23]. Lv et al. found that downregulation of ADAM17 expression suppressed the migration and invasion of NSCLC cells [21]. In the present study, we identified that ADAM17 was a target of miR-152 in NSCLC cells, and supplement of ADAM17 remarkably attenuated the tumor suppressive effects of miR-152 on NSCLC cells. In addition, ADAM17 was inversely correlated with miR-152 in NSCLC tissues.
Y. Su et al. / FEBS Letters 588 (2014) 1983–1988
1987
Fig. 5. miR-152 suppressed NSCLC progression partially by targeting ADAM17. (A) A549 cells were transfected with miR-152 with/without ADAM17 overexpression plasmid, and MTT assay was performed. (B) Colony formation assay. (C) Migration assay. (D) Invasion assay. (E) Protein level of ADAM17. Experiments were performed in triplicate. ⁄ P < 0.05, ⁄⁄P < 0.01 compared with control; #P < 0.05, compared with miR-152 transfected group.
Fig. 6. miR-152 was negatively correlated with ADAM17 in NSCLC tissues. (A) The expression of ADAM17 in 30 pairs of NSCLC tissues was measured by qRT-PCR. (B) ADAM17 mRNA level was inversely correlated with miR-152 level in NSCLC tissues (Spearman’s correlation analysis, r = 0.377, P = 0.04). ⁄P < 0.05 compared with control.
In conclusion, we demonstrate in the present study that miR152 acts as a tumor suppressor in NSCLC cells, which are largely mediated through inhibiting of ADAM17 expression. These findings provide novel insight into the mechanisms of NSCLC.
Conflict of interest The authors declare that they have no conflicts of interest concerning this article.
References [1] Siegel, R., Naishadham, D. and Jemal, A. (2012) Cancer statistics, 2012. CA Cancer J. Clin. 62, 10–29. [2] Nakamura, H., Taniguchi, Y., Miwa, K., Adachi, Y., Fujioka, S., Haruki, T., Takagi, Y. and Yurugi, Y. (2011) Comparison of the surgical outcomes of thoracoscopic lobectomy, segmentectomy, and wedge resection for clinical stage I non-small cell lung cancer. Thorac. Cardiovasc. Surg. 59, 137–141. [3] Aryal, B., Rotllan, N. and Fernandez-Hernando, C. (2014) Noncoding RNAs and atherosclerosis. Curr. Atheroscler. Rep. 16, 407. [4] Xin, C., Zhang, H. and Liu, Z. (2014) MiR-154 suppresses colorectal cancer cell growth and motility by targeting TLR2. Mol. Cell. Biochem. 387, 271–277.
1988
Y. Su et al. / FEBS Letters 588 (2014) 1983–1988
[5] Kolesnikoff, N., Attema, J.L., Roslan, S., Bert, A.G., Schwarz, Q.P., Gregory, P.A. and Goodall, G.J. (2014) Specificity protein 1 (Sp1) maintains basal epithelial expression of the miR-200 family: implications for epithelial–mesenchymal transition. J. Biol. Chem. 289, 11194–11205, http://dx.doi.org/10.1074/ jbc.M113.529172. [6] Yang, H., Li, Q., Zhao, W., Yuan, D., Zhao, H. and Zhou, Y. (2014) MiR-329 suppresses the growth and motility of neuroblastoma by targeting KDM1A. FEBS Lett. 588, 192–197. [7] Ke, Y., Zhao, W., Xiong, J. and Cao, R. (2013) Downregulation of miR-16 promotes growth and motility by targeting HDGF in non-small cell lung cancer cells. FEBS Lett. 587, 3153–3157. [8] Wang, X., Yu, H., Lu, X., Zhang, P., Wang, M. and Hu, Y. (2014) MiR-22 suppresses the proliferation and invasion of gastric cancer cells by inhibiting CD151. Biochem. Biophys. Res. Commun. 