摘要
目的探究miR-29a-3p/SGMS2轴对人类卵巢颗粒样肿瘤细胞系(human ovarian granulosa-like tumor cell line,KGN)炎症反应的调控机制。方法通过构建并验证过表达和敲低miR-29a-3p及SGMS2转染KGN细胞模型,双荧光素酶报告基因实验检测miR-29a-3p与SGMS2的结合;MTT法检测各组细胞的增殖;免疫荧光法观察增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)和Ki67蛋白荧光强度;流式细胞术检测细胞凋亡;酶联免疫吸附法检测细胞上清液中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白介素(interleukin,IL)-6、IL-1β和鞘磷脂的表达;Western blotting检测细胞中Caspase-3、cleaved-Caspase-3、SGMS2和p-p65蛋白表达。结果miR-29a-3p与SGMS2之间存在位点结合,且能够负向调控SGMS2的表达水平。KGN细胞中过表达SGMS2能够增高其吸光度值(P=0.007),并增强其PCNA和Ki67蛋白的免疫荧光强度(均P<0.001);过表达SGMS2能够降低KGN细胞的凋亡率(P=0.001),升高炎症因子TNF-α、IL-6、IL-1β和鞘磷脂的表达水平(均P<0.001)。过表达SGMS2时KGN细胞中cleaved-Caspase-3、p-p65和SGMS2蛋白表达量升高(P=0.001,P<0.001,P<0.001)。而敲低SGMS2时,以上结果具有与过表达SGMS2相反的变化趋势。结论miR-29a-3p与SGMS2之间存在靶向负向调节关系,SGMS2能够促进KGN细胞的炎症反应,可为多囊卵巢综合征等妇科内分泌疾病的治疗提供靶点。
Objective To investigate the regulatory mechanism of miR-29a-3p/SGMS2 axis on the inflammatory response of ovarian granulosa cells(KGN).Methods By constructing and validating overexpression and knockdown of miR-29a-3p and SGMS2 transfected KGN cell models,dual luciferase reporter gene assay was used to detect the binding of miR-29a-3p to SGMS2.MTT assay was used for detecting the cell proliferation.Fluorescence intensity of proliferating cell nuclear antigen(PCNA)and Ki67 protein were detected by immunofluorescence.Apoptosis was detected by flow cytometry.The expression levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-6,IL-1βand sphingomyelin in cell supernatant were detected by enzyme-linked immunosorbent assay.The protein expression levels of Caspase-3,cleaved-Caspase-3,SGMS2 and p-p65 in KGN were detected by Western blotting.Results miR-29a-3p had site-specific binding to SGMS2 and can negatively regulated SGMS2 expression level.Overexpression of SGMS2 in KGN cells was able to increase their levels of absorbance(P=0.007)and enhanced the immunofluorescence intensity of the protein of PCNA and Ki67(all P<0.001).Overexpression of SGMS2 could reduce the apoptosis rate of KGN cells(P=0.001).Overexpression of SGMS2 increased the expression levels of TNF-α,IL-6,IL-1βand sphingomyelin(all P<0.001).The expressions of cleaved-Caspase-3,p-p65 and SGMS2 proteins were elevated in KGN cells when SGMS2 was overexpressed(P=0.001,P<0.001,P<0.001),while knock down SGMS2,the above results had a trend of change opposite to overexpression of SGMS2.Conclusion There is a targeted negative regulatory relationship between miR-29a-3p and SGMS2,which can promote the inflammatory response of KGN and may provide a target for the treatment of polycystic ovary syndrome.
作者
徐芳
李晓珂
潘紫萌
梁锦玲
马明
匡洪影
孙淼
Xu Fang;Li Xiaoke;Pan Zimeng;Liang Jinling;Ma Ming;Kuang Hongying;Sun Miao(Department of Gynecology,the First Affiliated Hospital,Heilongjiang University of Chinese Medicine,Harbin 150040,China;Department of Obstetrics and Gynecology,Shunyi Hospital,Beijing Hospital of Chinese Medicine,Beijing 101300,China;Department of First Clinical Medical College,Heilongjiang University of Chinese Medicine,Harbin 150040,China;Department of Surgery,the First Affiliated Hospital,Heilongjiang University of Chinese Medicine,Harbin 150040,China)
出处
《中华生殖与避孕杂志》
CAS
CSCD
北大核心
2023年第10期1032-1040,共9页
Chinese Journal of Reproduction and Contraception
基金
国家自然科学基金(82004401,81804138,82174422)
黑龙江省中医药管理局科研项目(ZHY2023-018)。
