miR-128-3p靶向Lin28B增加肝癌细胞对奥沙利铂的敏感性
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摘要
目的研究miR-128-3p与肝癌(hepatocellular carcinoma,HCC)细胞对奥沙利铂敏感性的影响,并探讨其作用机制.方法采用qRT-PCR法检测人正常肝细胞HL-7702、HCC细胞BEL-7402和Hep-3B中miR-128-3p、Lin28B的表达;将对数生长期的HCC细胞BEL-7402、Hep-3B随机分成miR-128-3pmimics组(转染miR-128-3Pmimics)、miR-NC组(未转染细胞)、Lin28B-3'UTR WT组(载体psiCHECK2-Lin28B-3'UTR WT和miR-128-3p共转染)、Lin28B-3'UTRMUT组(载体psiCHECK2-Lin28B-3'UTRMUT和miR-NC共转染)和miR-128-3p+Lin28B组(miR-128-3p和Lin28B共转染)、si-Lin28B组(转染si-Lin28B)和si-NC组(转染沉默对照),均以脂质体转染.采用MTT法检测各组细胞的存活率和活力; Western Blot检测各组细胞的蛋白表达.结果与人正常肝细胞相比, HCC细胞BEL-7402和Hep-3B中miR-128-3p的表达较显著降低(P<0.01), Lin28B的表达较显著升高(P<0.01),且过表达miR-128-3p、沉默Lin28B均可抑制HCC细胞增殖,促进凋亡,增加HCC细胞对奥沙利铂的敏感性.Lin28B为mi R-128-3p的靶标,且回补Lin28B可逆转mi R-128-3p增加HCC细胞对奥沙利铂敏感性的作用.结论miR-128-3p可增加HCC细胞对奥沙利铂的敏感性,其作用机制可能与靶向Lin28B有关,提示miR-128-3p可作为抗奥沙利铂耐药性的潜在靶点.
AIM To investigate the effect of miR-128-3 p on the sensitivity of hepatocellular carcinoma(HCC) cells to oxaliplatin, and explore the underlying mechanism.METHODS q RT-PCR was used to detect the expression of miR-128-3 p and Lin28 B in human liver cells(HL-7702) and human HCC cells(BEL-7402 and Hep-3 B). BEL-7402 and Hep-3 B cells as well as oxaliplatin resistant BEL-7402 and Hep-3 B cells in logarithmic growth phase were randomly divided into a mi R-128-3 p mimic group(transfected with miR-128-3 p mimics), a miR-NC group(untransfected cells), a Lin28 B-3' UTR WT group(psiCHECK2-Lin28 B-3'UTR WT and miR-128-3 p co-transfection), a Lin28 B-3'-UTR MUT(psiCHECK2-Lin28 B-3' UTR MUT and miRNC co-transfection), a miR-128-3 p + Lin28 B group(miR-128-3 p and Lin28 B co-transfection), a si-Lin28 B group(transfected with si-Lin28 B) and a si-NC(transfected with silencing control). All cells were transfected via liposomes. The survival rate and viability of each group were detected by MTT assay, and the protein expression was detected by Western blot.RESULTS Compared with human hepatocytes, the expression of miR-128-3 p in HCC cells(BEL-7402 and Hep-3 B) wassignificantly decreased, and the expression of Lin28 B was significantly increased. Overexpression of miR-128-3 p or silencing Lin28 B increased the sensitivity of HCC cells to oxaliplatin. Lin28 B is a target of miR-128-3 p, and overexpression of Lin28 B could reverse the effect of mi R-128-3 p in increasing the sensitivity of HCC cells to oxaliplatin.CONCLUSION Mi R-128-3 p can increase the sensitivity of HCC cells to oxaliplatin possibly via a mechanism related to targeting Lin28 B, suggesting that mi R-128-3 p could be used as a potential target for treatment of oxaliplatin resistance.
