MSI2对DHT诱导的KGN细胞增殖凋亡失衡的影响

Effect of MSI2 on proliferation and apoptosis imbalance of KGN cells induced by DHT

目的探究Musashi-2(MSI2)过表达对二氢睾酮(DHT)诱导的人卵巢颗粒细胞系(KGN)细胞增殖和凋亡失衡的影响。方法对人卵巢颗粒细胞(GCs)原代培养进程中的基因表达谱进行统计分析,筛选其中的差异表达基因。构建pc DNA3.1-MSI2真核表达质粒,瞬时转染至KGN细胞中,并利用实时荧光定量PCR(RT-qPCR)和Western blot技术检测MSI2的过表达效果。在DHT诱导的KGN细胞中过表达MSI2后,然后分别利用四唑盐(MTT)比色法和5-乙炔基-2’脱氧尿嘧啶核苷(Ed U)染色法检测各组细胞的增殖情况,并进一步利用流式细胞术(FCM)检测各组细胞的凋亡情况。结果MSI2的mRNA表达水平在人卵巢GCs原代培养过程中逐渐下降。成功构建pc DNA3.1-MSI2真核表达质粒,转染至KGN细胞中能够提高MSI2的mRNA和蛋白表达水平。MTT、Ed U和FCM结果显示与空白组相比,DHT诱导能够明显降低KGN细胞的增殖率,同时提高其凋亡率(P<0.05),而在MSI2过表达后,KGN细胞增殖率又有所提高,且凋亡率也有所降低(P<0.05),均接近于空白组。结论MSI2过表达能够有效缓解DHT诱导的KGN细胞增殖凋亡失衡现象,表明其在GCs生长和卵泡发育过程中发挥着重要作用。

Objective To study the effect of Musashi-2(MSI2)overexpression on the imbalance of proliferation and apoptosis of human ovarian granulosa cell line(KGN)induced by dihydrotestosterone(DHT).Methods The gene expression profiles of human ovarian granulosa cells(GCs)in primary culture were statistically analyzed to screen the differentially expressed genes.pcDNA3.1-MSI2 eukaryotic expression plasmid was constructed and transiently transfected into the KGN cells,and the overexpression effect of MSI2 was detected by Quantitative Real-time PCR(RT-qPCR)and Western blot.After overexpressing MSI2 in DHT induced KGN cells,MTT colorimetry and Edu staining were used to detect the proliferation of cells in each group,and flow cytometry(FCM)was further used to detect the apoptosis of cells in each group.Results The mRNA expression level of MSI2 gradually decreased during the primary culture of human ovarian GCs.And pcDNA3.1-MSI2 was successfully constructed and transfected into KGN cells to improve the mRNA and protein expression levels of MSI2.Then MTT,EdU and FCM results showed that compared with the blank group,DHT induction could significantly reduce the proliferation rate and increase the apoptosis rate of KGN cells(P<0.05).However,after MSI2 overexpression,the proliferation rate of KGN cells increased and the apoptosis rate decreased(P<0.05),which were close to the blank group.Conclusion Overexpression of MSI2 can effectively alleviate the imbalance of proliferation and apoptosis of KGN cells induced by DHT,indicating that MSI2 plays an important role in GCs growth and follicle development.