miR-489-3p靶向PTEN/PI3K/Akt信号通路对膀胱癌细胞增殖、凋亡和侵袭的影响

Effects of miR-489-3p targeting PTEN/PI3K/Akt signaling pathway on proliferation,apoptosis and invasion of bladder cancer cells

目的探讨微小RNA(miR)-489-3p靶向第10号染色体缺失的磷酸酶及张力蛋白同源基因(PTEN)/磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)信号通路对膀胱癌(BC)细胞增殖、凋亡和侵袭的影响。方法2021年1-7月于河北北方学院附属第一医院进行细胞实验,利用火山图从癌症基因组图谱(TCGA-BLCA)数据库筛选BC差异表达miRNA。以110例BC患者的癌组织及其癌旁组织和购买的人膀胱上皮永生化细胞SV-HUC-1及人BC细胞系5637、J82、T24为研究对象,采用实时荧光定量PCR测定miR-489-3p和PTEN mRNA的表达。根据miR-489-3p的相对表达量均值将BC患者分为低表达组(≤0.36)70例和高表达组(>0.36)40例,并分析miR-489-3p表达在BC患者不同临床病理特征中的变化。双荧光素酶实验验证miR-489-3p和PTEN的靶向关系。选择miR-489-3p表达最低的T24细胞进行转染实验,T24细胞随机分组为对照组、miR-NC(阴性对照)组、miR-489-3p mimics(模拟物)组、miR-489-3p mimics+pcDNA组、miR-489-3p mimics+pc-PTEN组。MTT和克隆形成实验、Transwell实验、流式细胞术分别检测细胞增殖、侵袭和凋亡;Western-blot检测PTEN、PI3K、Akt蛋白表达。结果通过火山图筛选出BC组织中显著下调的miR-489-3p。BC癌组织中miR-489-3p表达低于癌旁组织,PTEN mRNA表达高于癌旁组织(t=186.168,510.107,P均<0.001);BC细胞系miR-489-3p表达低于SV-HUC-1细胞,PTEN mRNA表达高于SV-HUC-1细胞(F=883.826,807.220,P均<0.001)。miR-489-3p低表达组患者TNM分期T_(3~4)、有淋巴结转移、肿瘤低分化比例高于高表达组(P<0.05)。双荧光素酶实验显示,miR-489-3p靶向负调控PTEN表达。miR-489-3p mimics组凋亡率及PI3K、Akt蛋白表达水平高于miR-NC组,PTEN蛋白表达水平、OD值(24 h、48 h、72 h)、克隆数目和细胞侵袭数低于miR-NC组(P均<0.01);miR-489-3p mimics+pc-PTEN组凋亡率及PI3K、Akt蛋白表达水平低于miR-489-3p mimics组,PTEN蛋白表达水平、OD值(24 h、48 h、72 h)、克隆数目和细胞侵袭数高于miR-489-3p mimics组(P<0.01)。结论miR-489-3p可能通过靶向下调PTEN表达,激活PI3K/Akt信号通路,进而抑制BC细胞增殖、侵袭,促进凋亡。

Objective To investigate the effect of microRNA(miR)-489-3p targeting chromosome 10 deleted phosphatase and tensin homolog gene(PTEN)/phosphatidylinositol 3 kinase(PI3K)/protein kinase B(Akt)signaling pathway in the bladder Effects of cancer(BC)cell proliferation,apoptosis and invasion.Methods From June 2018 to July 2021,experiments were conducted at the First Affiliated Hospital of Hebei North University,using volcano plots to screen BC differentially expressed miRNAs from The Cancer Genome Atlas(TCGA-BLCA)database.The cancer tissues and adjacent tissues of 110 BC patients,purchased human bladder epithelial immortalized cells SV-HUC-1 and human BC cell lines 5637,J82,and T24 were used as the research objects,and real-time quantitative PCR was used to determine miR-489-3p and PTEN mRNA expression.According to the relative expression of miR-489-3p,BC patients were divided into 70 cases with low expression group(≤0.36)and 40 cases in high expression group(>0.36).changes in characteristics.The dual-luciferase experiment verified the targeting relationship between miR-489-3p and PTEN.T24 cells with the lowest expression of miR-489-3p were selected for transfection experiments.T24 cells were randomly divided into control group,miR-NC(negative control)group,miR-489-3p mimics(mimetic)group,and miR-489-3p mimics+pcDNA group and miR-489-3p mimics+pc-PTEN group.MTT and colony formation assay,Transwell assay,and flow cytometry were used to detect cell proliferation,invasion and apoptosis,respectively;Western-blot was used to detect the protein expressions of PTEN,PI3K and Akt.Results The significantly down-regulated miR-489-3p in BC tissues was screened by volcano plot.The expression of miR-489-3p in BC cancer tissues was lower than that in adjacent tissues,and the expression of PTEN mRNA was higher than that in adjacent tissues(t=186.168,510.107,P<0.001);the expression of miR-489-3p in BC cell lines was lower than that in SV-The expression of PTEN mRNA in HUC-1 cells was higher than that in SV-HUC-1 cells(F=883.826,807.220,both P<0.001).The proportion of patients with low miR-489-3p expression in TNM stage T_(3-4),lymph node metastasis,and poorly differentiated tumors was higher than that in the high expression group.Dual-luciferase experiments revealed that miR-489-3p targets negatively regulate PTEN expression.The apoptosis rate and PI3K and Akt protein expression levels in the miR-489-3p mimics group were higher than those in the miR-NC group.The miR-489-3p mimics+pc-PTEN group had lower apoptotic rate and PI3K and Akt protein expression levels than the miR-489-3p mimics group,and the OD values(24 h,48 h,72 h),the number of clones and the number of cell invasion were higher than those in the miR-489-3p mimics group(P<0.01).Conclusion miR-489-3p may target down-regulate PTEN expression and activate PI3K/Akt signaling pathway,thereby inhibiting BC cell proliferation,invasion,and promoting apoptosis.