Downregulation of HNRNPU regulates tumor-associated macrophage polarization and chemotaxis by inhibiting chemokine ligand 2 secretion in bladder cancer cells
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.Methods Totally,10 cases of cancer tis-sues 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 blad-der cancer tissues and paracancerous normal tissues was analyzed using real-time quantitative reverse tran-scriptase-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.Results The 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 expres-sion of M2 phenotypic markers in macrophages co-cultured with T24 cells(Arg1∶3.21±0.76 vs.1.05± 0.11,t=6.761,P＜0.05 and CSF1R:4.37±0.47 vs.1.80±0.25,t=6.432,P＜0.05)and 5637 cells(Arg1∶2.76±0.47vs.1.68±0.51,t=12.470,P＜0.05 and CSF1R:3.91±0.27 vs.1.03± 0.11,t=23.470,P＜0.05).The co-cultured macrophages showed significantly lower expression of M2 phenotypic markers.Conclusion HNRNPU was highly expressed in bladder cancer tissues;down-regula-tion of HNRNPU inhibited the secretion of CCL2 in bladder cancer cells and suppressed chemotaxis and M2 polarization in co-cultured macrophages.
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