EZH2通过抑制LncRNA而抑制烧伤后皮肤成纤维细胞增殖的机制研究

EZH2 Inhibits the Proliferation of Fibroblasts after Burn Injury by Inhibiting LncRNA

目的:探讨EZH2抑制长链非编码RNA H19对烧伤后皮肤成纤维细胞增殖的影响及机制。方法:采用RT-PCR检测8例成熟皮肤、8例成熟烧伤皮肤长链非编码RNA H19和EZH2的表达;用脂质体转染体外培养的人烧伤后皮肤成纤维细胞,分为空白组(未转染)、阴性对照组(转染非特异性siRNA)、siRNA H19组、siRNA-EZH2组和siRNA H19+siRNA-EZH2组,验证lncRNA-H19 siRNA的转染效率;MTT法检测转染lncRNA-H19 siRNA后成纤维细胞的增殖,Western blot检测转染后血管内皮生长因子(VEGF),采用RNA pull-down和免疫共沉淀的方法检测H19与EZH2的相互作用关系。结果:初步检测到H19在成熟皮肤细胞中的表达较低,而在烧伤皮肤中表达较高。与空白组和阴性对照组相比,siRNA H19组、siRNA-EZH2组和siRNA H19+siRNAEZH2组成纤维细胞的增殖明显受到抑制更为明显(P<0.05),VEGF表达水平明显降低(P<0.05);RNA pull-down与免疫共沉淀的结果显示H19可与EZH2特异性结合。结论:EZH2和H19相互作用抑制烧伤后皮肤成纤维细胞增殖更为明显,H19可能通过特异性结合EZH2调节烧伤后皮肤成纤维细胞的增殖。

Objective To investigate the effect and mechanism of EZH2 on the proliferation of skin fibroblasts after burn injury.Methods RT-PCR was used to detect the expression of long-chain noncoding RNA H19 and EZH2 in 8 cases of mature skin and 8 cases of mature burn skin.The cultured human burn skin fibroblasts were transfected with liposomes and divided into blank group(without siRNA and liposome),negative control group and lncRNA-h19 siRNA transfection group.To verify the transfection effi ciency of lncRNA-h19 siRNA.MTT assay was used to detect the proliferation of fi broblasts transfected with lncRNA-h19 siRNA.After transfection,VEGF was detected by Western blot.The interaction between H19 and EZH2 was detected by RNA pull-down and immunocoprecipitation.Results It was found that the expression of H19 in mature skin cells was lower than that in burn skin cells.Compared with the blank control group and the negative control group,the proliferation of fibroblasts in the lncRNA-h19 siRNA transfection group was signifi cantly inhibited(P<0.05),and the expression level of VEGF was signifi cantly reduced(P<0.05).The results of RNA pull-down and immunoprecipitation showed that H19 could specifi cally bind to EZH2.Conclusion The interaction of EZH2 and H19 inhibited the proliferation of skin fibroblasts after burn more obviously.H19 may regulate the proliferation of skin fibroblasts after burn through specifi ccombination of EZH2.