色素上皮衍生因子对子宫肌瘤平滑肌细胞增殖和分化的影响

Effect of pigment epithelial-derived factor on proliferation and differentiation of smooth muscle cells of uterine fibroid

目的探讨色素上皮衍生因子(PEDF)对子宫肌瘤平滑肌细胞增殖和分化的影响。方法通过Western blot和qRT-PCR检测30例子宫肌瘤组织以及邻近正常子宫肌层组织中的PEDF表达。分离、培养、鉴定子宫平滑肌细胞后,将子宫平滑肌细胞分为5组:对照组、pLenti6.3-NC组、PEDF-pLenti6.3组、shRNA-NC组、shRNA-PEDF组。对照组细胞不进行转染,pLenti6.3-NC组细胞转染阴性对照慢病毒(pLenti6.3-NC),PEDF-pLenti6.3组细胞转染过表达PEDF的重组慢病毒(PEDF-pLenti6.3),shRNA-NC组细胞转染阴性对照shRNA(shRNA-NC),shRNA-PEDF组细胞转染靶向PEDF基因序列的短发夹RNA(shRNA-PEDF)。通过Western blot和qRT-PCR检测子宫平滑肌细胞中的PEDF表达。采用细胞计数试剂盒8(CCK-8)和EdU染色试剂盒检测细胞增殖,使用一步法TUNEL细胞凋亡检测试剂盒检测细胞凋亡。通过qRT-PCR或Western blot检测PEDF、ERα、ERβ、SM22α、α-SMA、Bcl-2、cleaved Caspase-3、PI3K、total-AKT和p-AKT的表达。结果与正常子宫肌层组织和细胞相比,子宫肌瘤组织和细胞中PEDF的mRNA和蛋白水平均降低(P<0.05)。与pLenti6.3-NC组相比,PEDF-pLenti6.3组的细胞相对活力和EdU阳性率降低,TUNEL阳性率升高(P<0.05),Bcl-2蛋白相对表达量降低(P<0.05),cleaved Caspase-3蛋白相对表达量升高(P<0.05)。与pLenti6.3-NC组相比,PEDF-pLenti6.3组子宫肌瘤平滑肌细胞SM22α、α-SMA、ERα、ERβ、PI3K和p-AKT蛋白相对表达量均降低(P<0.05)。与shRNA-NC组相比,shRNA-PEDF组细胞的相对活力和EdU阳性率升高(P<0.05),TUNEL阳性率降低(P<0.05),Bcl-2蛋白相对表达量升高(P<0.05),cleaved Caspase-3蛋白相对表达量降低(P<0.05)。与shRNA-NC组相比,shRNA-PEDF组SM22α、α-SMA、ERα、ERβ、PI3K和p-AKT蛋白相对表达量均升高(P<0.05)。各组total-AKT蛋白相对表达量无显著差异(P>0.05)。结论PEDF在子宫肌瘤中被抑制,上调PEDF可抑制子宫肌瘤平滑肌细胞的增殖和分化,并诱导细胞凋亡,其机制部分是通过PI3K/AKT信号通路介导的。

Objective To reveal the effect of pigment epithelium-derived factor(PEDF)on the proliferation and differentiation of ute-rine fibroid smooth muscle cells.Methods Western blot and qRT-PCR were used to detect the expression of PEDF in 30 cases of uterine fibroids and normal adjacent myometrium tissues.Uterine smooth muscle cells were isolated,cultured and identified,and then the uterine smooth muscle cells were divided into five groups:control group,pLenti6.3-NC group,PEDF-pLenti6.3 group,shRNA-NC group,and shRNA-PEDF group.The cells in control group were not transfected,the cells in pLenti6.3-NC group were transfected with negative control lentivirus(pLenti6.3-NC),the cells in PEDF-pLenti6.3 group were transfected with PEDF-overexpressing recombinant lentivirus(PEDF-pLenti6.3),the cells in shRNA-NC group were transfected with negative control shRNA(shRNA-NC),and the cells in shRNA-PEDF group were transfected with short hairpin RNA targeting PEDF gene sequence(shRNA-PEDF).PEDF expression in uterine smooth muscle cells was detected by Western blot and qRT-PCR.Cell counting kit 8(CCK-8)and EdU staining kit were used to detect the cell proliferation,and one-step TUNEL cell apoptosis detection kit was used to detect the cell apoptosis.The expression levels of PEDF,ERα,ERβ,SM22α,α-SMA,Bcl-2,cleaved Caspase-3,PI3K,total-AKT and p-AKT were detected by qRT-PCR or Western blot.Results Compared with normal myometrium tissues and cells,PEDF mRNA and protein levels in uterine fibroid tissues and cells were reduced(P<0.05).Compared with pLenti6.3-NC group,the relative cell viability and EdU positive rate in PEDF-pLenti6.3 group were decreased(P<0.05),TUNEL positive rate was increased(P<0.05),the relative expression of Bcl-2 protein was decreased,and the cleaved Caspase-3 was increased(P<0.05).Compared with pLenti6.3-NC group,the relative expression levels of SM22α,α-SMA,ERα,ERβ,PI3K and p-AKT proteins in the smooth muscle cells of uterine fibroids in PEDF-pLenti6.3 group were all decreased(P<0.05).Compared with shRNA-NC group,the relative cell viability and EdU positive rate were increased in shRNA-PEDF group(P<0.05),TUNEL positive rate was decreased(P<0.05),the relative expression of Bcl-2 protein was increased(P<0.05),and the cleaved Caspase-3 was decreased(P<0.05).Compared with shRNA-NC group,the relative expression levels of SM22α,α-SMA,ERα,ERβ,PI3K and p-AKT proteins in shRNA-PEDF group were increased(P<0.05).There was no significant difference in the relative expression of total-AKT protein between groups(P>0.05).Conclusion PEDF is inhibited in uterine fibroids.Up-regulation of PEDF can inhibit the proliferation and differentiation of uterine fibroid smooth muscle cells and induce the cell apoptosis,which is partly mediated by PI3K/AKT signaling pathway.