微小RNA-495-3p靶向高迁移率族蛋白B1对膀胱癌细胞增殖、周期和凋亡的影响

Effects of microRNA-495-3p targeting high mobility group protein B1 on proliferation,cycle and apoptosis of bladder cancer cells

目的观察微小RNA(miR)-495-3p靶向高迁移率族蛋白B1(HMGB1)对膀胱癌细胞增殖、周期和凋亡的影响。方法选取2018年1月到2023年1月驻马店市中心医院收治的76例膀胱癌组织和癌旁组织作为研究对象, 采用荧光定量聚合酶链反应(PCR)分析肿瘤和癌旁组织miR-495-3p表达水平。人膀胱癌细胞T24采用采用对号miRNA和miR-495-3p过表达慢病毒感染, 建立对照miRNA和miR-495-3p组细胞系, 采用细胞计数试剂盒(CCK-8)和克隆形成实验测定两组细胞增殖能力, 采用小鼠体内成瘤实验分析两组细胞的成瘤能力, 采用流式细胞术分析两组细胞的周期和凋亡水平;生物信息学和双荧光素酶测定miR-495-3p的靶基因;蛋白质免疫印迹分析肿瘤组织和细胞系中靶基因的表达水平。组间比较采用t检验。结果癌旁组织中miR-495-3p表达水平(1.04±0.18)明显高于肿瘤组织水平(0.64±0.12), 差异有统计学意义(t=16.060, P<0.05)。对照组细胞CCK-8吸光度(A)值(1.99±0.09)明显高于miR-495-3p组(1.55±0.11), 差异有统计学意义(t=7.716, P<0.05)。对照组细胞克隆形成数量[(136.17±17.77)个]明显高于miR-495-3p组[(108.67±15.89)个], 差异有统计学意义(t=2.825, P<0.05)。对照组细胞体内成瘤体积[(785.50±121.44) mm^(3)]明显高于miR-495-3p组[(464.50±93.71) mm^(3)], 差异有统计学意义(t=5.126, P<0.05)。对照组细胞G0/G1期百分比[(29.83±3.76)%]明显低于miR-495-3p组细胞[(35.17±3.19)%], 差异有统计学意义(t=2.648, P<0.05)。对照组细胞S期百分比[(32.83±3.76)%]明显高于miR-495-3p组细胞[(36.33±2.94)%], 差异有统计学意义(t=3.332, P<0.05)。对照组细胞凋亡比例[(4.45±1.08)%]明显低于miR-495-3p组细胞[(16.92±1.60)%], 差异有统计学意义(t=15.890, P<0.05)。HMGB1是miR-495-3p的靶基因。癌旁组织中HMGB1表达水平(1.45±0.22)明显低于肿瘤组织表达水平(2.14±0.25), 差异有统计学意义(t=16.478, P<0.05)。对照组细胞HMGB1表达水平(1.22±0.10)明显高于miR-495-3p组(0.56±0.05), 差异有统计学意义(t=14.390, P<0.05)。结论 miR-495-3p在膀胱癌组织中低表达, 通过调节膀胱癌细胞HMGB1表达水平, 进而调节膀胱癌细胞的增殖、周期和凋亡等细胞生物学过程。

Objective To investigate the effects of microRNA(miR)-495-3p targeting high mobili-ty group protein B1(HMGB1)on the proliferation,cycle and apoptosis of bladder cancer cells.Methods A total of 76 cases of bladder cancer tissues and adjacent tissues from January 2018 to January 2023 in our hospital were selected as research objects,and the expression level of miR-495-3p in tumor and adjacent tissues was analyzed by fluorescence quantitative polymerase chain reaction(PCR).The human bladder cancer cell T24 was infected with Lentivirus overexpressing miRNA and miR-495-3p,and the cell lines of control miRNA and miR-495-3p groups were established.The proliferation ability of the two groups cells was measured by cell counting kit-8(CCK-8)and clone formation assays.The tumorigenicity of the two groups was analyzed by mouse in vivo tumorigenesis test.The cell cycle and apoptosis level of the two groups were analyzed by flow cytometry.Bioinformatics and double Luciferase were used to determine the target gene of miR495-3p.The target gene expression levels in tumor tissues and cell lines were analyzed by Western blotting.The comparison of measurement data between groups was conducted using t-test.Results The expression level of miR-495-3p in adjacent cancer tissues(1.04±0.18)was significantly higher than that in tumor tissues(0.64±0.12,t=16.060,P<0.05).The absorbance(A)value of CCK-8 cells in the control group(1.99±0.09)was significantly higher than that in the miR-495-3p group(1.55±0.11,t=7.716,P<0.05).The number of cell clones formed in the control group[(136.17±17.77)cells]was significantly greater than that in the miR-495-3p group[(108.67±15.89)cells,t=2.825,P<0.05].The tumor formation volume in the control group[(785.50±121.44)mm^(3)]was significantly greater than that in the miR-495-3p group[(464.50±93.71)mm^(3),t=5.126,P<0.05].The percentage of Go/G,phase cells in the control group[(29.83±3.76)%]was significantly lower than that in the miR-495-3p group[(35.17±3.19)%,t=2.648,P<0.05].The percentage of S phase cells in the control group[(32.83±3.76)%]was significantly higher than that in the miR-495-3p group[(36.33±2.94)%,t=3.332,P<0.05].The apoptosis rate in the control group[(4.45±1.08)%]was significantly lower than that in the miR-495-3p group[(16.92±1.60)%,t=15.890,P<0.05].The expression level of HMGB1 in adjacent cancer tissues(1.45±0.22)was significantly lower than that in tumor tissues(2.14±0.25,t=16.478,P<0.05).The expression level of HMGB1 in the control group(1.22±0.10)was significantly higher than that in the miR495-3p group(0.56±0.05,t=14.390,P<0.05).Conclusion The low expression of miR495-3p in bladder cancer can regulate the proliferation,cell cycle and apoptosis of bladder cancer cells by regulating the expression of HMGBI in bladder cancer cells.