miR-145-5p通过SMAD4基因对食管鳞状细胞癌细胞增殖与凋亡影响  

EFFECT OF MIR-145-5P ON THE PROLIFERATION AND APOPTOSIS OF ESOPHAGEAL SQUAMOUS CELL CARCINOMA CELLS BY TARGETING THE SMAD4 GENE

作　　者：赵红丹[1] 郭康[1] 宋士军[1] 高明[2] ZHAO Hongdan;GUO Kang;SONG Shijun;GAO Ming(Department of Oncology,the Third Affiliated Hospital of Xinxiang Medical University,Xinxiang 453000,China)

机构地区：[1]新乡医学院第三附属医院肿瘤内二科,河南新乡453000 [2]郑州大学第一附属医院肿瘤科

出　　处：《青岛大学学报（医学版）》2022年第4期577-583,共7页Journal of Qingdao University(Medical Sciences)

基　　金：河南省医学科技攻关计划普通项目(201602007)。

摘　　要：目的探究miR-145-5p靶向调控SMAD4基因对食管鳞状细胞癌细胞增殖和凋亡的作用及其机制。方法收集68例食管鳞状细胞癌病人癌组织及其癌旁正常组织,采用免疫组织化学方法检测SMAD4蛋白阳性表达率,应用实时定量聚合酶链反应(qRT-PCR)技术检测食管鳞状细胞癌及其癌旁正常组织中miR-145-5p和SMAD4表达水平。筛选合适食管鳞状细胞癌细胞株分为Blank组、NC组、miR-145-5pmimic组、miR-145-5pinhibitor组、siRNA-SMAD4组和miR-145-5pinhibitor+siRNA-SMAD4组。生物学网站和双荧光素酶实验验证miR-145-5p和SMAD4的靶向关系。qRT-PCR检测miR-145-5p、SMAD4、TGF-β、Bcl-2和Bax的mRNA表达水平,WesternBlot分别检测SMAD4、TGF-β、Bcl-2和Bax的蛋白的表达水平。CCK8方法检测细胞增殖活力;流式细胞仪检测细胞凋亡率。结果免疫组织化学检测显示,SMAD4阳性颗粒主要表达于细胞浆或细胞核,且与癌旁正常组织相比,食管鳞状细胞癌组织中SMAD4阳性率明显升高( χ^(2)=14.251,P<0.05);而qRT-PCR显示miR-145-5p表达水平显著降低(t=109.800,P<0.05)。相比其他细胞株,ECA-109细胞中的miR-145-5p表达量最低(F=48.000,P<0.05),选用于后续实验。双荧光素酶报告基因实验显示,miR-145-5p可靶向调控SMAD4(t=21.820,P<0.05)。与Blank组相比,NC组的各项指标无显著差异(P均>0.05);与Blank组和NC组相比,miR-145-5pmimic组miR-145-5p和Bax表达增加,SMAD4、TGF-β和Bcl-2表达降低,细胞增殖能力明显下降,细胞凋亡率显著上升(P均<0.05);siRNA-SMAD4组miR-145-5p表达无明显变化,Bax表达增加,SMAD4、TGF-β和Bcl-2表达降低,细胞增殖能力明显下降,细胞凋亡率显著上升(P均<0.05);miR-145-5pinhibitor组miR-145-5p和Bax表达降低,SMAD4、TGF-β和Bcl-2表达上升,细胞增殖能力明显上升,细胞凋亡率显著下降(P均<0.05);而miR-145-5pinhibitor+siRNA-SMAD4组与Blank组和NC组相比组间差异无显著性(P均>0.05)。结论miR-145-5p高表达可通过靶向抑�Objective To investigate the effect of miR-145-5p on the proliferation and apoptosis of esophageal squamous cell carcinoma cells through targeted regulation of the SMAD4 gene and its mechanism.Methods Cancerous tissue samples and adjacent tissue samples were collected from 68 patients with esophageal squamous cell carcinoma.Immunohistochemistry was used to measure the positive expression rate of SMAD4 protein,and quantitative real-time PCR was used to measure the expression level of miR-145-5p and SMAD4 in esophageal squamous cell carcinoma tissue and adjacent tissue.Suitable esophageal squamous cell carcinoma cell lines were screened out and divided into Blank group,NC group,miR-145-5p mimic group,miR-145-5p inhibitor group,siRNA-SMAD4 group,and miR-145-5p inhibitor+siRNA-SMAD4 group.Biological websites and dual-luciferase reporter assay were used to verify the targeting relationship between miR-145-5p and SMAD4.Quantitative real-time PCR was used to measure the mRNA expression levels of miR-145-5p, SMAD4,TGF-β,Bcl-2 ,and Bax,and Western Blot was used to measure the protein expression levels of SMAD4,TGF-β,Bcl-2,and Bax.CCK8 assay was used to measure cell proliferation activity,and flow cytometry was used to measure cell apoptosis rate.Results Immunohistochemistry showed that SMAD4 was mainly expressed in cytoplasm or nucleus,and the positive rate of SMAD4 in esophageal squamous cell carcinoma tissue was significantly higher than that in normal adjacent tissue( χ^(2)=14.251,P<0.05),while quantitative real-time PCR showed a significant reduction in the expression level of miR-145-5p(t=109.800,P<0.05).Compared with the other cell lines,ECA-109 cells had the lowest expression level of miR-145-5p(F=48.000,P<0.05)and were then selected for subsequent experiments.Dual-luciferase reporter assay showed that miR-145-5p had a targeted regulatory effect on SMAD4(t=21.820,P<0.05).There were no significant differences in related indices between the Blank group and the NC group(all P>0.05);compared with the Blank group and

关 键 词：食管鳞癌 微RNAS Smad4蛋白质 TGF-β/Smad4信号通路 细胞增殖 细胞凋亡 

分 类 号：R735.1[医药卫生—肿瘤] R342.2[医药卫生—临床医学]