MiR-148 b-3 p/FOXO 4轴对乳腺癌细胞自噬及生长的影响  

Effects of miR-148 b-3p/FOXO4 axis on autophagy and growth of breast cancer cells

作　　者：郭玲 陈敬信 GUO Ling;CHEN Jingxin(Department of Pathology,First People’s Hospital of Xianyang City,Xianyang Shaanxi 712000;Department of Pathology,Sengong Hospital of Shaanxi Province,Xi’an 710300,China)

机构地区：[1]咸阳市第一人民医院病理科,陕西咸阳712000 [2]陕西省森工医院病理科,西安710300

出　　处：《临床与病理杂志》2023年第9期1621-1630,共10页Journal of Clinical and Pathological Research

基　　金：陕西省教育厅专项科研计划项目(17JK0209)。

摘　　要：目的:MiR-148b-3p通过调节叉头框蛋白O4(forkhead box O4,FOXO4)在乳腺癌(breast cancer,BC)进展中的作用还未完全阐明。本研究探究miR-148b-3p调节FOXO4表达对BC细胞增殖、凋亡和自噬的影响。方法:采用实时反转录聚合酶链反应(real-time reverse transcription polymerase chain reaction,real-time RT-PCR)检测BC患者的癌组织标本(BC组织)和邻近的非肿瘤组织标本(正常组织)、人正常乳腺上皮细胞MCF-10A和人BC细胞(MCF7、SKBR3、MDA-MB-231细胞)中miR-148b-3p和FOXO4的表达情况。对MDA-MB-231细胞进行转染并将其分为对照组(Ctrl组)、inhibitor-NC组、miR-148b-3p inhibitor组、si-NC组、si-FOXO4组、miR-148b-3p inhibitor+si-NC组、miR-148b-3p inhibitor+si-FOXO4组。采用细胞计数试剂盒8(cell counting kit 8,CCK-8)法检测细胞增殖;流式细胞术检测细胞凋亡;透射电镜观察细胞自噬;蛋白质印迹法检测细胞FOXO4、增殖、凋亡和自噬相关蛋白表达;双荧光素酶报告基因检测(double luciferase reporter gene assay,DLR)和miRNA下拉实验验证miR-148b-3p与FOXO4的靶向作用关系。结果:与正常组织比较,BC组织中miR-148b-3p表达升高,FOXO4 mRNA表达降低(P<0.05);Pearson相关性分析结果显示BC患者癌组织中miR-148b-3p的表达水平与FOXO4 mRNA的水平呈负相关(r=−0.774,P<0.01)。与MCF-10A细胞比较,MCF-7细胞、SKBR3细胞和MDA-MB-231细胞中miR-148b-3p表达均升高,FOXO4蛋白和mRNA表达均降低(均P<0.05),且MDA-MB-231细胞中升高或降低更多。miR-148b-3p靶向负调控FOXO4的表达。敲减miR-148b-3p后MDA-MB-231细胞活力、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)蛋白水平降低,凋亡率、自噬小体数量、FOXO4蛋白和mRNA水平、cleaved-caspase-3、cleaved-PARP-1、LC3-II/LC3-I、Beclin-1蛋白水平均升高(均P<0.05),而敲减FOXO4后与之相反;敲减FOXO4可减弱敲减miR-148b-3p对MDA-MB-231细胞增殖、凋亡和自噬的影响。结论:miR-148b-3p可能通过靶向FOXO4促进Objective:The role of miR-148b-3p in breast cancer(BC)progression through the regulation of forkhead box O4(FOXO4)remains incompletely elucidated.This study investigates the effects of miR-148b-3p-mediated regulation of FOXO4 expression on BC cell proliferation,apoptosis,and autophagy.Methods:The expression of miR-148b-3p and FOXO4 in BC patient tumor specimens(BC tissues)and adjacent non-tumor specimens(normal tissues),as well as in normal human breast epithelial cells(MCF-10A)and human BC cells(MCF7,SKBR3,MDA-MB-231 cells)were assessed by real-time reverse transcription polymerase chain reaction(real-time RT-PCR).MDA-MB-231 cells were transfected and divided into a control group,a inhibitor-NC group,a miR-148b-3p inhibitor group,a si-NC group,a si-FOXO4 group,a miR-148b-3p inhibitor+si-NC group,and a miR-148b-3p inhibitor+si-FOXO4 group.Cell proliferation was detected by cell counting kit 8(CCK-8)assay.Apoptosis was detected by flow cytometry.Autophagy was observed by transmission electron microscope.The expressions of FOXO4,proliferation,apoptosis,and autophagy-related proteins were detected by Western blotting.Dual luciferase reporter gene assay and miRNA pull-down assay were used to verify the targeting relationship between miR-148b-3p and FOXO4.Results:Compared to normal tissues,miR-148b-3p expression was upregulated,and FOXO4 mRNA expression was downregulated in BC tissues(P<0.05).Pearson correlation analysis revealed a negative correlation between miR-148b-3p and FOXO4 mRNA levels in BC patient tumor tissues(r=−0.774,P<0.01).Compared to MCF-10A cells,miR-148b-3p expression increased,while FOXO4 protein and mRNA expression decreased in MCF-7,SKBR3,and MDA-MB-231 cells(P<0.05),with MDA-MB-231 cells showing more significant changes.MiR-148b-3p negatively regulated the expression of FOXO4.Knocking down miR-148b-3p in MDA-MB-231 cells reduced cell viability and proliferating cell nuclear antigen(PCNA)protein levels,increased apoptosis rate,autophagosome number,FOXO4 mRNA and protein levels,as well as cleaved

关 键 词：miR-148b-3p 叉头框蛋白O4 乳腺癌 增殖 凋亡 自噬 

分 类 号：R737.9[医药卫生—肿瘤]