miR-30c-5p对前列腺癌细胞增殖、迁移和侵袭的抑制作用及其机制  

Inhibitory effect of miR-30c-5p on proliferation,migration,and invasion of prostate cancer cells and its mechanism

作　　者：赵斌[1] 杨金叶 李支尧[2] 毕城伟 杨李波 施致裕 李心 张建朋 施远龙 杨勇[1] 张国颖[1] ZHAO Bin;YANG Jinye;LI Zhiyao;BI Chengwei;YANG Libo;SHI Zhiyu;LI Xin;ZHANG Jianpeng;SHI Yuanlong;YANG Yong;ZHANG Guoying(Department of Urology,Yunnan Cancer Hospital,Third Affiliated Hospital,Kunming Medical University,Peking University Cancer Hospital Yunnan Hospital,Yunnan Province,Kunming 650118,China;Department of Ultrasound Medicine,Yunnan Cancer Hospital,Third Affiliated Hospital,Kunming Medical University,Peking University Cancer Hospital Yunnan Hospital,Yunnan Province,Kunming 650118,China)

机构地区：[1]云南省肿瘤医院、昆明医科大学第三附属医院、北京大学肿瘤医院云南医院泌尿外科,云南昆明650118 [2]云南省肿瘤医院、昆明医科大学第三附属医院、北京大学肿瘤医院云南医院超声医学科,云南昆明650118

出　　处：《吉林大学学报（医学版）》2024年第6期1632-1643,共12页Journal of Jilin University：Medicine Edition

基　　金：云南省科技厅-昆明医科大学应用基础研究联合专项基金资助项目(202001AY070001-069);云南省教育厅科学研究项目(2020J0207);昆明医科大学第三附属医院2023年教学联盟本科教育教学研究项目(JXYJ20230211);昆明医科大学2024年研究生教育创新基金(2024S341,2024S342)。

摘　　要：目的:探讨微小RNA(miR)-30c-5p对人前列腺癌细胞(LNCap)增殖、迁移和侵袭的影响,并阐明其可能的机制。方法:LNCap细胞根据转染质粒不同分为LNCap组(无转染质粒)、miR-30c-5p mimic组(转染miR-30c-5p mimic)、mimic NC组(转染miR-30c-5p mimic NC)、sh-DNA损伤诱导转录因子4(DDIT4)组(转染sh-DDIT4)、sh-NC组(转染sh-DDIT4 NC)、miR-30c-5p mimic+pc-DNA3.1-NC组(共转染miR-30c-5p mimic和pc DNA3.1-DDIT4空载质粒)和miR-30c-5p mimic+pc-DNA3.1-DDIT4组(共转染miR-30c-5pmimic和pc-DNA3.1-DDIT4过表达质粒),RWPE-1细胞正常培养。实时荧光定量PCR(RT-qPCR)法检测各组细胞中miR-30c-5p和DDIT4mRNA表达水平,Western blotting法检测各组细胞中DDIT4蛋白表达水平,CCK-8法检测各组LNCap细胞增殖率,Transwell小室实验检测各组LNCap细胞侵袭细胞数,划痕实验检测各组LNCap细胞划痕愈合率,双荧光素酶报告基因实验验证miR-30c-5p与DDIT4的靶向关系。体内裸鼠成瘤实验,18只雄性BALB/c裸鼠随机分为空白组、agomiR-NC组(转染agomiR-30c-5p NC)和agomiR-30c-5p组(转染agomiR-30c-5p),每组6只。agomiR-NC组和agomiR-30c-5p组裸鼠皮下注射LNCap细胞,空白组裸鼠注射等量生理盐水,检测各组小鼠肿瘤体积。HE染色观察各组小鼠前列腺癌组织形态表现,RT-qPCR法和免疫荧光染色法检测各组小鼠前列腺癌组织中miR-30c-5p和DDIT4 mRNA表达水平及DDIT4蛋白荧光强度。结果:体外前列腺癌细胞实验,与人正常前列腺上皮RWPE-1细胞比较,前列腺癌LNCap细胞中miR-30c-5p表达水平明显降低(P<0.01),DDIT4 mRNA和蛋白表达水平明显升高(P<0.05或P<0.01);转染48 h后,与LNCap组和mimic NC组比较,miR-30c-5p mimic组LNCap细胞中miR-30c-5p表达水平明显升高(P<0.01)。与LNCap组和sh-NC组比较,sh-DDIT4组LNCap细胞中DDIT4 mRNA表达水平明显降低(P<0.01);与miR-30c-5p mimic组和miR-30c-5p mimic+pcDNA3.1 NC组比较,miR-30c-5p mimic+pc-DNA3.1-DDIT4组LNCap细胞中miR-30c-5p表达水平明显降低(Objective:To discuss the effect of microRNA(miR)-30c-5p on the proliferation,migration,and invasion of the human prostate cancer cells(LNCap),and to clarify its possible mechanism.Methods:The LNCap cells were divided into LNCap group(without plasmid transfection),miR-30c-5p mimic group(transfected with miR-30c-5p mimic),mimic NC group(transfected with miR-30c-5p mimic NC),sh-DNA damage inducible transcript 4(DDIT4)group(transfected with sh-DDIT4),sh-NC group(transfected with sh-DDIT4 NC),miR-30c-5p mimic+pc-DNA3.1-NC group(co-transfected with miR-30c-5p mimic and pc-DNA3.1 empty vector),and miR-30c-5p mimic+pc-DNA3.1-DDIT4 group(co-transfected with miR-30c-5p mimic and pc-DNA3.1-DDIT4 over-expression plasmid).The RWPE-1 cells were cultured normally.Real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of miR-30c-5p and DDIT4 mRNA in the cells in various groups;Western blotting method was used to detect the expression levels of DDIT4 protein in the cells in various groups;CCK-8 method was used to detect the proliferation rates of the LNCap cells in various groups;Transwell assay was used to detect the numbers of the invasion LNCap cells in various groups;Scratch assay was used to detect the scratch healing rates of LNCap cells in various groups;dual-luciferase reporter assay was used to detect the targeting relationship between miR-30c-5p and DDIT4.In the in vivo tumor formation experiment,18 male BALB/c nude mice were divided randomly into blank group,agomiR-NC group(transfected with agomiR-30c-5p NC),and agomiR-30c-5p group(transfected with agomiR-30c-5p);there were six mice in each group.The mice in agomiR-NC group and agomiR-30c-5p group were subcutaneously injected with LNCap cells,while the mice in blank group were given an equal volume of physiological saline.The volumes of tumor of the mice in various groups were detected.HE staining was used to observe the morphology of prostate cancer tissue the mice of in various groups;RT-qPCR method and immunofluorescence stai

关 键 词：前列腺肿瘤 微小RNA-30c-5p DNA损伤诱导转录因子4 细胞增殖 细胞迁移 

分 类 号：R737.25[医药卫生—肿瘤]