TRPM8激活Calcineurin-NFATc3通路调节结肠癌细胞免疫逃逸

目的 探讨TRPM8调节结肠癌细胞免疫逃逸的机制.方法 采用qRT-PCR和Western blot检测结肠癌细胞系SW620和正常人结肠上皮细胞系CCD 841 CoN中TRPM8的表达水平.将干涉和过表达TRPM8载体TRPM8siRNA和pcDNA3.1-TRPM8转染至SW620细胞,并设置相应对照组(Control siRNA组和pcDNA3.1组),用CCK-8、集落形成实验和流式细胞术检测细胞增殖和凋亡,酶标仪检测Calcineurin活性,Western blot检测Calcineurin-NFATc3信号通路相关蛋白的表达.采用CCK-8检测CD8+T细胞与SW620细胞共孵育的细胞活力,Western blot检测Calcineurin特异性抑制剂FK506处理SW620细胞后NFATc3和PD-L1蛋白表达.结果 与CCD 841 CoN细胞相比,TRPM8 mRNA和蛋白在SW620细胞中的表达均增加(P<0.01).与Control siRNA组比较,干涉TRPM8表达后SW620细胞活力、细胞增殖能力、Calcineurin活性,以及TRPM8、PD-L1和NFATc3蛋白表达均降低(P<0.01),而细胞凋亡率和p-NFATc3蛋白表达均升高(P<0.01);过表达TRPM8可增强细胞活力、细胞增殖能力和Calcineurin活性,上调TRPM8、PD-L1及NFATc3蛋白表达(P<0.01),下调p-NFATc3蛋白表达(P=0.002).CD8+T细胞与干涉TRPM8表达的SW620细胞共孵育后总细胞活力降低(P=0.002),而与过表达SW620细胞共孵育后总细胞活力增强(P=0.005).FK506处理可抑制SW620细胞Calcineurin活性,下调NFATc3、PD-L1蛋白表达(P<0.01).结论 TRPM8过表达可能通过激活Calcineurin-NFATc3信号通路而促进PD-L1表达,从而增强结肠癌细胞免疫逃逸能力,促进结肠癌细胞增殖.

Immune evasion of colon cancer cells regulated by TRPM8-activtaed Calcineurin-NFATc3 signaling pathway

Objective　To investigate the underlying mechanism of TRPM8 regulating the immune evasion of colon cancer cells. Methods The expression of TRPM8 in colon cancer cell line SW620 and human colon epithelial cell line CCD 841 CoN was detected by qRT-PCR and Western blot. SW620 cells were transfected with TRPM8 siRNA and pcDNA3.1-TRPM8，respectively，and the corresponding control groups（Control siRNA group and pcDNA3.1 group）were established. The CCK-8，colony formation experiment and flow cytometry were used to detect cell proliferation and apoptosis，microplate reader was used to detect Calcineurin activity，and Western blot was used to detect the expression of related proteins in Calcineurin-NFATc3 signaling pathway. The cell viability after co-incubation of CD8⁺ T cells and transfected SW620 cells was detected by CCK-8，and the expression of NFATc3 and PD-L1 proteins in SW620 cells treated with the Calcineurin inhibitor FK506 was detected by Western blot. Results The expressions of TRPM8 mRNA and protein in SW620 cells were up-regulated compared with CCD 841 CoN cells（P<0.01）. Compared with the Control siRNA group，the cell viability，cell proliferation，Calcineurin activity，and the expression of TRPM8，PD-L1 and NFATc3 proteins in SW620 cells after interference with TRPM8 expression decreased（P<0.01），while the cell apoptosis rate and p-NFATC3 protein expression increased（P<0.01）；overexpression of TRPM8 could enhance cell viability，cell proliferation and Calcineurin activity，up-regulate the expression of TRPM8，PD-L1 and NFATc3 proteins （P<0.01），and down-regulate the expression of p-NFATc3 proteins（P=0.002）. After CD8⁺ T cells co-incubation with SW620 cells that transfected with TRPM8 siRNA，the total cell viability decreased （P=0.002），but increased after CD8⁺ T cells co-incubation with SW620 cells transfected with pcDNA3.1-TRPM8（P=0.005）. FK506 could inhibit Calcineurin viability of SW620 cells and down-regulate the expression of NFATc3 and PD-L1 proteins（P<0.01）. Conclusion TRPM8 overexpression may promote the expression of PD-L1 by activating Calcineurin-NFATc3 signaling pathway，thereby enhancing the immune evasion ability of colon cancer cells and promoting its proliferation.