慢病毒介导的靶向MAT2B的短发夹RNA干扰可诱导肝癌细胞生长抑制和凋亡。

Lentivirus mediated shRNA interference targeting MAT2B induces growth-inhibition and apoptosis in hepatocelluar carcinoma.

作者信息

Wang Qun, Liu Quan-Yan, Liu Zhi-Su, Qian Qun, Sun Quan, Pan Ding-Yu

机构信息

Department of General Surgery, Zhongnan Hospital of Wuhan University, No. 160 Dong hu Road, Wuhan, Hubei Province, China.

出版信息

World J Gastroenterol. 2008 Aug 7;14(29):4633-42. doi: 10.3748/wjg.14.4633.

DOI:10.3748/wjg.14.4633

PMID:18698677

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC2738787/

Abstract

AIM

To investigate the effects of lentivirus vector mediated short hairpin RNA interference targeting methionine adenosyltransferase 2beta gene (LV-shMAT2B) on hepatocelluar carcinoma (HCC) cells.

METHODS

We constructed four plasmids of RNA interference targeting the MAT2B gene. After LV-shMAT2B was transfected with L-02 cells and two kinds of HCC cells, cell viability and proliferation were measured with MTT and [3H]thymidine assays respectively. Flow cytometry was used to assess cell apoptosis. The level of S-adenosyl methionine (SAMe) in HepG2 cells was evaluated. The expressions of cyclin D1, cyclin D2, bcl-x(L) and bcl-x(S) were detected with western blot.

RESULTS

We constructed LV-shMAT2B successfully. LV-shMAT2B was safe for human normal liver cells. LV-shMAT2B caused dramatic reduction in proliferation compared with controls in HCC cells Bel-7402 (P = 0.054) and HepG2 (P = 0.031). Flow cytometry analysis showed that cell apoptosis caused by LV-shMAT2B was greater in HCC cells Bel-7402 and HepG2 than in control induced by scrambled siRNA (P = 0.047), but apoptosis rates in L-02 induced by LV-shMAT2B and scrambled siRNA respectively had no significant difference. Moreover, LV-shMAT2B significantly suppressed expression of MAT2B leading to growth-inhibition effect on HCC cells by down-regulating cyclin D1. Apoptosis induced by LV-shMAT2B was involved in down-regulating bcl-x(L) and up- regulating bcl-x(S).

CONCLUSION

LV-shMAT2B can induce cell apoptosis and growth-inhibition in HCC cells. MAT2B may be a therapy target in HCC in the future.

摘要

目的

研究慢病毒载体介导的靶向蛋氨酸腺苷转移酶2β基因的短发夹RNA干扰（LV-shMAT2B）对肝癌（HCC）细胞的影响。

方法

构建了四种靶向MAT2B基因的RNA干扰质粒。将LV-shMAT2B转染L-02细胞和两种肝癌细胞后，分别用MTT法和[3H]胸腺嘧啶掺入法检测细胞活力和增殖情况。采用流式细胞术评估细胞凋亡。检测HepG2细胞中S-腺苷甲硫氨酸（SAMe）的水平。用蛋白质免疫印迹法检测细胞周期蛋白D1、细胞周期蛋白D2、bcl-x(L)和bcl-x(S)的表达。

结果

成功构建了LV-shMAT2B。LV-shMAT2B对人正常肝细胞安全。与对照组相比，LV-shMAT2B使肝癌细胞Bel-7402（P = 0.054）和HepG2（P = 0.031）的增殖显著降低。流式细胞术分析显示，LV-shMAT2B诱导的肝癌细胞Bel-7402和HepG2的细胞凋亡率高于乱序siRNA诱导的对照组（P = 0.047），但LV-shMAT2B和乱序siRNA分别诱导的L-02细胞凋亡率无显著差异。此外，LV-shMAT2B显著抑制MAT2B的表达，通过下调细胞周期蛋白D1对肝癌细胞产生生长抑制作用。LV-shMAT2B诱导的凋亡与下调bcl-x(L)和上调bcl-x(S)有关。