SNHG12在乙肝病毒X蛋白诱导肝癌发生中的作用研究

doi:10.3969/j.issn.1000-8535.2023.01.003

摘要/Abstract

摘要： 目的探究长链非编码RNA SNHG12在乙肝病毒X蛋白(HBx)诱导肝癌发生过程中的作用。方法把课题组构建的肝前体细胞14-19、EGFP-14-19、HBx-EGFP-14-19通过小鼠肝门静脉注射到体内;采用 qRT-PCR、Western blot 方法检测30 d,90 d,180 d,360 d小鼠肝脏组织及细胞模型中HBx、SNHG12以及下游调节基因的mRNA和蛋白表达情况;使用si-SNHG12干扰其表达,并通过CCK-8、划痕实验、transwell实验、流式细胞术观察其对体外表型影响;检测SNHG12及下游的 mRNA 和蛋白表达;HE染色观察小鼠肝组织切片。结果 qRT-PCR结果表明SNHG12、Notch1、Hes1在HBx-EGFP-14-19细胞及各时间段的小鼠肝组织中均上调(F=48.808,P<0.000 1;F=13.322,P<0.000 1);Western blot结果显示在HBx过表达细胞及动物模型中,受HBx诱导SNHG12表达升高后Notch1信号通路被激活,促凋亡因子Bax下调,抗凋亡因子Bcl-2上调,细胞周期因子CDK2和Cyclin E上调;抑制SNHG12表达后,qRT-PCR、Western blot实验结果显示SNHG12(t=22.746,P<0.000 1)及其上述基因表达均扭转,CCK-8实验显示细胞增殖受到明显抑制,流式细胞术检测显示细胞凋亡增多,划痕及transwell实验表明细胞迁移及侵袭减弱。结论 HBx通过上调SNHG12诱导Notch1表达,导致细胞增殖、周期和凋亡异常,从而促进肝癌的发生。

关键词: 乙肝病毒X蛋白, SNHG12, 肝癌, 动物模型

Abstract: Objective In order to explore the role of long non-coding RNA SNHG12 in hepatitis B virus X protein (HBx) induced hepatocarcinogenesis. Methods The liver precursor cells 14-19, EGFP-14-19 and HBx-EGFP-14-19 constructed by the research group was injected into the body through the hepatic portal vein of KM mice; qRT-PCR and Western blot were used to detect the mRNA and protein expression of HBx, SNHG12 and downstream regulatory genes in liver tissues of 30 d, 90 d, 180 d and 360 d mice and cell models. Si-SNHG12 was used to interfere with SNHG12 expression in vitro, the mRNA and protein expression of SNHG12 and downstream genes were detected, and its effect on phenotype in vitro was observed by CCK-8, flow cytometry, scratch test and transwell test. HE staining was used to observe the liver sections of mice. Results qRT-PCR showed that SNHG12, Notch1 and Hes1 were up-regulated in HBx overexpression cells and mouse liver tissue at each time point (F=48.808,P<0.000 1;F=13.322,P<0.000 1); the results of Western blot showed that in HBx over-expressing cells and animal models, the expression of SNHG12 was increased induced by HBx, resulting in the activation of Notch1 signal pathway, the down regulation of pro-apoptotic factor Bax, anti-apoptotic factor Bcl-2, and the up-regulation of cell cycle factors CDK2, cyclin E. After inhibiting the expression of SNHG12, the results of qRT-PCR and Western blot showed that SNHG12 (t=22.746,P<0.000 1) and the above genes were inhibited; CCK-8 experiment showed that cell proliferation was significantly inhibited, flow cytometry showed that cell apoptosis increased, scratch experiment and transwell experiment showed that cell migration and invasion decreased. Conclusions HBx induced Notch1 expression by up regulating SNHG12, resulting in abnormal cell proliferation, cycle and apoptosis, so as to promote the occurrence of liver cancer.

Key words: HBx, SNHG12, liver cancer, animal model

吴勇, 况钦, 周梦瑶, 杜彬, 毋楠, 冯涛. SNHG12在乙肝病毒X蛋白诱导肝癌发生中的作用研究[J]. 广州医药, 2023, 54(1): 16-24.

WU Yong, KUANG Qin, ZHOU Mengyao, DU Bin, WU Nan, FENG Tao. Study on the role of SNHG12 in the occurrence of hepatocarcinoma induced by hepatitis B virus X protein[J]. Guangzhou Medical Journal, 2023, 54(1): 16-24.

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