HMGA1在膀胱癌细胞中的作用及其机制研究

The role and mechanism of HMGA1 in bladder cancer cells

目的探讨高迁移率族蛋白A1(High Mobility Group Protein A1,HMGA1)对人膀胱移行细胞癌细胞(T24)迁移和化疗耐药的作用及其分子机制。方法采用实时荧光定量PCR(RT-PCR)和免疫蛋白印迹(Western blot)的方法检测HMGA1在人膀胱上皮永生化细胞(SV-HUC-1)和人膀胱癌T24细胞中的表达;用脂质体将构建好的HMGA1 siRNA瞬时转染入T24细胞中,CCK-8法检测沉默HMGA1后膀胱癌细胞增殖能力的变化;平板划痕和Transwell实验检测沉默HMGA1后膀胱癌细胞迁移能力的变化;CCK-8法和流式细胞术检测沉默HMGA1后膀胱癌细胞化疗敏感性和存活率的变化;Western blot检测沉默HMGA1后下游相关基因的改变。结果RT-PCR实验表明,相比于SV-HUC-1细胞中HMGA1 mRNA的表达水平(0.98±0.01),HMGA1 mRNA在人膀胱癌T24细胞中的表达上调(3.55±0.13,P<0.001);T24细胞中HMGA1蛋白表达量明显高于SV-HUC-1细胞(P<0.05);相比于阴性对照组,沉默HMGA1后,膀胱癌细胞的增殖水平明显降低(P<0.001);且膀胱癌细胞的迁移能力也显著下调(P<0.01);另外,膀胱癌细胞的化疗敏感性和凋亡率也显著增加(P<0.01)。随着化疗药物浓度增加,PI3K和AKT表达不断降低;且在降低HMGA1表达水平后,PI3K和AKT表达水平也降低。结论HMGA1在膀胱癌细胞中表达上调,高水平HMGA1可能通过调控PI3K/AKT通路促进膀胱癌细胞的转移和化疗耐药,有望为临床膀胱癌的治疗提供新的分子靶点和理论依据。

Objective To investigate the effect and molecular mechanism of high mobility group protein A1(HMGA1)on the migration and chemotherapy resistance of human bladder cancer cells(T24).Methods The expression of HMGA1 in human bladder epithelial immortalized cells(SV-HUC-1)and human bladder cancer T24 cells was detected by real-time quantitative PCR and Western blot.The established HMGA1 siRNA was transfected into human bladder cancer T24 cells by liposome 3000.The proliferation capacity of bladder cancer cells after HMGA1 silencing was detected by CCK-8 methods.Changes of migration in bladder cancer cell after HMGA1 silencing were detected by plate scratch and Transwell assay.CCK-8 methods and flow cytometry were used to detect changes in bladder cancer cell survival and chemical sensitivity after silencing HMGA1.Finally,Western blot was used to detect changes in downstream related genes after silencing HMGA1.Results Compared with SV-HUC-1(0.98±0.01),HMGA1 mRNA was up-regulated in human bladder cancer T24 cells(3.55±0.13,P<0.0001).The HMGA1 protein expression in T24 cells was significantly higher than that in SV-HUC-1 cells(P<0.05).Compared with the negative control group,HMGA1 silencing decreased the proliferation capacity of bladder cancer cells(P<0.001).HMGA1 silencing inhibits migration of bladder cancer cells(P<0.01).In addition,the chemotherapy sensitivity and apoptosis rate of bladder cancer cells also increased significantly after HMGA1 silencing(P<0.01).PI3K and AKT expression decreased as the concentration of chemotherapy drugs increased,and PI3K and AKT levels decreased after decreasing HMGA1 expression levels.Conclusion HMGA1 is upregulated in bladder cancer cells.High levels of HMGA1 may promote metastasis and chemotherapy resistance of bladder cancer cells by modulating the PI3K/AKT pathway,which is expected to provide new molecular targets and theoretical basis for the treatment of clinical bladder cancer.