下调HNRNPU通过抑制膀胱癌细胞趋化因子配体2分泌调节肿瘤相关巨噬细胞极化和趋化  

Downregulation of HNRNPU regulates tumor-associated macrophage polarization and chemotaxis by inhibiting chemokine ligand 2 secretion in bladder cancer cells

作　　者：汤玉麒 胡伟民 程帆[1] Tang Yuqi;Hu Weimin;Cheng Fan(Department of Urology,Renmin Hospital of Wuhan University,Wuhan 420060,China)

机构地区：[1]武汉大学人民医院泌尿外科,武汉430060

出　　处：《中华实验外科杂志》2024年第3期529-532,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(82170775)。

摘　　要：目的探究HNRNPU在膀胱癌中的表达以及下调膀胱癌细胞HNRNPU表达对于趋化因子配体2(CCL2)分泌和肿瘤相关巨噬细胞趋化和极化的影响。方法收集武汉大学人民医院2023年7月至10月膀胱癌根治术癌组织10例,癌旁组织10例,使用实时定量反转录聚合酶链反应(RT-qPCR)和免疫组织化学分析膀胱癌组织和癌旁正常组织HNRNPU的表达;通过小干扰RNA(siRNA)敲低人膀胱癌细胞系T24和5637的HNRNPU表达。通过酶联免疫吸附法分析细胞上清中的CCL2含量;将不同处理的T24和5637细胞与巨噬细胞在小室中共培养;通过趋化实验分析巨噬细胞的趋化能力并通过流式细胞术和RT-qPCR分析巨噬细胞M2表型标志物表达,组间比较采用配对样本或非配对样本t检验。结果膀胱癌中HNRNPU的mRNA水平高于正常癌旁组织(0.94±0.41比1.54±0.28,t=5.800,P<0.05)。敲低HNRNPU后T24细胞[(230.56±18.62)pg/ml比(105.67±22.10)pg/ml,t=20.830,P<0.05]和5637细胞[(165.83±27.27)pg/ml比(93.94±18.06)pg/ml,t=4.120,P<0.05]细胞中CCL2的含量显著降低;趋化实验结果显示敲低HNRNPU后向T24细胞(110±16比38±7,t=12.630,P<0.05)和5637细胞(92±12比41±8,t=9.578,P<0.05)趋化的巨噬细胞数量显著降低;流式细胞术结果显示与T24细胞[(2.31±0.13)%比(0.41±0.03)%,t=56.600,P<0.05]和5637细胞[(1.75±0.01)%比(0.71±0.02)%,t=99.300,P<0.05]共培养的巨噬细胞表面CD206表达显著降低;RT-qPCR结果显示与T24细胞(Arg1:3.21±0.76比1.05±0.11,t=6.761,P<0.05和CSF1R:4.37±0.47比1.80±0.25,t=6.432,P<0.05)和5637细胞(Arg1:2.76±0.47比1.68±0.51,t=12.470,P<0.05和CSF1R:3.91±0.27比1.03±0.11,t=23.470,P<0.05)共培养的巨噬细胞M2表型标志物表达显著降低。结论HNRNPU在膀胱癌组织中高表达;下调HNRNPU能够抑制膀胱癌细胞CCL2的分泌并抑制共培养的巨噬细胞的趋化和M2极化。Objective To investigate the expression of HNRNPU in bladder cancer and the effects of down-regulation of HNRNPU expression in bladder cancer cells on chemokine ligand 2(CCL2)secretion and tumor-associated macrophage chemotaxis and polarization.MethodsTotally,10 cases of cancer tissues from radical cystectomy of bladder cancer and 10 cases of paracancerous tissues were collected from Renmin Hospital of Wuhan University from July to October 2023,and the expression of HNRNPU in bladder cancer tissues and paracancerous normal tissues was analyzed using real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)and immunohistochemistry.HNRNPU expression was knocked down by small interfering RNA(siRNA)in human bladder cancer cell lines T24 and 5637.CCL2 content in cell supernatants was analyzed by enzyme-linked immunosorbent assay.T24 and 5637 cells with different treatments were co-cultured with macrophages in small chambers.Macrophage chemotaxis ability was analyzed by chemotaxis assay and macrophage M2 phenotypic marker expression was analyzed by flow cytometry and RT-qPCR.ResultsThe mRNA level of HNRNPU was higher in bladder cancer tissues than in normal paracancerous tissues(0.94±0.41 vs.1.54±0.28,t=5.800,P<0.05).The content of CCL2 in T24 cells[(230.56±18.62)vs.(105.67±22.10)pg/ml,t=20.830,P<0.05]and 5637 cells[(165.83±27.27)vs.(93.94±18.06)pg/ml,t=4.120,P<0.05]was significantly decreased after knockdown of HNRNPU.The results of chemotaxis assay showed a significant decrease in the number of macrophages chemotaxed toward T24 cells(110±16 vs.38±7,t=12.630,P<0.05)and 5637 cells(92±12 vs.41±8,t=9.578,P<0.05)after knockdown of HNRNPU.The flow cytometry results showed significantly lower CD206 expression on the surface of macrophages co-cultured with T24 cells[(2.31±0.13)%vs.(0.41±0.03)%,t=56.600,P<0.05]and 5637 cells[(1.75±0.01)%vs.(0.71±0.02)%,t=99.300,P<0.05].RT-qPCR results showed a significant decrease in the expression of M2 phenotypic markers in macrophages co-cultured with T2

关 键 词：膀胱癌 巨噬细胞 趋化因子配体2 

分 类 号：R737.14[医药卫生—肿瘤]