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
赵斌 1杨金叶 1李支尧 2毕城伟 1杨李波 1施致裕 1李心 1张建朋 1施远龙 1杨勇 1张国颖1
作者信息
- 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
- 折叠
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-5p mimic 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DDIT4 mRNA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(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DDIT4 mRNA表达水平明显升高(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DDIT4蛋白表达水平明显升高(P<0.05).CCK-8法检测,与LNCap组和mimic NC组比较,miR-30c-5p mimic组LNCap细胞增殖率明显降低(P<0.01);与LNCap组和 sh-NC组比较,sh-DDIT4 组LNCap细胞增殖率明显降低(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细胞增殖率明显升高(P<0.01).Transwell小室实验检测,与LNCap组和mimic NC组比较,miR-30c-5p mimic组LNCap细胞侵袭细胞数明显减少(P<0.01);与LNCap组和sh-NC组比较,sh-DDIT4 组LNCap细胞侵袭细胞数明显减少(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细胞侵袭细胞数明显增加(P<0.01).划痕实验检测,与LNCap组和mimic NC组比较,miR-30c-5p mimic组LNCap细胞划痕愈合率明显降低(P<0.01);与LNCap组和sh-NC组比较,sh-DDIT4组细胞划痕愈合率明显降低(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组细胞划痕愈合率明显升高(P<0.01).双荧光素酶实验检测,与共转染WT-DDIT4和mimic NC的LNCap细胞比较,共转染WT-DDIT4和miR-30c-5p mimic的LNCap细胞中荧光素酶活性明显降低(P<0.01).体内裸鼠成瘤实验,细胞注射后第 3、6、9、12和 15天,与空白组和agomiR-NC组比较,agomiR-30c-5p组裸鼠肿瘤体积明显减小(P<0.05).HE染色观察,空白组和agomiR-NC组小鼠前列腺癌组织中细胞核增大,核仁突出且畸形;agomiR-30c-5p组小鼠前列腺癌组织部分区域细胞排列整齐,细胞核恢复正常形态.RT-qPCR法和免疫荧光染色法检测,与agomiR-NC组比较,agomiR-30c-5p组小鼠前列腺癌组织中miR-30c-5p表达水平明显升高(P<0.01);与空白组和agomiR-NC组比较,agomiR-30c-5p组小鼠前列腺癌组织中DDIT4 mRNA表达水平明显降低(P<0.01);DDIT4蛋白主要在细胞质表达,与空白组和agomiR-NC组比较,agomiR-30c-5p组小鼠前列腺癌组织中DDIT4蛋白荧光强度明显降低(P<0.01).结论:miR-30c-5p在前列腺癌LNCap细胞中表达水平明显降低,其可通过靶向下调DDIT4抑制前列腺癌细胞增殖、迁移和侵袭,从而参与前列腺癌的发生发展.
