circRAF1调节人卵巢颗粒细胞的增殖与凋亡

circRAF1 regulates the proliferation and apoptosis of human ovarian granulosa cells

目的探讨circRAF1在早发性卵巢功能不全(primary ovarian insufficiency,POI)中的表达及对人卵巢颗粒细胞(GCs)系(KGN细胞)增殖、凋亡的影响。方法RT-qPCR检测正常卵巢储备功能的患者(n=50)和POI患者(n=50)卵巢GCs及血清中circRAF1的表达,并分析其与卵巢储备功能指标的相关性;构建针对circRAF1的小干扰RNA(small interfering RNA,siRNA),转染至KGN细胞;利用CCK-8、EdU实验检测细胞增殖,JC-1、Tunel实验检测细胞凋亡;RT-qPCR和WB检测细胞增殖与凋亡相关基因(FSHR、PCNA、Bcl-2、Casp-9、Bax)的表达。结果circRAF1在POI患者GCs及血清中表达下降,circRAF1的表达与血清中基础E2和AMH水平呈正相关(P<0.001),而与血清中基础FSH、LH水平呈负相关(P<0.001),同时,circRAF1的表达与AFC呈正相关(P<0.001)。干扰circRAF1的表达可抑制KGN细胞的增殖,促进其凋亡。结论POI患者GCs及血清中的circRAF1表达下调,且与卵巢储备功能下降有关;干扰circRAF1可以抑制GCs增殖,促进其凋亡。

Objective To investigate the expression of circRAF1 in primary ovarian insufficiency(POI)and explore its effect on cell proliferation and apoptosis of human ovarian granulosa cells(GCs)line(KGN cells).Methods The expression of circRAF1 in GCs and serum of patients with normal ovarian reserve function(n=50)and patients with POI(n=50)were detected with RT-qPCR.The correlation of circRAF1 with ovarian reserve function indexes was analyzed.Small interfering RNA(siRNA)targeting circRAF1 was constructed and transfected into KGN cells,with the cell proliferation detected by CCK-8 and EdU assay,and the cell apoptosis detected by JC-1 and Tunel assay.The mRNA and protein levels of genes related to cell proliferation and apoptosis(FSHR,PCNA,Bcl-2,Casp-9,Bax)were detected by RT-qPCR and WB.Results The expression of circRAF1 decreased in GCs and serum of POI patients.The expression of circRAF1 was positively correlated with serum E2 and AMH levels(P<0.001),but negatively correlated with serum FSH and LH levels(P<0.001).At the same time,the expression of circRAF1 was positively correlated with AFC(P<0.001).Interfering with the expression of circRAF1 could inhibit the proliferation of KGN cells and promote their apoptosis.Conclusion The expression of circRAF1 in the GCs and serum of POI patients is down-regulated,which is correlated with the decline of ovarian reserve function.Interfering with circRAF1 can inhibit the proliferation of GCs and promote their apoptosis.