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人肝癌细胞系中miR-20a-5p的表达及对肝癌细胞Bel-7402增殖及凋亡的影响
郭瑛,李君,李宗芳,孙晋,张健,田静,王亚利,孔光耀
0
(西安交通大学第二附属医院生物诊断治疗国家地方联合工程研究中心;西安交通大学第二附属医院干部病房普通外科;西安交通大学第二附属医院肿瘤科)
摘要:
【摘要】 目的 探讨miR20a5p在人肝癌细胞系中表达及其对肝癌细胞Bel7402增殖及凋亡的影响。方法 通过反转录实时定量聚合酶链反应(RTqPCR)考察miR20a在肝细胞L02和7种肝癌细胞中的表达;构建过表达或沉默miR20a5p的人肝癌Bel7402细胞株;通过噻唑蓝(MTT)实验考察过表达或沉默miR20a5p后对细胞增殖的影响;通过AnnexinVFITC/PI流式实验考察对细胞凋亡的影响,并进一步通过蛋白质免疫印迹试验考察对凋亡相关基因蛋白表达水平的影响。结果 与人肝细胞L02相比,6种细胞系的miR20a5p表达水平较低。过表达miR20a5p后细胞增殖能力显著下降(P=0.011),沉默后显著上升(P=0.007)。高表达miR20a5p后细胞凋亡和早期凋亡比例均显著增加(P=0.009、0.017),沉默miR20a5p均明显降低(P=0.012、0.007)。高表达miR20a5p后细胞中凋亡抑制蛋白XIAP、Survivin、Bcl2表达下调,凋亡促进蛋白Bax表达上调;沉默miR20a5p后XIAP、Survivin、Bcl2表达上调,Bax表达下调。结论 miR20a5p在6种肝癌细胞中低表达,可促进肝癌细胞Bel7402凋亡,抑制细胞增殖能力。
关键词:  微小RNA-20a-5p  肝癌  增殖  凋亡
DOI:
基金项目:国家自然科学基金青年科学基金项目(81603159);中央高校基本科研业务费专项资金资助(XJJ2015102);西安交通大学第二附属医院科研基金青年项目(YJ(QN)201502)
The expression of miR-20a-5p in different human hepatocellular carcinoma cell lines and its effects on proliferation and apoptosis in hepatocellular carcinoma Bel-7402 cell line
GUO Ying,LI Jun,LI Zongfang,SUN Jin,ZHANG Jian,TIAN Jing,WANG Yali,SUN Guangyao
(National & Local Joint Engineering Research Center of Biodiagnostics and Biotherapy, The Second Affiliated Hospital;General Surgery of Cadre Ward, The Second Affiliated Hospital, Xi’an Jiaotong University;Department of Oncology, The Second Affiliated Hospital, Xi’an Jiaotong University)
Abstract:
【Abstract】 Objective To explore the expression of miR20a5p in different human hepatocellular carcinoma cell lines and investigate its effects on proliferation and apoptosis in hepatocellular carcinoma Bel7402 cell line. Methods Reverse transcriptase Realtime quantitative polymerase chain reaction (RTqPCR) was used to detect the expression level of miR20a5p in human hepatic cells L02 and 7 hepatocellular carcinoma cell lines. Bel7402 cell lines overexpressing or lowexpressing miR20a5p were constructed by using Lipo3000. MTT assay and AnnexinVFITC/PI flow cytometry assay were used to examine the effects of miR20a5p on proliferation and apoptosis in Bel7402 cells. Immunoblotting was carried out to detect the effects of miR20a5p on expression levels of apoptosis related protein. Results The results of RTqPCR showed that the expressions of miR20a5p in 6 human hepatocellular carcinoma cell lines were lower than that in L02. After miR20a5p overexpressing or silencing, MTT assay showed the proliferation ability was significantly inhibited or promoted(P=0.011, 0.007)and AnnexinVFITC/PI flow cytometry assay showed the rates of apoptosis and early apoptosis were significantly enhanced or reduced(P=0.009、0.017), as compared with the control group. Immunoblotting assay showed the overexpression of miR20a5p downregulated the expression of antiapoptosis protein XIAP, Survivin, Bcl2 and upregulated the apoptosis protein Bax and vice versa. Conclusion The expression level of miR20a5p in 6 hepatocellular carcinoma cell lines was lower than that in hepatic cells and miR20a5p can induce the apoptosis and inhibit the proliferation capability of Bel7402 cells.
Key words:  MicroRNA-20a-5p  Hepatocellular carcinoma  Proliferation  Apoptosis

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