精母细胞来源外泌体中的piR-1207/2107靶向调控精囊上皮细胞精囊凝胶蛋白1基因表达的体外实验研究  

piR-1207/2107 in exosomes from spermatocyte targeted-regulates expression of seminal vesicle gel protein 1 gene in seminal vesicle epithelial cells

作　　者：邬燕倩 沈鹭 蒋莹 洪叶挺 WU Yan-qian;SHEN Lu;JIANG Ying;HONG Ye-ting(School of Laboratory Medicine and Bioengineering,Hangzhou Medical College,Hangzhou 311300;Key Laboratory of Biomarkers and In Vitro Diagnosis Translation of Zhejiang Province,Hangzhou 310053;The First Affiliated Hospital of Zhejiang Chinese Medical University,Hangzhou 310006)

机构地区：[1]杭州医学院检验医学院生物工程学院,杭州311300 [2]浙江省生物标志物与体外诊断转化重点实验室,杭州310053 [3]浙江中医药大学附属第一医院,杭州310006

出　　处：《生殖医学杂志》2023年第8期1197-1207,共11页Journal of Reproductive Medicine

基　　金：国家自然科学基金资助项目(81801513);浙江省高校基本科研经费(KYZD202003)。

摘　　要：目的探究精母细胞来源外泌体中的piR-1207/2107对精囊上皮细胞(HSVEpiC)精囊凝胶蛋白1(SEMG1)基因表达影响。方法采用超高速离心法提取精母细胞上清液中的外泌体,运用透射电镜、纳米激光粒度分析仪、外泌体表征蛋白鉴定精母细胞来源的外泌体;采用实时荧光定量PCR(qRT-PCR)检测外泌体中piR-1207/2107的表达情况;采用蛋白免疫印迹(Western blot)检测精母细胞来源的外泌体中PIWIL1蛋白的表达情况;通过生物信息学软件RNAhybrid预测piR-1207和piR-2107可共同调控的潜在靶基因。在HSVEpiC中分别转染piR-1207/2107模拟物(piR-NC-mimic、piR-1207-mimic、piR-2107-mimic)或piR-1207/2107抑制剂(Anti-piR-NC、Anti-piR-1207、Anti-piR-2107),Western blot检测转染后各组SEMG1蛋白的表达情况。在精母细胞中分别转染Anti-piR-NC、Anti-piR-1207、Anti-piR-2107,提取外泌体与HSVEpiC共培养24 h,采用qRT-PCR检测各组piR-1207/2107的表达情况,并采用Western blot检测SEMG1蛋白的表达情况。结果透射电子显微镜结果显示,精母细胞来源外泌体呈圆形或椭圆形膜性小囊泡;纳米激光粒度分析仪检测结果显示,外泌体的直径主要分布在100~200 nm之间。qRT-PCR检测结果显示,精母细胞来源的外泌体含有piR-1207/2107;Western blot检测结果显示,精母细胞来源的外泌体中未检测到PIWIL1蛋白。应用生物信息学软件RNAhybrid预测结果显示,piR-1207和piR-2107可以共同靶向结合SEMG1 mRNA的CDS区。HSVEpiC转染piR-1207/2107模拟物后,与piR-NC相比,piR-1207/2107表达量显著升高(P<0.001),而SEMG1蛋白表达水平显著降低(P<0.01);HSVEpiC转染piR-1207/2107抑制剂后,与Anti-piR-NC相比,piR-1207/2107表达量显著降低(P<0.001),而SEMG1蛋白表达水平显著升高(P<0.05)。精母细胞转染Anti-piRNAs后提取外泌体与HSVEpiC共培养,piR-1207/2107表达量显著降低,而SEMG1蛋白表达水平显著增加(P<0.05)。结论精母细胞来源外泌体中的piR-1207/2107可�Objective:To investigate the effect of piR-1207/2107 in exosomes from spermatocyte[GC-2spd(ts)]on seminal vesicle gel protein 1(SEMG1)gene expression in seminal vesicle epithelial cells(HSVEpiC).Methods:The exosomes were extracted from the supernatant of GC-2spd(ts)by ultrahigh-speed centrifugation.The exosomes from GC-2spd(ts)were identified by transmission electron microscopy,nano-laser particle size analyzer and exosome characterization protein.The expression of piR-1207/2107 was detected by qRT-PCR,and the expression of PIWIL1 protein in exosomes was detected and Western blot.Bioinformatics software RNAhybrid was used to predict the potential target genes co-regulated by piR-1207 and piR-2107.The piR-1207/2107 analogs(piR-NC-mimic,piR-1207-mimic,piR-2107-mimic)or piR-1207/2107 inhibitors(Anti-piR-NC,Anti-piR-1207,Anti-piR-2107)were transfected to HSVEpiC.SEMG1 protein expression in each group after transfection were detected by Western blot.GC-2spd(ts)were transfected with Anti-piR-NC,Anti-piR-1207 and Anti-piR-2107,and the exosomes were extracted and co-cultured with HSVEpiC for 24 hours.The expression of piR-1207/2107 was detected by qRT-PCR,and the expression of SEMG1 protein was detected by Western blot.Results:The results of transmission electron microscopy showed that the exocrine bodies of GC-2spd(ts)were round or oval membranous vesicles.The results of nano laser particle size analyzer showed that the diameter of exosomes mainly distributed between 100-200 nm.The results of qRT-PCR showed that the exosome from GC-2spd(ts)contained piR-1207/2107.Western blot analysis showed that PIWIL1 protein was not detected in the exosomes from GC-2spd(ts).The results of bioinformatics software RNAhybrid showed that piR-1207 and piR-2107 jointly targeted the CDS region of SEMG1 mRNA.After transfection of piR-1207/2107 analogs to HSVEpiC,the expression level of piR-1207/2107 was significantly increased(P<0.001),while the expression of SEMG1 protein was significantly decreased compared with that of piR-NC(P<0.01).Afte

关 键 词：精母细胞 外泌体 PIRNA 精囊上皮细胞 精囊凝胶蛋白1 

分 类 号：R339.21[医药卫生—人体生理学]