IL-22通过激活STAT3信号轴损害NK细胞功能并促进膀胱癌细胞顺铂耐药OA

Objective:To investigate the mechanism by which IL-2 promotes bladder cancer cell resistance by impairing NK cell function via the STAT3 signalling axis.Methods:Bladder cancer T24 cells were routinely cultured,and cisplatin-resistant T24/DDP cells were established through stepwise dose-escalation method.Cells were divided into the following groups:control,DDP,IL-22,IL-22+DDP,IL-22+anti-IL-22,and IL-22+DDP+Stattic(a STAT3 inhibitor).The mRNA expression of IL-22,cyclin D1,and BCL2 was detected using qRT-PCR.Protein expression of BAX,BCL2,and phosphorylated STAT3(p-STAT3)was analyzed using WB.Cell proliferation and apoptosis were assessed using the CCK-8 assay and flow cytometry,respectively.The levels of lactate dehydrogenase(LDH),TNF-α,IFN-γ,granzyme B(GzmB),and perforin(PRF)in the supernatant were measured using enzyme-linked immunosorbent assay(ELISA).Results:T24/DDP cells exhibited significantly reduced sensitivity to DDP(P<0.05),accompanied by markedly elevated expression levels of drug resistance-associated genes(P-glycoprotein[P-gp],lung drug resistance protein[LRP],and multidrug resistance-associated protein 1[MRP1]),as well as IL-22 and its receptor(all P<0.05),indicating successful establishment of DDP-resistant cells.Compared with the control group,the DDP group showed decreased proliferation,increased apoptosis,upregulated BAX protein expression,and downregulated Bcl-2 protein expression in T24 cells(all P<0.05).Compared with the DDP group,the IL-22+DDP group showed significantly increased proliferative activity,decreased apoptosis rate,downregulated BAX,and upregulated BCL expression(all P<0.05),suggesting that IL-22 promotes DDP resistance in T24 cells by modulating BAX/BCL2 expression.Compared with the control group,IL-22 stimulation significantly increased total and nuclear p-STAT3 expression in T24 cells(all P<0.05),and this increase was significantly attenuated by pre-treatment with an IL-22 neutralizing antibody(IL-22+anti-IL-22 group)(P<0.05),indicating that IL-22 activates STAT3 phosphorylation and promotes its nuclear translocation in T24 cells.In the T24-NK92 co-culture system,the levels of LDH,TNF-α,IFN-γ,GzmB,and PRF in the supernatant were significantly increased in the DDP group compared with the control group(all P<0.05).Co-treatment with IL-22 and DDP significantly reduced the levels of these cytotoxicity-related factors compared to the DDP group(all P<0.05).Furthermore,IL-22 treatment alone significantly decreased the levels of these factors compared to the control group(all P<0.05),while the addition of the STAT3 inhibitor Stattic(IL-22+Stattic group)reversed this suppression,leading to significant elevations in these factors(all P<0.05).These findings indicate that IL-22 diminishes the cytotoxicity of NK92 cells against T24 cells,which can be reversed by STAT3 inhibition.Regarding chemoresistance,T24 cell proliferative activity was significantly higher in the IL-22+DDP group than in the DDP group(P<0.05).This enhancement was abolished by Stattic,as evidenced by significantly lower activity in the IL-22+DDP+Stattic group compared to the IL-22+DDP group(P<0.05).Consistently,the apoptosis rate was significantly decreased in the IL-22+DDP group compared with the DDP group(P<0.05),and Stattic co-treatment significantly increased the apoptosis rate compared to the IL-22+DDP group(P<0.05).These findings indicate that IL-22 regulates both DDP resistance in T24 cells and NK cell-mediated immune function via the STAT3 pathway.Conclusion:IL-22 promotes DDP resistance in T24 bladder cancer cells and suppresses NK cell function via activating the STAT3 signaling axis.