piR-3127964经PIWIL-3/载唾液酸蛋白途径调控滋养细胞侵袭力的机制  

Mechanism for regulation of trophoblast invasion through piR-3127964 via PIWIL-3/sialophorin pathway

作　　者：康翼 袁雨[1,2] 林俐 谭彬 漆洪波[1,3] Yi Kang;Yu Yuan;Li Lin;Bin Tan;Hongbo Qi(Department of Obstetrics,the First Affiliated Hospital of Chongqing Medical University(Chongqing Key Laboratory of Maternal and Fetal Medicine,Joint International Research Laboratory of Reproduction and Development of Ministry of Education of the People Republic of China),Chongqing 400016,China;Department of Gynecology,People's Hospital of Shapingba District of Chongqing,Chongqing 400030,China;Department of Obstetrics and Gynecology,Women and Children's Hospital of Chongqing Medical University,Chongqing 401147,China)

机构地区：[1]重庆医科大学附属第一医院产科(母胎医学重庆市重点实验室,教育部生殖与发育国际合作联合实验室),重庆400016 [2]重庆市沙坪坝区人民医院妇科,重庆400030 [3]重庆医科大学附属妇女儿童医院妇产科,重庆401147

出　　处：《中华围产医学杂志》2023年第4期305-314,共10页Chinese Journal of Perinatal Medicine

基　　金：国家自然科学基金(82001573);国家自然科学基金联合基金(U21A20346);高等学校学科创新引智计划(外专办发【2016】404)。

摘　　要：目的研究子痫前期与非病理妊娠胎盘组织之间差异表达的piR-3127964调控滋养细胞侵袭力的分子机制。方法收集2020年11月至2021年8月于重庆医科大学附属第一医院产科就诊的非病理性早产(对照组,n=12)与子痫前期孕妇(子痫前期组,n=10)经剖宫产分娩的胎盘以及早孕期人工流产者的绒毛组织(早孕期人工流产组,n=10)。提取对照组和子痫前期组胎盘组织的总RNA进行测序,利用实时荧光定量聚合酶链反应(real-time quantitative-polymerase chain reaction,qRT-PCR)检测piR-3127964在各类组织中的相对表达量。利用蛋白质印迹法检测3组中PIWIL-1、PIWIL-2和PIWIL-3的表达量。外源性调节HTR-8/SVneo细胞内piR-3127964的水平,通过qRT-PCR检测下游候选靶分子,即鸟嘌呤核苷酸结合蛋白样3-样蛋白(guanine nucleotide-binding protein-like 3-like,GNL3L)和载唾液酸蛋白(sialophorin,SPN)mRNA的相对表达量,利用qRT-PCR检测GNL3L和SPN mRNA在对照组和子痫前期组胎盘组织中的相对表达量。进一步通过双荧光素酶报告基因试验验证上述两者与piR-3127964的相互作用。通过荧光原位杂交和免疫荧光结合的方式明确piR-3127964和SPN在早孕期人工流产组绒毛组织中的定位。通过Transwell细胞侵袭实验探究piR-3127964对HTR-8/SVneo细胞侵袭能力的影响和潜在机制。采用独立样本t检验及单因素方差分析及LSD事后检验对数据进行统计学分析。结果(1)差异表达的piR-3127964预测的下游靶基因的富集通路与细胞运动相关;piR-3127964在早孕期人工流产组绒毛、对照组和子痫前期组胎盘组织中差异有统计学意义(分别为2.950±0.853、1.036±0.303与0.254±0.155,F=27.35,P<0.05);且piR-3127964定位于早孕期人工流产组绒毛的绒毛外滋养细胞(extravillous cytotrophoblasts,EVTs)中。(2)子痫前期组胎盘组织中PIWIL-3蛋白表达量明显低于对照组胎盘组织和早孕期人工流产组绒毛组织(0.810±0.400与3.Objective To investigate the molecular mechanism for regulation of trophoblast invasion by piR-3127964,which is differentially expressed in placental tissues of preeclamptic and healthy pregnant women.Methods Placenta samples of healthy(control group,n=12)and preeclamptic pregnant(PE group,n=10)women who delivered by caesarean section and chorionic villi specimens of patients undergoing artificial abortion were collected in the Department of Obstetrics of the First Affiliated Hospital of Chongqing Medical University during November 2020 to August 2021.Total RNA was extracted from placenta samples and sequenced and the expression of piR-3127964 in different tissues was determined by real-time quantitative-polymerase chain reaction(qRT-PCR).The expressions of PIWI proteins including PIWIL-1,PIWIL-2 and PIWIL-3 in different tissues were detected by Western blot.The expressions of two candidate targets,guanine nucleotide-binding protein-like 3-like(GNL3L)mRNA and sialophorin(SPN)mRNA were evaluated by qRT-PCR after exogenous treating HTR-8/SVneo cells with mimics,inhibitor or negative control of piR-3127964,respectively.qRT-PCR was also used to detect the relative expression of GNL3L and SPN at mRNA level in placentas of all women.The interactions between GNL3L/SPN and piR-3127964 were analyzed by double luciferase reporter gene detection.The localization of piR-3127964 and SPN in chorionic villi was detected by fluorescence in situ hybridization and immunofluorescence.Transwell assay was performed to analyze the influence of piR-3127964 on the invasion of HTR-8/SVneo cells and the possible mechanism.Independent sample t-test,analysis of variance,and LSD post test were used for analysis Results(1)Enrichment pathways of candidate targets predicted by differentially expressed piR-3127964 were associated with cell motility.There were statistically significant differences in piR-3127964 expression in villi,healthy and preeclamptic placentas(2.950±0.853 vs 1.036±0.303 vs 0.254±0.155,F=27.35,P<0.05),and piR-3127964 was

关 键 词：子痫前期 RNA 小分子干扰 Argonaute蛋白质类 Leukosialin RNA结合蛋白质类 

分 类 号：R714.244[医药卫生—妇产科学]