MiR-200b通过靶向调控SMYD2/p53信号通路抑制肝癌细胞增殖  

Effect of miRNA-200b on the proliferation of liver cancer cells via targeting SMYD2/p53 signaling pathway

作　　者：方伟进 宋立莹[1] 李佐军[1] 孟佩佩 左珊如 刘世坤[1] FANG Weijin;SONG Liying;LI Zuojun;MENG Peipei;ZUO Shanru;LIU Shikun(Department of Pharmacy,Third Xiangya Hospital,Central South University,Changsha 410013;Department of Pharmacy,Women and Children’s Health Care Hospital of Linyi,Linyi Shandong 276000,China)

机构地区：[1]中南大学湘雅三医院药学部,长沙410013 [2]临沂市妇幼保健院,山东临沂276000

出　　处：《中南大学学报（医学版）》2022年第10期1303-1314,共12页Journal of Central South University ：Medical Science

基　　金：the Natural Science Foundation of Hunan Province(2018JJ3795),China.

摘　　要：目的:本课题组前期研究证实组蛋白甲基转移酶2(SET and MYND domain-containing protein 2,SMYD2)高水平表达在肝癌细胞进展过程中发挥重要作用。MiR-200b作为肿瘤抑制基因参与多种肿瘤的发生和发展。本研究旨在探究miR-200b与SMYD2在肝癌中的作用机制。方法:采用实时反转录聚合酶链反应(real-time reverse transcription-polymerase chain reaction,RT-PCR)及蛋白质印迹法分别检测癌组织及癌旁组织中SMYD2和miR-200b的表达水平。通过双荧光素酶报告验证SMYD2与miR-200b的靶向关系。通过沉默及过表达miR-200b研究其对SMYD2及下游p53/CyclinE1通路的影响,并通过沉默SMYD2研究其对肝癌细胞株的增殖及细胞周期蛋白(Cyclin)E1的影响,进一步证实SMYD2与p53/CyclinE1的关系。结果:与癌旁组织相比,肝癌组织中miR-200b表达显著减少,而SMYD2表达显著增加(均P<0.05),SMYD2与miR-200b呈负相关(P<0.01)。双荧光素酶报告结果显示:在工具细胞HEK 293T细胞中,miR-200b直接靶向结合SMYD2并抑制其表达。在肝癌细胞株MHCC-97L中下调miR-200b可增加SMYD2的表达(P<0.01)并促进肝癌细胞的增殖(P<0.05),上调miR-200b则得出相反的结果。在MHCC-97L细胞株中沉默SMYD2可抑制细胞的增殖(P<0.01),并导致p53水平的升高和CyclinE1表达的下降(均P<0.05)。结论:MiR-200b通过靶向结合SMYD2及调控p53/CyclinE1通路参与肝癌的进展,有望成为治疗肝癌细胞的新靶点。Objective:Our previous study has verified that high level of SET and MYND domain containing protein 2(SMYD2)plays an important role in acquiring aggressive ability for liver cancer cells in hepatocellular carcinoma.MiR-200b as a tumor suppressor gene involves in a variety of cancers.This study aims to investigate the correlation between miR 200b and SMYD2 in hepatocellular carcinoma and the underlying mechanism.Methods:Firstly,the levels of SMYD2 and miR-200b in hepatocellular carcinoma tissues and matched adjacent non-tumor liver tissues were tested with real-time reverse transcription-polymerase chain reaction(RT-PCR)and Western blotting.Secondly,we evaluated the interaction between miR-200b and SMYD2 using dual-luciferase reporter assay.Thirdly,we elucidated the effect of miR-200b on SMYD2 and its downstream targets p53/CyclinE1.Finally,we silenced SMYD2 in hepatocellular carcinoma cell lines to investigate its effect on tumor proliferation and cell cycle progression,and further confirmed the correlation among SMYD2 and p53/CyclinE1.Results:Compared with the matched adjacent non-tumor liver tissues,miR-200b was obviously decreased,and SMYD2 was significantly increased in hepatocellular carcinoma(both P<0.05).Spearman’s rank correlation revealed that miR-200b expression was negatively correlated with SMYD2(P<0.01).Computer algorithm and dual-luciferase reporter assay revealed that miR-200b directly targeted and suppressed SMYD2 in HEK 293T cells.The down-regulated miR-200b expression promoted hepatoma cell proliferation(P<0.05)and increased SMYD2 expression(P<0.01),while the up-regulated expression of miR-200b had an opposite effect.The knockdown of SMYD2 suppressed the proliferation of MHCC-97L cells(P<0.01),down-regulated CyclinE1,and up-regulated p53 expression(both P<0.05).Conclusion:MiR-200b is involved in hepatocellular carcinoma progression via targeting SMYD2 and regulating SMYD2/p53/CyclinE1 signaling pathway and may be used as a potential target for hepatocellular carcinoma treatment.

关 键 词：肝细胞癌 miR-200b 组蛋白甲基转移酶2 P53 细胞周期蛋白E1 

分 类 号：R735.7[医药卫生—肿瘤]