445, 175–179. [9] Lei, L., Huang, Y. and Gong, W. (2013) MiR-205 promotes the growth, metastasis and chemoresistance of NSCLC cells by targeting PTEN. Oncol. Rep. 30, 2897–2902. [10] Wan, L., Zhang, L., Fan, K. and Wang, J. (2014) MiR-27b targets LIMK1 to inhibit growth and invasion of NSCLC cells. Mol. Cell. Biochem. 390, 85–91, http:// dx.doi.org/10.1007/s11010-013-1959-1. [11] Zheng, X., Chopp, M., Lu, Y., Buller, B. and Jiang, F. (2013) MiR-15b and miR152 reduce glioma cell invasion and angiogenesis via NRP-2 and MMP-3. Cancer Lett. 329, 146–154. [12] Tsuruta, T. et al. (2011) MiR-152 is a tumor suppressor microRNA that is silenced by DNA hypermethylation in endometrial cancer. Cancer Res. 71, 6450–6462. [13] Sanfiorenzo, C., Ilie, M.I., Belaid, A., Barlesi, F., Mouroux, J., Marquette, C.H., Brest, P. and Hofman, P. (2013) Two panels of plasma microRNAs as noninvasive biomarkers for prediction of recurrence in resectable NSCLC. PLoS One 8, e54596.
[14] Huang, J., Wang, Y., Guo, Y. and Sun, S. (2010) Down-regulated microRNA-152 induces aberrant DNA methylation in hepatitis B virus-related hepatocellular carcinoma by targeting DNA methyltransferase 1. Hepatology 52, 60–70. [15] Azizi, M., Teimoori-Toolabi, L., Arzanani, M.K., Azadmanesh, K., Fard-Esfahani, P. and Zeinali, S. (2014) MicroRNA-148b and microRNA-152 reactivate tumor suppressor genes through suppression of DNA methyltransferase-1 gene in pancreatic cancer cell lines. Cancer Biol. Ther. 15. [16] Zhu, C. et al. (2013) MiR-152 controls migration and invasive potential by targeting TGFalpha in prostate cancer cell lines. Prostate 73, 1082–1089. [17] Primakoff, P. and Myles, D.G. (2000) The ADAM gene family: surface proteins with adhesion and protease activity. Trends Genet. 16, 83–87. [18] Ding, X., Yang, L.Y., Huang, G.W., Wang, W. and Lu, W.Q. (2004) ADAM17 mRNA expression and pathological features of hepatocellular carcinoma. World J. Gastroenterol. 10, 2735–2739. [19] Yoshimura, T., Tomita, T., Dixon, M.F., Axon, A.T., Robinson, P.A. and Crabtree, J.E. (2002) ADAMs (a disintegrin and metalloproteinase) messenger RNA expression in Helicobacter pylori-infected, normal, and neoplastic gastric mucosa. J. Infect. Dis. 185, 332–340. [20] Blanchot-Jossic, F., Jarry, A., Masson, D., Bach-Ngohou, K., Paineau, J., Denis, M.G., Laboisse, C.L. and Mosnier, J.F. (2005) Up-regulated expression of ADAM17 in human colon carcinoma: co-expression with EGFR in neoplastic and endothelial cells. J. Pathol. 207, 156–163. [21] Lv, X., Li, Y., Qian, M., Ma, C., Jing, H., Wen, Z. and Qian, D. (2014) ADAM17 silencing suppresses the migration and invasion of non-small cell lung cancer. Mol. Med. Rep. 9, 1935–1940, http://dx.doi.org/10.3892/mmr.2014.2029. [22] Zheng, X., Jiang, F., Katakowski, M., Lu, Y. and Chopp, M. (2012) ADAM17 promotes glioma cell malignant phenotype. Mol. Carcinog. 51, 150–164. [23] Zheng, X. et al. (2007) Inhibition of ADAM17 reduces hypoxia-induced brain tumor cell invasiveness. Cancer Sci. 98, 674–684.
