Lin28对肝癌HepG2细胞5-Fu敏感性的影响及其分子机制  

Effect and molecular mechanism of Lin28 on 5-Fu sensitivity of hepatocellular carcinoma HepG2 cells

作　　者：陈少坚[1] 林拥华[1] 吴友谊[1] 魏剑锋[1] 廖政戎 CHEN Shaojian;LIN Yonghua;WU Youyi;WEI Jianfeng;Liao Zhengrong(Department of General Surgery,the Second Affiliated Hospital of Fujian Medical University,Quanzhou 362000,Fujian,China)

机构地区：[1]福建医科大学附属第二医院普外科,福建泉州362000

出　　处：《中国肿瘤生物治疗杂志》2020年第3期261-266,共6页Chinese Journal of Cancer Biotherapy

基　　金：福建省中青年教师教育科研资助项目(No.JA15193)。

摘　　要：目的:探讨RNA结合蛋白Lin28对肝癌HepG2细胞5-氟尿嘧啶(5-Fu)敏感性的影响及其机制。方法:应用pcDNA3.1-Lin28或si-Lin28转染HepG2细胞,采用q PCR及WB法检测转染后HepG2细胞Lin28的表达水平,CCK-8法检测5-Fu作用后转染细胞增殖活性变化并计算5-Fu对细胞作用的IC50,流式细胞术检测5-Fu处理后细胞凋亡情况、WB法检测凋亡相关蛋白的表达变化,qPCR法检测转染后耐药相关miRNA(let-7a和let-7b)及肿瘤干细胞标记物(Oct4、Nanog和Sox2)的mRNA表达水平。结果:与空载对照组(HepG2/Vector)相比,HepG2/Lin28组细胞中Lin28 mRNA和蛋白的表达水平均显著升高(P<0.05或P<0.01),过表达Lin28可显著抑制HepG2细胞对5-Fu作用的敏感性(IC50明显升高,P<0.05)和增强细胞增殖活性、使细胞凋亡率和凋亡相关蛋白caspase-3表达明显降低(均P<0.01)。与阴性对照组(si-control)相比,si-Lin28细胞中Lin28表达水平显著下降(P<0.01);敲减Lin28可使细胞增殖活性及5-Fu的IC50显著降低(均P<0.01),凋亡率及凋亡蛋白表达明显增加(P<0.01)。与HepG2/Vector组比较,HepG2/Lin28细胞中let-7a、let-7b及肿瘤干细胞标记物(Oct4、Nanog和Sox2)的表达水平明显上调(均P<0.01),而si-Lin28细胞中let-7a、let-7b及Oct4、Nanog、Sox2的表达水平较si-control组明显下调(均P<0.01)。结论:Lin28可通过调节miRNAs的表达及肿瘤干细胞的形成从而调节肝癌HepG2细胞对化疗的敏感性,靶向调节Lin28表达有望成为提高肝癌化疗疗效的手段。Objective: To investigate the effect and mechanism of RNA binding protein Lin28 on the 5-fluorouracil(5-Fu) sensitivity of HepG2 cells. Methods: HepG2 cells were transfected with plasmid pcDNA3.1-Lin28 or si-Lin28(small interfering RNA of Lin28).qPCR and Western blotting were used to detect the expression of Lin28 in HepG2 cells after transfection. Changes of cell proliferation in transfected cells after 5-Fu treatment was detected by CCK8 assay and the 50% inhibitory concentration(IC50) was calculated. Flow cytometry was used to detect apoptotic rate after 5-Fu treatment and the expression of apoptosis-related protein was assayed by Western blotting. The mRNA expressions of drug-resistant miRNAs(let-7 a and let-7 b), as well as cancer stem cell markers(Oct4, Nanog and Sox2) after transfection were detected by qPCR. Results: As compared to the HepG2/Vector cells, the mRNA and protein expressions of Lin28 were significantly up-regulated in HepG2/Lin28 cells(P<0.05 or P<0.01). Over-expression of Lin28 significantly suppressed the sensitivity of HepG2 cells to 5-Fu(IC50 elevated obviously, P<0.05) and significantly increased cell proliferation while decreased apoptotic rate and expression of apoptotic-related protein caspase-3(all P<0.01). As compared to si-control group, expression of Lin28 in HepG2/si-Lin28 cells was significantly down-regulated(P<0.01). Lin28 knockdown significantly reduced cell proliferation and IC50 of 5-Fu(all P<0.01) but increased apoptotic rate and expression of apoptosis-related protein(P<0.01). Compared with HepG2/Vector group, expressions of let-7 a and let-7 b, as well as cancer stem cell markers(Oct4, Nanog and Sox2) were significantly increased in HepG2/Lin28 cells(all P<0.01);while these molecules were significantly decreased in HepG2/si-Lin28 cells as comparing to si-control group(all P<0.01). Conclusion: Lin28 can modulate the chemosensitivity of HepG2 cells by regulating the expression of miRNAs and the formation of cancer stem cells. Targeting Lin28 might be a promising approach t

关 键 词：肝细胞癌 HEPG2细胞 Lin28 5-氟尿嘧啶 化疗敏感性 肿瘤干细胞 凋亡 

分 类 号：R735.7[医药卫生—肿瘤] R730.5[医药卫生—临床医学]