JWA编码区PKC磷酸化位点突变对佛波酯诱导MCF-7细胞分化的影响

The effect of PKC phosphorylation sites mutation in JWA coding region on TPA-induced MCF-7 cell differentiation

目的探讨JWA蛋白PKC磷酸化位点对TPA诱导的MCF-7细胞分化的影响。方法利用连续PCR引入点突变技术构建JWA编码区PKC磷酸化位点1、2以及两位点同时突变的三种pEGFP-N1载体,脂质体稳定转染获得相应的三种MCF-7细胞株,油红O染色观察细胞分化状态。结果20μmol/L TPA处理MCF-7-N1(稳定转染pEGFP-N1载体的MCF-7细胞)、MCF-7-JWA(稳定转染pEGFP-N1-JWA载体的MCF-7细胞)、MCF-7-JWA-1、MCF-7-JWA-2、MCF-7-JWA-1+2细胞2d后,油红O染色阳性细胞百分比分别为48%、67%、69%、67%、70%。与MCF-7-N1相比,TPA诱导MCF-7- JWA、MCF-7-JWA-1、MCF-7-JWA-2、MCF-7-JWA-1+2细胞分化率(油红O染色阳性细胞百分比)显著升高(P<0.05),但MCF-7-JWA、MCF-7-JWA-1、MCF-7-JWA-2、MCF-7-JWA-1+2细胞分化率,差异无统计学意义(P>0.05)。结论稳定转染JWA能增强TPA诱导的MCF-7细胞分化;JWA编码区PKC磷酸化位点突变并不影响TPA诱导MCF-7细胞分化,可能存在其他的分子机制。

Objective To investigate the effect of PKC phosphorylation sites mutation in JWA coding region on TPA-induced MCF-7 cell differentiation. Methods Site directed gene mutation was used to construct one or two PKC sites mutations in pEGFP-N1-JWA vectors, and transfected into MCF-7 cells by polyfect reagent, and cell differentiation was characterized by accumulation of lipid droplet as indicated by positive Oil-red-O staining of cells. Results All these trasfected cell lines, MCF-7-N1 （transfected with pEGFP-N1 vector）, MCF-7-JWA（transfected with pEGFP-N1-JWA vector）, MCF-7-JWA-1 （transfected with PKC site 1 mutation pEGFP-N1-JWA vector）, MCF-7-JWA-2（transfected with PKC site 2 mutation pEGFP-N1-JWA vector）, MCF-7-JWA-1＋2（transfected with both PKC site 1 and 2 mutation pEGFP-N1-JWA vector） were treated with 20 nmol/ L TPA for 48 h, and the percentages of positive Oil-red-O staining of cells were 48%, 67%, 69%, 67% and 70% respectively. The percentages of cell differentiation in JWA containing vectors transfected cells treated with TPA were significantly higher those of MCF-7-N1 cells （vector only control）. However, there were no significant differences between mutated and unmutated cells. Conclusion JWA transfection enhanced MCF-7 cell differentiation induced by TPA significantly, and PKC sites mutation in JWA coding region has no obviously effect on TPA-induced MCF-7 cell differentiation.