miR-335-5p靶向调控Galectin-3调节前列腺癌细胞免疫逃逸的机制研究  

Study on the mechanism of miR-335-5p regulating the immune escape of prostate cancer cells through targeted regulation on Galectin-3

作　　者：周汝宝 郭宗华[1] 杨君[1] 徐娓 赵克栋[1] 饶志刚[1] 孔东波[1] 江波涛[1] ZHOU Rubao;GUO Zonghua;YANG Jun;XU Wei;ZHAO Kedong;RAO Zhigang;KONG Dongbo;JIANG Botao(Department of Urology,Xianning Central Hospital(The First Affiliated Hospital of Hubei University of Science and Technology),Hubei Xianning 437199,China)

机构地区：[1]咸宁市中心医院(湖北科技学院附属第一医院)泌尿外科,湖北咸宁437199

出　　处：《现代肿瘤医学》2023年第13期2431-2437,共7页Journal of Modern Oncology

基　　金：湖北省卫生健康委2021-2022年度面上项目(编号:WJ2021M091)。

摘　　要：目的:探讨miR-335-5p靶向调控半乳糖凝集素-3(Galectin-3)对前列腺癌细胞免疫逃逸的影响。方法:qRT-PCR检测正常前列腺上皮细胞WPMY-1及前列腺癌细胞LNCaP、PC-3、DU145中miR-335-5p的表达;将PC-3细胞分为:NC组、mimics NC组、miR-335-5p mimics组、miR-335-5p mimics+pcDNA组、miR-335-5p mimics+pcDNA-Galectin-3组,qRT-PCR检测各组细胞中miR-335-5p表达;western blot检测各组细胞中Galectin-3蛋白表达。将上述5组细胞分别与自然杀伤细胞NK-92MI共培养后,ELISA法检测共培养细胞上清液中肿瘤坏死因子α(TNF-α)、干扰素γ(IFN-γ)水平;CCK-8法检测NK-92MI细胞的免疫杀伤率;流式细胞术检测NK-92MI细胞凋亡率;双荧光素酶报告基因、RNA下拉、RNA结合蛋白免疫沉淀(RIP)实验验证miR-335-5p与Galectin-3的靶向关系。结果:与正常前列腺上皮细胞WPMY-1比较,前列腺癌细胞LNCaP、PC-3、DU145中miR-335-5p的表达水平显著降低(P<0.05),且PC-3细胞中miR-335-5p的表达水平最低,因此选用PC-3细胞为后续研究对象;与NC组比较,miR-335-5p mimics组PC-3细胞中miR-335-5p表达水平显著升高,Galectin-3蛋白表达显著降低(P<0.05);与NC共培养组比较,miR-335-5p mimics共培养组细胞上清液中TNF-α、IFN-γ水平及NK-92MI细胞免疫杀伤率显著升高,NK-92MI细胞凋亡率降低(P<0.05);与miR-335-5p mimics组比较,miR-335-5p mimics+pcDNA-Galectin-3组PC-3细胞中miR-335-5p表达水平差异无统计学意义(P>0.05),Galectin-3蛋白表达显著升高(P<0.05);与miR-335-5p mimics共培养组比较,miR-335-5p mimics+pcDNA-Galectin-3共培养组细胞上清液中TNF-α、IFN-γ水平及NK-92MI细胞免疫杀伤率显著降低,NK-92MI细胞凋亡率升高(P<0.05);miR-335-5p靶向负调控Galectin-3表达。结论:过表达miR-335-5p通过靶向下调Galectin-3表达来抑制PC-3细胞免疫逃逸。Objective:To investigate the effect of miR-335-5p on the immune escape of prostate cancer cells through targeted regulation on Galectin-3.Methods:qRT-PCR was used to detect the expression of miR-335-5p in normal prostate epithelial cells WPMY-1 and prostate cancer cells LNCaP,PC-3,DU145.PC-3 cells were divided into:NC group,mimics NC group,miR-335-5p mimics group,miR-335-5p mimics+pcDNA group,and miR-335-5p mimics+pcDNA-Galectin-3 group,qRT-PCR was performed to measure the expression of miR-335-5p of cells in each group.Western blot was performed to measure the expression of Galectin-3 protein of cells in each group.After the cells in above five groups were co-cultured with the natural killer cell NK-92MI,ELISA method was performed to measure the levels of tumor necrosis factor alpha(TNF-α)and interferon gamma(IFN-γ)in the supernatant of co-cultured cells.CCK-8 method was performed to measure the immune killing rate of NK-92MI cells.The apoptosis rate of NK-92MI cells was detected by flow cytometry.The targeting relationship between miR-335-5p and Galectin-3 was verified by dual-luciferase reporter gene,RNA pull-down,and RNA-binding protein immunoprecipitation(RIP)experiments.Results:Compared with normal prostate epithelial cells WPMY-1,the expression level of miR-335-5p in prostate cancer cells LNCaP,PC-3,and DU145 was significantly reduced(P<0.05),and the expression level of miR-335-5p in PC-3 cells was the lowest,so PC-3 cells were selected as the follow-up research object.Compared with the NC group,the expression level of miR-335-5p in PC-3 cells in the miR-335-5p mimics group was significantly increased,and the expression of Galectin-3 protein was significantly decreased(P<0.05).Compared with the NC co-culture group,the levels of TNF-αand IFN-γin the cell supernatant and the immune killing rate of NK-92MI cells in the miR-335-5p mimics co-culture group were significantly increased,reduced apoptosis rate of NK-92MI cells(P<0.05).Compared with the miR-335-5p mimics group,there was no significant difference

关 键 词：miR-335-5p 前列腺癌 半乳糖凝集素-3 免疫逃逸 

分 类 号：R735.25[医药卫生—肿瘤]