LHX3基因调控Sonic hedgehog信号通路影响原发性肝癌发生和发展的研究  

Influence of LHX3 gene on occurrence and development of primary liver cancer by regulating Sonic hedgehog signaling pathway

作　　者：李荣军[1] 游忠岚 徐春霞[1] 王健[1] 杨小丽[1] 向洪志 杨伟兴 LI Rongjun;XU Chunxia;WANG Jian;YANG Xiaoli;XIANG Hongzhi;YOU Zhonglan;YANG Weixing(Department of spleen,stomach and liver diseases,Luzhou Hospital of Traditional Chinese Medicine,Luzhou 646000,China;Department of Infectious Diseases,Chongqing Key Laboratory for Reasearch of Infectious Diseases,First Affiliated Hospital of PLA Army Medical University,Chongqing 400038,China;Department of Spleen and Stomach Diseases,Affiliated Hospital of Traditional Chinese Medicine,Southwest Medical University,Luzhou 646000,China)

机构地区：[1]泸州市中医医院脾胃病肝病科,646000 [2]中国人民解放军陆军军医大学第一附属医院感染科感染病研究重庆市重点研究室,重庆400038 [3]西南医科大学附属中医医院脾胃病科,四川泸州646000

出　　处：《国际消化病杂志》2022年第2期97-104,共8页International Journal of Digestive Diseases

基　　金：重庆市基础研究与前沿探索项目(重庆市自然科学基金)(cstc2018jcyjAX0473)。

摘　　要：目的探究LHX3基因在原发性肝癌(以下简称肝癌)发生和发展中的作用及机制。方法收集2017年1月至2019年1月在泸州市中医医院接受手术治疗的54例肝癌患者的肿瘤组织及癌旁组织(距肿瘤边缘>3 cm),采用免疫组织化学染色法检测组织中LHX3表达水平。将HepG2细胞随机分为空白对照组(正常培养细胞)、NC-shRNA组(转染阴性对照NC-shRNA慢病毒质粒)、LHX3-shRNA组(转染LHX3-shRNA慢病毒质粒)和LHX3-shRNA+Pur组(转染LHX3-shRNA慢病毒质粒后用Purmorphamine试剂处理24 h)。采用实时荧光定量PCR(real-time qPCR)和蛋白质印迹法(Western blotting)检测细胞中LHX3表达水平,Western blotting法检测Sonic hedgehog(Shh)信号通路相关蛋白Shh、Gli-l的表达,CCK-8法检测细胞增殖能力,Transwell实验检测细胞侵袭和迁移能力。选取30只SPF级雌性BALB/c裸鼠随机分为对照组、NC-shRNA组和LHX3-shRNA组,分别接种空白对照组、NC-shRNA组和LHX3-shRNA组HepG2细胞,以构建移植瘤裸鼠模型。在接种后第21天处死裸鼠,采用H-E、TUNEL染色法观察各组肿瘤组织的病理变化,免疫组织化学染色法检测Gli-l表达,Western blotting法检测Shh、Gli-l蛋白表达。结果肝癌患者肿瘤组织中LHX3阳性表达率较癌旁组织显著升高(P<0.05)。与空白对照组比较,沉默LHX3表达的LHX3-shRNA组HepG2细胞中LHX3 mRNA和蛋白相对表达量均显著下降(P均<0.05),Shh、Gli-l蛋白相对表达量均显著下降(P<0.05),细胞增殖能力显著减弱(P<0.05),细胞侵袭数量与迁移数量均显著减少(P均<0.05)。与LHX3-shRNA组比较,LHX3-shRNA+Pur组细胞的增殖能力显著增强(P<0.05),细胞侵袭数量与迁移数量均显著增加(P均<0.05)。与对照组比较,LHX3-shRNA组裸鼠肿瘤组织体积显著减小、湿重显著降低(P均<0.05),肿瘤组织细胞密度降低,细胞凋亡率显著升高(P<0.05),Shh和Gli-l蛋白相对表达量均显著下降(P均<0.05),Gli-l蛋白阳性表达率显著下降(P<0.05)。结Objective This paper attempts to explore the role and mechanism of LHX3 gene in the occurrence and development of primary liver cancer(hereafter referred to as liver cancer).Methods The tumor tissues and adjacent tissues(>3 cm from the tumor edge)of 54 patients with liver cancer who received surgical treatment in Luzhou Hospital of Traditional Chinese Medicine from January 2017 to January 2019 were collected,and the expression of LHX3 in the tissues was detected by immunohistochemical staining.HepG2 cells were randomly divided into the blank control group(normal cultured cells),the NC-shRNA group(transfected with negative control NC-shRNA lentiviral plasmid),the LHX3-shRNA group(transfected with LHX3-shRNA lentiviral plasmid),and the LHX3-shRNA+Pur group(treated with Purmorphamine for 24 h after transfection with LHX3-shRNA lentiviral plasmid).The expression levels of LHX3 in cells were detected by real-time qPCR and Western blotting.The expressions of Sonic hedgehog(Shh)signaling pathway-related proteins Shh and Gli-l were detected by Western blotting.Cell proliferation was detected by CCK-8 assay.Cell invasion and migration were detected by Transwell assay.Thirty SPF female BALB/c nude mice were randomly divided into the control group,the NC-shRNA group,and the LHX3-shRNA group,and were inoculated with HepG2 cells to construct the transplanted tumor nude mouse model.The nude mice were sacrificed on day 21 after inoculation.The pathological changes of tumor tissues in each group were observed by H-E and TUNEL staining,while the expression of Gli-1 was detected by immunohistochemical staining,and the protein expressions of Shh and Gli-1 were detected by Western blotting.Results The positive expression rate of LHX3 in tumor tissues of patients with liver cancer is significantly higher than that in adjacent tissues(P<0.05).Compared with the blank control group,the relative expressions of LHX3 mRNA and protein in HepG2 cells in the LHX3-shRNA group with silenced LHX3 expression are significantly decreased(P<0.05),th

关 键 词：肝癌 LHX3 Sonic hedgehog信号通路 肿瘤生长 

分 类 号：R735.7[医药卫生—肿瘤]