STAT3 miR-146-b反馈回路抑制IL-6的NF-κB信号轴调控结直肠癌细胞的干性机制

Mechanism by STAT3 miR-146-b Feedback Loop Inhibits NF-κB Signal Axis of IL-6 in Regulating Stemness of Colorectal Cancer Cells

目的:探究STAT3 miR-146-b反馈回路抑制IL-6的NF-κB信号轴调控结直肠癌细胞的干性机制。方法:用Dulbecco改良的Eagle培养基(DMEM)培养结直肠癌细胞HCT116和正常人结肠上皮细胞CCD 841 CoN细胞。通过免疫荧光检测IL-6和NF-κB信号轴。通过Western blot检测p-STAT3、p-JAK2和miR-146-b蛋白表达水平。通过Transwell分析法检测细胞迁移能力,通过TUNEL染色检测细胞凋亡。通过CCK-8评估不同处理组细胞活力。通过Western blot检测EMT间充质标志物波形蛋白(Vimentin,VIM),SNAIL和ZEB1及上皮标志物E-钙粘蛋白(CDH1)水平。通过PCR检测CRC标志物CD44、CD133、NANOG和Lgr5mRNA的表达。结果:与对照组相比,CRC组IL-6和NF-κB表达水平均升高(P<0.05),而IL-6抑制剂组较CRC组IL-6和NF-κB表达水平均降低(P<0.05)。与对照组相比,CRC组p-STAT3、p-JAK2和miR-146-b蛋白含量均升高(P<0.05),而IL-6抑制剂组较CRC组p-STAT3、p-JAK2和miR-146-b蛋白含量均降低(P<0.05)。与对照组相比,CRC组细胞迁移能力增加,细胞凋亡降低(P<0.05),而IL-6抑制剂组较CRC组细胞迁移能力降低,细胞凋亡增加(P<0.05)。与对照组相比,CRC组0 h细胞活力无差异(P>0.05),24、48和72 h细胞活力均增加(P<0.05),而IL-6抑制剂组较CRC组0 h细胞活力无差异(P>0.05),24、48和72 h细胞活力均降低(P<0.05)。与对照组相比,CRC组VIM、SNAIL和ZEB1蛋白含量均升高,CDH1蛋白含量降低(P<0.05),而IL-6抑制剂组较CRC组VIM、SNAIL和ZEB1蛋白含量均降低,CDH1蛋白含量升高(P<0.05)。与对照组相比,CRC组CD44、CD133、NANOG和Lgr5 mRNA的表达均升高(P<0.05),而IL-6抑制剂组较CRC组CD44、CD133、NANOG和Lgr5 mRNA的表达均降低(P<0.05)。结论:STAT3 miR-146-b反馈回路抑制IL-6的NF-κB信号轴调控结直肠癌细胞的干性。IL-6/STAT3/miR-146-b轴可以作为CRC的诊断标记,并为结肠癌治疗提供治疗靶标。

Objective:To explore the mechanism by STAT3 miR-146-b feedback loop inhibits the NF-κB signal axis of IL-6 in regulating the stemness of colorectal cancer cells.Method:Colorectal cancer cell HCT116 and Normal human colonic epithelial cells CCD 841 CoN cells were cultured with Dulbecco’s modified Eagle medium (DMEM).The IL-6 and NF-κB signal axis were detected by immunofluorescence.The expression levels of p-STAT3,p-JAK2 and miR-146-b protein were detected by Western blot.The cell migration ability was detected by Transwell analysis method,and cell apoptosis was detected by TUNEL staining.The cell viabilities of different treatment groups were evaluated by CCK-8.The levels of EMT mesenchymal markers vimentin (Vimentin,VIM),SNAIL and ZEB1 and epithelial marker E-cadherin (CDH1) were detected by Western blot.The expressions of CRC markers CD44,CD133,NANOG and Lgr5 mRNA were detected by PCR.Result:Compared with the control group,the expression levels of IL-6 and NF-κB in the CRC group were increased (P<0.05),while the expression levels of IL-6 and NF-κB in the IL-6 inhibitor group were decreased compared with the CRC group (P<0.05).Compared with the control group,the protein contents of p-STAT3,p-JAK2 and miR-146-b in the CRC group were all increased (P<0.05),while the protein levels of p-STAT3,p-JAK2 and miR-146-b in the IL-6 inhibitor group were decreased compared with the CRC group (P<0.05).Compared with the control group,the CRC group had increased cell migration ability and decreased cell apoptosis (P<0.05),while the IL-6 inhibitor group had decreased cell migration ability and increased cell apoptosis compared with the CRC group (P<0.05).Compared with the control group,there was no difference in cell viability of the CRC group at 0 h (P>0.05),and increased cell viability at 24,48 and 72 h (P<0.05),while the IL-6 inhibitor group had no difference in cell viability at 0 h compared with the CRC group (P>0.05),24,48 and 72 h cells reduced vitality (P<0.05).Compared with the control group,the protein contents of VIM,SNAIL and ZEB1 in the CRC group were increased,and the CDH1 protein content was decreased (P<0.05),while the IL-6 inhibitor group had decreased VIM,SNAIL and ZEB1 protein content and increased CDH1 protein content compared with the CRC group (P<0.05).Compared with the control group,the expression of CD44,CD133,NANOG and Lgr5 mRNA in the CRC group were all increased (P<0.05),while the expression of CD44,CD133,NANOG and Lgr5 mRNA in the IL-6 inhibitor group were decreased compared with the CRC group (P<0.05).Conclusion:STAT3 miR-146-b feedback loop inhibits NF-κB signal axis of IL-6 to regulate the stemness of colorectal cancer cells.IL-6/STAT3/miR-146-b axis can be used as a diagnostic marker for CRC and provide a therapeutic target for the treatment of colon cancer.