长链非编码RNA HAGLROS调控miR-26b/JAK2/STAT3通路对肝癌细胞上皮间质转化的影响  

Effects of long non-coding RNA HAGLROS on epithelial-mesenchymal transition of hepatocellular carcinoma cells by targeting miR-26b/JAK2/STAT3

作　　者：张坤 朱强 庄雅峰 游悦凯 张帅帅 ZHANG Kun;ZHU Qiang;ZHUANG Yafeng;YOU Yuekai;ZHANG Shuaishuai(Department of Hepatobiliary Surgery, Xiang'an Hospital Affiliated to Xiamen University, Xiamen 361000, China)

机构地区：[1]厦门大学附属翔安医院肝胆外科,福建厦门361000 [2]福建医科大学福总临床医学院肝胆外科,350025

出　　处：《临床肿瘤学杂志》2022年第4期331-338,共8页Chinese Clinical Oncology

摘　　要：目的探讨长链非编码RNA(lncRNA)HAGLR互补链lncRNA(HAGLROS)能否通过微小RNA-26b(miR-26b)/Janus激酶2(JAK2)/转录激活因子3(STAT3)轴来调控肝癌增殖、迁移、侵袭和上皮间质转化(EMT)。方法通过实时荧光定量PCR(qPCR)检测正常肝细胞系LO2和肝癌细胞(Hep3B、MHCC-LM3、Huh7、SMMC-7721)的HAGLROS和miR-26b水平。用双荧光素酶报告实验鉴定HAGLROS与miR-26b的相互作用。分别转染HAGLROS干扰序列si-HAGLROS和miR-26b抑制剂(inhibitor)至SMMC-7721细胞。挽救实验通过转染miR-26b inhibitor同时干扰HAGLROS表达。将SMMC-7721细胞分为si-NC组、si-HAGLROS组、miR-26b inhibitor组和si-HAGLROS+miR-26b inhibitor组。分别利用MTT法、划痕实验和Transwell实验检测SMMC-7721细胞增殖、迁移和侵袭情况。通过检测E-钙黏蛋白(E-cad)、N-钙黏蛋白(N-cad)和纤维粘连蛋白(FN)水平以评估EMT过程。qPCR和Western blotting检测B细胞淋巴瘤2(Bcl-2)、磷酸化JAK2(p-JAK2)和磷酸化STAT3(p-STAT3)的水平。结果与LO2细胞相比,肝癌细胞的HAGLROS水平升高,而miR-26b水平降低(P<0.05);在线生物信息学预测和荧光素酶报告分析证实miR-26b能够与HAGLROS靶向结合。细胞功能丧失实验表明,与si-NC组相比,SMMC-7721细胞的增殖、迁移和侵袭能力及N-cad、FN、Bcl-2、p-JAK2和p-STAT3水平在si-HAGLROS组中降低而在miR-26b inhibitor组中增加,E-cad水平则在si-HAGLROS组中升高而在miR-26b inhibitor组中降低,上述差异均有统计学意义(P<0.05);挽救实验表明,与si-HAGLROS组相比,si-HAGLROS+miR-26b inhibitor组SMMC-7721细胞的增殖、迁移和侵袭能力及N-cad、FN、Bcl-2、p-JAK2和p-STAT3水平增加,而E-cad水平降低,上述差异均有统计学意义(P<0.05)。结论HAGLROS通过靶向miR-26b促进肝癌细胞迁移、侵袭和EMT过程并调控JAK2/STAT3信号通路活性,为HAGLROS作为肝癌的干预治疗的新靶点提供新思路。Objective To explore the effects of long non-coding RNA(lncRNA)HAGLR opposite strand lncRNA(HAGLROS)on the proliferation,migration,invasion and epithelial-mesenchymal transition(EMT)of hepatocellular carcinoma cells by microRNA-26b(miR-26b)/Janus-activated kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3)axis.Methods Levels of HAGLROS and miR-26b in normal liver cell line LO2 and HCC cell lines(Hep3B,MHCC-LM3,Huh7,SMMC-7721)were detected by quantitative real-time polymerase chain reaction(qPCR).The molecular interaction between HAGLROS and miR-26b was identified by luciferase reporter assay.The small interfering RNA(siRNA)sequence targeting HAGLROS(si-HAGLROS)and miR-26b inhibitor were transfected into SMMC-7721 cells,respectively.The rescue experiments were performed in SMMC-7721 cells treated with miR-26b inhibitor and HAGLROS knockdown.The cells were allocated into si-NC group,si-HAGLROS group,miR-26b inhibitor group and si-HAGLROS+miR-26b inhibitor group.MTT assay,wound healing assay and Transwell assay were used for investigating SMMC-7721 cell proliferation,migration and invasion,respectively.Levels of E-cadherin(E-cad),N-cadherin(N-cad)and fibronectin(FN)were measured to evaluate EMT progression of SMMC-7721 cells.Moreover,levels of B-cell lymphoma-2(Bcl-2),phosphorylated JAK2(p-JAK2)and phosphorylated STAT3(p-STAT3)were investigated by qPCR and Western blotting.Results Compared with LO2 cells,HAGLROS were remarkably increased and miR-26b was remarkably decreased in the hepatocellular carcinoma cells(P<0.05).Online bioinformatic prediction and luciferase reporter assay revealed that miR-26b targeted with HAGLROS.Loss-of-function assays indicated that compared with si-NC group,the proliferation,migration and invasion ability of SMMC-7721 cells and levels of N-cad,FN,Bcl-2,p-JAK2 and p-STAT3 were decreased in si-HAGLROS group but increased in miR-26b inhibitor group,while E-cad level increased in si-HAGLROS group but decreased in miR-26b inhibitor group(P<0.05).Rescue experiments indic

关 键 词：肝癌 HAGLR互补链lncRNA 微小RNA-26b 迁移 侵袭 上皮间质转化 

分 类 号：R735.7[医药卫生—肿瘤]