RNF181与Hippo/YAP对脑胶质瘤SHG44细胞恶性程度的影响及其可能机制

RNF181 promotes malignancy of glioma cells along Hippo / Yap signaling pathway

目的探讨环指蛋白RNF181与Hippo/YAP对脑胶质瘤SHG44细胞恶性程度的影响及其可能机制。方法选取人脑胶质瘤细胞系SHG44细胞体外培养,腺病毒表达载体转染过表达RNF181,对比分析RNF181过表达对肿瘤细胞生长增殖能力、细胞干性和体外侵袭及转移能力;通过RT-PCR和Western blot实验检测RNF181及Hippo/YAP的表达,明确RNF181与Hippo/YAP的关系;最后通过免疫荧光共定位分析RNF181和YAP在SHG44细胞表达情况。结果转染后SHG44中RNF181 mRNA及蛋白相对表达量1.95±0.06、0.39±0.06,明显高于对照组1.05±0.03、0.23±0.06和Negative组1.03±0.04、0.24±0.07;YAP mRNA及蛋白相对表达量2.63±0.12、0.71±0.06,与对照组1.02±0.04、0.29±0.05和Negative组1.03±0.05、0.31±0.07比较,表达水平明显升高,差异有统计学意义(P<0.05)。MTT、免疫荧光和Transwell实验证实,RNF181过表达组细胞数、细胞增值率及SHG-44穿膜细胞数明显高于对照组和Negative组,差异均有统计学意义(P<0.05)。免疫荧光共定位检测表明RNF181和YAP在SHG44细胞核内共表达。结论RNF181过表达增加胶质瘤细胞的增殖和侵袭能力,可能是通过Hippo/YAP信号途径实现的。

Objective To explore the effect of overexpression of RNF181 on the malignancy of human glioma SHG44 cells and the possible mechanism.Methods Human glioma cell line SHG44 was cultured in vitro.The adenovirus expression vector was transfected to overexpress RNF181.The effects of overexpression of RNF181 on the growth and proliferation of tumor cells,cell stemness,invasion and metastasis in vitro were compared.The expressions of RNF181 and hippo/Yap were detected by Western blot,and the relationship between RNF181 and hippo/Yap was clarified.Finally,the expressions of RNF181 and Yap in SHG44 cells were analyzed by immunofluorescence co-localization.Results After transfection,the relative expression levels of RNF181 mRNA and protein in SHG44 were[(1.95±0.06)and(0.39±0.06)],which were much higher than those of the control group[(1.05±0.03)and(0.23±0.06)]and the negative group[(1.03±0.04)and(0.24±0.07)],and the difference was statistically significant(P<0.05).The relative expression levels of YAP mRNA and protein were(1.02±0.04),and(0.29±0.05)in the control group,compared with[(1.03±0.05)and(0.31±0.07)]in the negative group,and[(2.63±0.12)and(0.71±0.06)]in the overexpression group,so the difference was statistically significant(P<0.05).MTT,immunofluorescence and Transwell assay confirmed that the proliferation rate of the RNF181 overexpression group was significantly higher than that of the control group and the negative group.The cell number of the RNF181 overexpression group was significantly larger than that of control group and negative group,so was the number of SHG-44 transmembrane cells.Immunofluorescence co-localization showed that RNF181 and YAP were co-expressed in the nucleus of SHG44.Conclusions Overexpression of RNF181 may contribute to the proliferation and invasion of glioma cells along the Hippo/Yap signaling pathway.