AIM To investigate the effect of miR-128-3 p on the sensitivity of hepatocellular carcinoma(HCC) cells to oxaliplatin, and explore the underlying mechanism.METHODS q RT-PCR was used to detect the expression of miR-128-3 p and Lin28 B in human liver cells(HL-7702) and human HCC cells(BEL-7402 and Hep-3 B). BEL-7402 and Hep-3 B cells as well as oxaliplatin resistant BEL-7402 and Hep-3 B cells in logarithmic growth phase were randomly divided into a mi R-128-3 p mimic group(transfected with miR-128-3 p mimics), a miR-NC group(untransfected cells), a Lin28 B-3' UTR WT group(psiCHECK2-Lin28 B-3'UTR WT and miR-128-3 p co-transfection), a Lin28 B-3'-UTR MUT(psiCHECK2-Lin28 B-3' UTR MUT and miRNC co-transfection), a miR-128-3 p + Lin28 B group(miR-128-3 p and Lin28 B co-transfection), a si-Lin28 B group(transfected with si-Lin28 B) and a si-NC(transfected with silencing control). All cells were transfected via liposomes. The survival rate and viability of each group were detected by MTT assay, and the protein expression was detected by Western blot.RESULTS Compared with human hepatocytes, the expression of miR-128-3 p in HCC cells(BEL-7402 and Hep-3 B) wassignificantly decreased, and the expression of Lin28 B was significantly increased. Overexpression of miR-128-3 p or silencing Lin28 B increased the sensitivity of HCC cells to oxaliplatin. Lin28 B is a target of miR-128-3 p, and overexpression of Lin28 B could reverse the effect of mi R-128-3 p in increasing the sensitivity of HCC cells to oxaliplatin.CONCLUSION Mi R-128-3 p can increase the sensitivity of HCC cells to oxaliplatin possibly via a mechanism related to targeting Lin28 B, suggesting that mi R-128-3 p could be used as a potential target for treatment of oxaliplatin resistance.
引文
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7 Liang L,Wong CM,Ying Q,Fan DN,Huang S,Ding J,Yao J,Yan M,Li J,Yao M,Ng IO,He X.MicroRNA-125b suppressesed human liver cancer cell proliferation and metastasis by directly targeting oncogene LIN28B2.Hepatology 2010;52:1731-1740[PMID:20827722 DOI:10.1002/hep.23904]
8 Yu D,Green B,Marrone A,Guo Y,Kadlubar S,Lin D,Fuscoe J,Pogribny I,Ning B.Suppression of CYP2C9 by microRNAhsa-miR-128-3p in human liver cells and association with hepatocellular carcinoma.Sci Rep 2015;5:8534[PMID:25704921 DOI:10.1038/srep08534]
9韩子午,周明杰,范春雷,田男.Lin28与肝癌细胞紫杉醇耐药关系的研究.浙江中医药大学学报2014;38:885-888[DOI:10.16466/j.issn1005-5509.2014.07.030]
10 Panella M,Mosca N,Di Palo A,Potenza N,Russo A.Mutual suppression of miR-125a and Lin28b in human hepatocellular carcinoma cells.Biochem Biophys Res Commun 2018;500:824-827[PMID:29689270 DOI:10.1016/j.bbrc.2018.04.167]
11 Viswanathan SR,Powers JT,Einhorn W,Hoshida Y,Ng TL,Toffanin S,O’Sullivan M,Lu J,Phillips LA,Lockhart VL,Shah SP,Tanwar PS,Mermel CH,Beroukhim R,Azam M,Teixeira J,Meyerson M,Hughes TP,Llovet JM,Radich J,Mullighan CG,Golub TR,Sorensen PH,Daley GQ.Lin28 promotes transformation and is associated with advanced human malignancies.Nat Genet 2009;41:843-848[PMID:19483683DOI:10.1038/ng.392]
