CXCL12/CXCR4轴通过诱导miR-125b促进肝癌细胞肿瘤干性和5-氟尿嘧啶抵抗的机制

CXCL12/CXCR4 axis promotes the tumor dry-type and 5-fluorouracil resistance of hepatocellular carcinoma cells by inducing miR-125b

目的探究趋化因子CXCL12/趋化因子受体CXCR4轴通过诱导miR-125b促进肝癌细胞肿瘤干性和5-氟尿嘧啶(5-FU)抵抗的机制。方法选择人肝细胞癌细胞系Huh7,分为7组:对照组(正常培养的肝细胞癌系),CXCR4转染组(CXCR4模拟转染超表达),CXCR4沉默组(CXCR4低表达或者不表达),miR-125b转染组(miR-125b超表达),CXCL12+125b抑制剂组(CXCL12超表达的+抑制miR-125b的表达),5-FU处理组(10μg/L的5-FU处理细胞),5-FU+miR-125b转染组(10μg/L的5-FU处理+miR-125b超表达的细胞)。聚合酶链式反应分析miR-125b mRNA表达;蛋白质印迹法检测E-钙粘蛋白、波形蛋白、CXCR4蛋白、Caspase-3、Bcl-2和Bax的蛋白表达;Transwell分析各组细胞中的细胞迁移、侵袭测定;MTT检测细胞存活力;细胞计数试剂盒8(CCK-8)评估细胞增殖。结果与对照组相比,CXCR4转染组miR-125b mRNA、波形蛋白、CXCR4的蛋白表达显著升高,E黏着蛋白表达显著降低(P<0.05);与CXCR4转染组相比,CXCR4沉默组miR-125b mRNA、波形蛋白、CXCR4的蛋白表达显著降低,E黏着蛋白表达显著升高(P<0.05)。与对照组相比,miR-125b转染组细胞迁移、侵袭数量、细胞活力均显著增加,细胞凋亡率显著降低(P<0.05);与miR-125b转染组相比,CXCL12+125b抑制剂组,细胞迁移、侵袭数量、细胞活均显著减少,细胞凋亡率显著升高(P<0.05)。与对照组相比,5-FU处理组细胞增殖率、Bcl-2表达显著降低,caspase-3、Bax的蛋白表达显著升高(P<0.05),与5-FU处理组相比,5-FU+miR-125b转染组细胞增殖率、Bcl-2表达显著升高,caspase-3、Bax的蛋白表达显著降低(P<0.05)。结论MiR-125b通过激活CXCL12/CXCR4轴而被上调,miR-125b增强CXCR4的表达,这在触发肿瘤侵袭和进展中形成了正反馈回路。这些结果为miR-125b在肝癌进程和化学抗性的发展中的作用提供了新的线索,为肝癌的治疗提供了潜在的治疗靶点。

Objective To the mechanism of CXCL12/CXCR4 axis by inducing miR-125b to promote tumor stemness and 5-fluorouracil(5-FU) resistance of liver cancer cells. Methods The human hepatocellular carcinoma cell line Huh7 was selected and divided into the following groups: control group(normally cultured hepatocellular carcinoma line),CXCR4 transfection group(CXCR4 mock transfection overexpression),CXCR4 silence group(CXCR4 low expression or no CXCR4 expression),miR-125b transfection group(miR-125b overexpression),CXCL12 + 125b inhibitor group(CXCL12 overexpression + inhibition of miR-125b expression),5-FU treatment group(10 μg/L of 5-FU Treated cells),5-FU + miR-125b transfection group(10 μg/L 5-FU treatment + miR-125b overexpressing cells). The expression of miR-125b mRNA was analyzed by polymerase chain reaction;the protein expression of E-cadherin,vimentin,CXCR4 protein,Caspase-3,Bcl-2 and Bax was detected by Western blotting;Cell migration and invasion were measured by Transwell analysis;cell viability was measured by MTT method;cell proliferation was evaluated by cell counting kit 8. Results Compared with the control group,the expression of miR-125b mRNA,vimentin and CXCR4 protein in the CXCR4 transfection group was significantly increased,and the expression of E adhesive protein was significantly decreased(P < 0. 05);compared with the CXCR4 transfection group,the expressions of miR-125b mRNA,vimentin,and CXCR4 in the CXCR4 silence group were significantly reduced,and the expression of E mucin was significantly increased(P < 0. 05). Compared with the control group,the miR-125b transfection group had significantly increased cell migration,invasion number,and cell viability,and the apoptosis rate was significantly reduced(P < 0. 05);compared with the miR-125b transfection group,CXCL12 + 125b inhibited in the drug group,cell migration,number of invasions,and cell viability were significantly reduced,and the rate of cell apoptosis was significantly increased(P < 0. 05).Compared with the control group,the cell proliferation rate and Bcl-2 expression in the 5-FU treatment group were significantly reduced,and the protein expression of caspase-3 and Bax were significantly increased(P < 0. 05). Compared with the 5-FU treatment group,the cell proliferation rate and Bcl-2 expression in the 5-FU + miR-125b transfection group were significantly increased,and the protein expression of caspase-3 and Bax was significantly decreased(P < 0. 05). Conclusion MiR-125b is up-regulated by activating the CXCL12/CXCR4 axis. MiR-125b enhances the expression of CXCR4,which forms a positive feedback loop in triggering tumor invasion and progression. These results provide new clues for the role of miR-125b in the process of HCC and the development of chemoresistance,and provide potential therapeutic targets for the treatment of HCC.