lncRNA ZFP36-AS1通过miR-221调控膀胱癌细胞增殖和免疫逃逸

lncRNA ZFP36-AS1 regulates proliferation and immune escape of bladder cancer cell through miR-221

目的探讨长链非编码RNA(lncRNA) ZFP36-AS1在膀胱癌中的表达及ZFP36-AS1/miR-221轴对膀胱癌细胞增殖和免疫逃逸的影响。方法通过cBioPortal数据库分析ZFP36-AS1在膀胱癌组织中的表达差异。采用实时荧光定量聚合酶链反应(RT-qPCR)分析ZFP36-AS1在膀胱癌细胞系(J82、RT-4、MGH-U3、5637)中的表达差异。MGH-U3细胞随机分为阴性对照(NC)组和ZFP36-AS1组,分别转染pcDNA3.1-NC质粒和pcDNA3.1-ZFP36-AS1质粒。集落形成实验、流式细胞术分别分析MGH-U3细胞增殖活力和细胞周期。T淋巴细胞与各组MGH-U3细胞共培养,酶联免疫吸附试验(ELISA)检测各组上清液中白细胞介素-10(IL-10)、γ-干扰素(IFN-γ)、白细胞介素-4(IL-4)水平。双荧光素酶报告基因实验验证ZFP36-AS1与miR-221的靶向关系。RT-qPCR检测ZFP36-AS1对MGH-U3细胞miR-221表达的影响。Western blotting法检测ZFP36-AS1/miR-221轴对MGH-U3细胞CDK3、Cyclin C、CDK5、Cyclin D1、Cyclin D3蛋白表达的影响。结果与正常膀胱组织相比,ZFP36-AS1在膀胱癌组织中呈异常低表达(P<0.01)。与SV-HUC-1细胞相比,ZFP36-AS1在膀胱癌细胞系(J82、RT-4、MGH-U3、5637)中呈异常低表达(P<0.01),且在MGH-U3细胞中表达最低(P<0.01)。NC组和ZFP36-AS1组MGH-U3细胞集落形成数分别为(220.80±34.65)个和(77.84±19.11)个,ZFP36-AS1组MGH-U3细胞集落形成数量显著下调,差异具有统计学意义(P<0.01)。NC组和ZFP36-AS1组G0/G1期细胞比例分别为(48.04±2.89)%和(72.89±3.46)%,S期细胞比例分别为(35.38±2.98)%和(20.62±2.56)%,G2/M期细胞比例分别为(16.59±1.46)%和(6.48±1.50)%,ZFP36-AS1组G0/G1期细胞比例上调(P<0.01),S期和G2/M期细胞比例均下调(P<0.01)。与NC组相比,ZFP36-AS1组IL-4和IFN-γ水平明显上调(P<0.01),IL-10水平明显下调(P<0.01)。ZFP36-AS1能够靶向结合miR-221(P<0.01)。NC组与ZFP36-AS1组miR-221相对表达量分别为6.84±1.35和1.00±0.21。与NC组相比,过表达ZFP36-AS1能显著抑制miR-221表达(P<0.01)。与NC组相比,ZFP36-AS1组细胞周期蛋白CDK3、Cyclin C、CDK5、Cyclin D1、Cyclin D3表达明显降低。结论 ZFP36-AS1在膀胱癌中呈异常低表达,其通过抑制miR-221表达降低膀胱癌细胞增殖活力并抑制其免疫逃逸。

Objective To investigate the expression of long non-coding RNA(lncRNA)ZFP36-AS1 in bladder cancer and the effect of ZFP36-AS1/miR-221 axis on the proliferation and immune escape of bladder cancer cells.Methods The expression difference of ZFP36-AS1 in bladder cancer tissues was analyzed by cBioPortal database.Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)was used to analyze the expression difference of ZFP36-AS1 in bladder cancer cell lines(J82,RT-4,MGH-U3,5637).MGH-U3 cells were randomly divided into negative control(NC)group and ZFP36-AS1 group,which were transfected with pcDNA3.1-NC plasmid and pcDNA3.1-ZFP36-AS1 plasmid,respectively.Colony formation assay and flow cytometry were used to analyze the proliferation activity and cell cycle of MGH-U3 cells,respectively.T lymphocytes were co-cultured with MGH-U3 cells in each group,and the levels of interleukin-10(IL-10),γ-interferon(IFN-γ),and interleukin-4(IL-4)in the supernatants of each group were detected by enzyme-linked immunosorbent assay(ELISA).The dual-luciferase reporter gene assay verified the targeting relationship between ZFP36-AS1 and miR-221.The effect of ZFP36-AS1 on the expression of miR-221 in MGH-U3 cells was detected by RT-qPCR.Western blotting was used to detect the effect of ZFP36-AS1/miR-221 axis on the protein expression of CDK3,Cyclin C,CDK5,Cyclin D1 and Cyclin D3 in MGH-U3 cells.Results Compared with normal bladder tissue,ZFP36-AS1 was abnormally low-expressed in bladder cancer tissue(P<0.01).Compared with SV-HUC-1 cells,ZFP36-AS1 was abnormally low-expressed in bladder cancer cell lines(J82,RT-4,MGH-U3,5637)(P<0.01),and the expression was lowest in MGH-U3 cells(P<0.01).The number of MGH-U3 cell colonies formed in the NC group and the ZFP36-AS1 group were(220.80±34.65)and(77.84±19.11),respectively,and the number of MGH-U3 cell colonies formed in the ZFP36-AS1 group was significantly down-regulated,the difference was statistically significant(P<0.01).The proportions of G0/G1 phase cells in NC group and ZFP36-AS1 group were(48.04±2.89)%and(72.89±3.46)%,respectively,and the proportion of S phase cells were(35.38±2.98)%and(20.62±2.56)%,respectively.The proportion of G2/M stage cells was(16.59±1.46)%and(6.48±1.50)%,respectively.The proportion of cells in G0/G1 phase were up-regulated in ZFP36-AS1 group(P<0.01),and the proportion of cells in S phase and G2/M phase were both down-regulated(P<0.01).Compared with the NC group,the levels of IL-4 and IFN-γin the ZFP36-AS1 group were significantly up-regulated(P<0.01),and the level of IL-10 was significantly down-regulated(P<0.01).ZFP36-AS1 can target miR-221(P<0.01).The relative expression of miR-221 in the NC group and the ZFP36-AS1 group was 6.84±1.35 and 1.00±0.21,respectively.Compared with the NC group,overexpression of ZFP36-AS1 could significantly inhibit the expression of miR-221(P<0.01).Compared with the NC group,the expressions of CDK3,Cyclin C,CDK5,Cyclin D1,and Cyclin D3 in the ZFP36-AS1 group were significantly decreased.Conclusion ZFP36-AS1 is abnormally low-expressed in bladder cancer,and it reduces the proliferation activity of bladder cancer cells and inhibits their immune escape by inhibiting the expression of miR-221.