12 Ma L,Zhao Q,Chen W,Zhang Y.Oncogene Lin28B increases chemosensitivity of colon cancer cells in a let-7-independent manner.Oncol Lett 2018;15:6975-6981[PMID:29725425 DOI:10.3892/ol.2018.8250]
13冯宇鹏,朱俊峰,黄薇,沈艳,叶向东,杨毅.Lin28B的高表达与膀胱癌的恶性程度及预后相关.河南外科学杂志2016;22:23-25[DOI:10.16193/j.cnki.hnwk.2016.03.012]
14 Wang X,Hu H,Liu H.RNA binding protein Lin28B confers gastric cancer cells stemness via directly binding to NRP-1.Biomed Pharmacother 2018;104:383-389[PMID:29787985 DOI:10.1016/j.biopha.2018.05.064]
15 Ren J,Fu J,Ma T,Yan B,Gao R,An Z,Wang D.LncRNA H19-elevated LIN28B promotes lung cancer progression through sequestering miR-196b.Cell Cycle 2018;17:1372-1380[PMID:29950144 DOI:10.1080/15384101.2018.1482137]
16 Zhang X,Liang W,Liu J,Zang X,Gu J,Pan L,Shi H,Fu M,Huang Z,Zhang Y,Qian H,Jiang P,Xu W.Long non-coding RNA UFC1 promotes gastric cancer progression by regulating mi R-498/Lin28b.J Exp Clin Cancer Res 2018;37:134[PMID:29970131 DOI:10.1186/s13046-018-0803-6]
17林万隆,陈强.奥沙利铂的药理作用及临床应用.中国肿瘤临床2000;27:872-874[DOI:10.3969/j.issn.1000-8179.2000.11.034]
18李建华,韩玲,杨志良.miR-125b通过靶向致癌基因LIN28B调控肝细胞癌细胞的增殖和侵袭行为.实用肿瘤杂志2018;33:128-132[DOI:10.13267/j.cnki.syzlzz.2018.02.007]
19陈超庭,沈永奇,黄汉生,斯韬,王志祥,谢华东,孔祥应,林海永.奥沙利铂单药治疗Child-Pugh B级中晚期肝癌的临床研究.中国热带医学2018;18:489-492;499[DOI:10.13604/j.cnki.46-1064/r.2018.05.19]
20郑美玲,华海清.奥沙利铂治疗肝细胞癌耐药的相关机制研究进展.临床肿瘤学杂志2018;22:369-374[DOI:10.3969/j.issn.1009-0460.2017.04.016]
21王作鹏,李凯.miRNA与肿瘤耐药性及逆转耐药策略.中华小儿外科杂志2013;34:140-142[DOI:10.3760/cma.j.issn.0253-3006.2013.02.016]
22 Kovalchuk O,Filkowski J,Meservy J,Ilnytskyy Y,Tryndyak VP,Chekhun VF,Pogribny IP.Involvement of micro RNA-451 in resistance of the MCF-7 breast cancer cells to chemotherapeutic drug doxorubicin.Mol Cancer Ther2008;7:2152-2159[PMID:18645025 DOI:10.1158/1535-7163.MCT-08-0021]
23杨涛,郑志敏,张献波,李振符,张国栓.m i R-93通过靶定PTEN基因增加肝癌细胞对奥沙利铂的耐药性.中国生物化学与分子生物学报2012;28:926-934[DOI:10.13865/j.cnki.cjbmb.2012.10.007]
24伍刚.Lin28B介导肝癌细胞中HBx相关Let-7沉默.现代肿瘤医学2017;25:2211-2215[DOI:10.3969/j.issn.1672-4992.2017.14.006]
2 Jiang J,Zheng X,Xu X,Zhou Q,Yan H,Zhang X,Lu B,Wu C,Ju J.Prognostic significance of miR-181b and miR-21 in gastric cancer patients treated with S-1/Oxaliplatin or Doxifluridine/Oxaliplatin.PLo S One 2011;6:e23271[PMID:21876743 DOI:10.1371/journal.pone.0023271]
3 Kurokawa K,Tanahashi T,Iima T,Yamamoto Y,Akaike Y,Nishida K,Masuda K,Kuwano Y,Murakami Y,Fukushima M,Rokutan K.Role of miR-19b and its target mRNAs in 5-fluorouracil resistance in colon cancer cells.JGastroenterol 2012;47:883-895[PMID:22382630 DOI:10.1007/s00535-012-0547-6]
4崔秀英,郭云杰,姚和瑞.耐药乳腺癌细胞株MCF-7/ADR中microRNA的分析.南方医科大学学报2008;28:1813-1815[DOI:10.3321/j.issn:1673-4254.2008.10.014]
5吕丽霞,薛静.mi RNA作为肿瘤治疗靶点的研究进展.山东医药2014;54:105-108[DOI:10.3969/j.issn.1002-266X.2014.40.046]
6 Huang CY,Huang XP,Zhu JY,Chen ZG,Li XJ,Zhang XH,Huang S,He JB,Lian F,Zhao YN,Wu GB.miR-128-3p suppresses hepatocellular carcinoma proliferation by regulating PIK3R1 and is correlated with the prognosis of HCC patients.Oncol Rep 2015;33:2889-2898[PMID:25962360DOI:10.3892/or.2015.3936]