Abstract
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 staining were used to detect the expression levels of miR-30c-5p and DDIT4 mRNA and the fluorescence intensities of DDIT4 protein in prostate cancer tissue of the mice in various groups.Results:The In vitro prostate cancer cell experiment results showed that compared with RWPE-1 cells,the expression level of miR-30c-5p in the prostate cancer LNCap cells was decreased(P<0.01),and the expression levels of DDIT4 mRNA and protein were increased(P<0.05 or P<0.01).After 48 of transfection,compared with LNCap group and mimic NC group,the expression level of miR-30c-5p in the LNCap cells in miR-30c-5p mimic group was increased(P<0.01).Compared with LNCap group and sh-NC group,the expression level of DDIT4 mRNA in the LNCap cells in sh-DDIT4 group was decreased(P<0.01).Compared with miR-30c-5p mimic group and miR-30c-5p mimic+pcDNA3.1 NC group,the expression level of miR-30c-5p in The LNCap cells in miR-30c-5p mimic+pc-DNA3.1-DDIT4 group was decreased(P<0.01);compared with miR-30c-5p mimic group and miR-30c-5p mimic+pcDNA3.1 NC group,the expression level of DDIT4 mRNA in the LNCap cells in miR-30c-5p mimic+pc-DNA3.1-DDIT4 group was increased(P<0.01);compared with miR-30c-5p mimic group and miR-30c-5p mimic+pcDNA3.1 NC group,the expression level of DDIT4 protein in the LNCap cells in miR-30c-5p mimic+pc-DNA3.1-DDIT4 group was increased(P<0.05).The CCK-8 method results showed that compared with LNCap group and mimic NC group,the proliferation rate of the LNCap cells in miR-30c-5p mimic group was decreased(P<0.01);compared with LNCap group and sh-NC group,the proliferation rate of the LNCap cells in sh-DDIT4 group was decreased(P<0.01);compared with miR-30c-5p mimic group and miR-30c-5p mimic+pcDNA3.1 NC group,the proliferation rate of the LNCap cells in miR-30c-5p mimic+pc-DNA3.1-DDIT4 group was increased(P<0.01).The Transwell assay results showed that compared with LNCap group and mimic NC group,the number of the invasion LNCap cells in miR-30c-5p mimic group was decreased(P<0.01);compared with LNCap group and sh-NC group,the number of invasion LNCap cells in sh-DDIT4 group was decreased(P<0.01);compared with miR-30c-5p mimic group and miR-30c-5p mimic+pcDNA3.1 NC group,the number of the invasion LNCap cells in miR-30c-5p mimic+pc-DNA3.1-DDIT4 group was increased(P<0.01).The scratch assay results showed that compared with LNCap group and mimic NC group,the scratch healing rate of the LNCap cells in miR-30c-5p mimic group was decreased(P<0.01);compared with LNCap group and sh-NC group,the scratch healing rate of the LNCap cells in sh-DDIT4 group was decreased(P<0.01);compared with miR-30c-5p mimic group and miR-30c-5p mimic+pcDNA3.1 NC group,the scratch healing rate of the LNCap cells in miR-30c-5p mimic+pc-DNA3.1-DDIT4 group was increased(P<0.01).The dual-luciferase reporter assay results showed that compared with the LNCap cells co-transfected with WT-DDIT4 and mimic NC,the luciferase activity of the LNCap cells co-transfected with WT-DDIT4 and miR-30c-5p mimic was decreased(P<0.01).The in vivo nude mouse tumor formation experiment results showed that on the 3 rd,6 th,9 th,12 th,and 15th days after cell injection,compared with blank group and agomiR-NC group,the tumor volumes of the nude mice in agomiR-30c-5p group were decreased(P<0.05).The HE staining results showed that in prostate cancer tissue of the mice in blank group and agomiR-NC group,the cell nuclei were enlarged,and nucleoli were prominent and deformed.In the mice in agomiR-30c-5p group,some regions of prostate cancer tissues results showed neatly arranged cells with normally shaped nuclei.The RT-qPCR and immunofluorescence staining showed that compared with agomiR-NC group,the expression level of miR-30c-5p in prostate cancer tissue of the mice in agomiR-30c-5p group was increased(P<0.01).Compared with blank group and agomiR-NC group,the expression level of DDIT4 mRNA in prostate cancer tissue of the mice in agomiR-30c-5p group was decreased(P<0.01).DDIT4 protein was mainly expressed in the cytoplasm.Compared with blank group and agomiR-NC group,the fluorescence intensity of DDIT4 protein in prostate cancer tissue of the mice in agomiR-30c-5p group was decreased(P<0.01).Conclusion:The expression level of miR-30c-5p in the prostate cancer LNCap cells is decreased,and it inhibits the proliferation,migration,and invasion of the prostate cancer cells by targeting downregulation of DDIT4,thereby participating in the occurrence and development of prostate cancer.
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Key words
Prostate tumor/Micro RNA-30c-5p/DNA damage-inducible transcript 4/Cell proliferation/Cell migration引用本文复制引用
出版年
2024
国家科技期刊平台
NSTL
万方数据