7 Liang L,Wong CM,Ying Q,Fan DN,Huang S,Ding J,Yao J,Yan M,Li J,Yao M,Ng IO,He X.MicroRNA-125b suppressesed human liver cancer cell proliferation and metastasis by directly targeting oncogene LIN28B2.Hepatology 2010;52:1731-1740[PMID:20827722 DOI:10.1002/hep.23904]
8 Yu D,Green B,Marrone A,Guo Y,Kadlubar S,Lin D,Fuscoe J,Pogribny I,Ning B.Suppression of CYP2C9 by microRNAhsa-miR-128-3p in human liver cells and association with hepatocellular carcinoma.Sci Rep 2015;5:8534[PMID:25704921 DOI:10.1038/srep08534]
9韩子午,周明杰,范春雷,田男.Lin28与肝癌细胞紫杉醇耐药关系的研究.浙江中医药大学学报2014;38:885-888[DOI:10.16466/j.issn1005-5509.2014.07.030]
10 Panella M,Mosca N,Di Palo A,Potenza N,Russo A.Mutual suppression of miR-125a and Lin28b in human hepatocellular carcinoma cells.Biochem Biophys Res Commun 2018;500:824-827[PMID:29689270 DOI:10.1016/j.bbrc.2018.04.167]
11 Viswanathan SR,Powers JT,Einhorn W,Hoshida Y,Ng TL,Toffanin S,O’Sullivan M,Lu J,Phillips LA,Lockhart VL,Shah SP,Tanwar PS,Mermel CH,Beroukhim R,Azam M,Teixeira J,Meyerson M,Hughes TP,Llovet JM,Radich J,Mullighan CG,Golub TR,Sorensen PH,Daley GQ.Lin28 promotes transformation and is associated with advanced human malignancies.Nat Genet 2009;41:843-848[PMID:19483683DOI:10.1038/ng.392]
12 Ma L,Zhao Q,Chen W,Zhang Y.Oncogene Lin28B increases chemosensitivity of colon cancer cells in a let-7-independent manner.Oncol Lett 2018;15:6975-6981[PMID:29725425 DOI:10.3892/ol.2018.8250]
13冯宇鹏,朱俊峰,黄薇,沈艳,叶向东,杨毅.Lin28B的高表达与膀胱癌的恶性程度及预后相关.河南外科学杂志2016;22:23-25[DOI:10.16193/j.cnki.hnwk.2016.03.012]
14 Wang X,Hu H,Liu H.RNA binding protein Lin28B confers gastric cancer cells stemness via directly binding to NRP-1.Biomed Pharmacother 2018;104:383-389[PMID:29787985 DOI:10.1016/j.biopha.2018.05.064]
15 Ren J,Fu J,Ma T,Yan B,Gao R,An Z,Wang D.LncRNA H19-elevated LIN28B promotes lung cancer progression through sequestering miR-196b.Cell Cycle 2018;17:1372-1380[PMID:29950144 DOI:10.1080/15384101.2018.1482137]
16 Zhang X,Liang W,Liu J,Zang X,Gu J,Pan L,Shi H,Fu M,Huang Z,Zhang Y,Qian H,Jiang P,Xu W.Long non-coding RNA UFC1 promotes gastric cancer progression by regulating mi R-498/Lin28b.J Exp Clin Cancer Res 2018;37:134[PMID:29970131 DOI:10.1186/s13046-018-0803-6]
17林万隆,陈强.奥沙利铂的药理作用及临床应用.中国肿瘤临床2000;27:872-874[DOI:10.3969/j.issn.1000-8179.2000.11.034]
18李建华,韩玲,杨志良.miR-125b通过靶向致癌基因LIN28B调控肝细胞癌细胞的增殖和侵袭行为.实用肿瘤杂志2018;33:128-132[DOI:10.13267/j.cnki.syzlzz.2018.02.007]
19陈超庭,沈永奇,黄汉生,斯韬,王志祥,谢华东,孔祥应,林海永.奥沙利铂单药治疗Child-Pugh B级中晚期肝癌的临床研究.中国热带医学2018;18:489-492;499[DOI:10.13604/j.cnki.46-1064/r.2018.05.19]
20郑美玲,华海清.奥沙利铂治疗肝细胞癌耐药的相关机制研究进展.临床肿瘤学杂志2018;22:369-374[DOI:10.3969/j.issn.1009-0460.2017.04.016]
21王作鹏,李凯.miRNA与肿瘤耐药性及逆转耐药策略.中华小儿外科杂志2013;34:140-142[DOI:10.3760/cma.j.issn.0253-3006.2013.02.016]
22 Kovalchuk O,Filkowski J,Meservy J,Ilnytskyy Y,Tryndyak VP,Chekhun VF,Pogribny IP.Involvement of micro RNA-451 in resistance of the MCF-7 breast cancer cells to chemotherapeutic drug doxorubicin.Mol Cancer Ther2008;7:2152-2159[PMID:18645025 DOI:10.1158/1535-7163.MCT-08-0021]
23杨涛,郑志敏,张献波,李振符,张国栓.m i R-93通过靶定PTEN基因增加肝癌细胞对奥沙利铂的耐药性.中国生物化学与分子生物学报2012;28:926-934[DOI:10.13865/j.cnki.cjbmb.2012.10.007]
24伍刚.Lin28B介导肝癌细胞中HBx相关Let-7沉默.现代肿瘤医学2017;25:2211-2215[DOI:10.3969/j.issn.1672-4992.2017.14.006]