精原细胞中泛素化修饰信号通路探究及其与生殖能力的关系
Study on ubiquitination modification signal pathway in spermatogonia and its relationship with reproductive ability
邹璇 1李进 1蒋韬1
作者信息
- 1. 随州市中心医院(湖北医药学院附属随州医院)生殖医学科(湖北随州 441300)
- 折叠
摘要
目的 探讨环指蛋白216(ring finger protein 216,RNF216)在精原细胞中表达和功能的分子机制.方法 将靶向RNF216的siRNA和pcDNA3.1-Flag-RNF216(RNF216过表达质粒)转染小鼠精原细胞系(GC-1细胞),以敲低或上调细胞中RNF216表达.通过细胞计数试剂盒8(cell counting kit-8,CCK-8)和结晶紫细胞增殖试验测定细胞增殖情况,Transwell实验测定细胞迁移能力,蛋白质印迹检测RNF216和Toll样受体4(toll-like receptor 4,TLR4)蛋白表达.结果 与si-NC组相比,si-RNF216#1组和si-RNF216#2组细胞克隆数和迁移数均显著减少(P＜0.001).与NC组相比,RNF216组GC-1细胞克隆数和迁移数均显著增加(P＜0.001).在用蛋白酶体抑制剂MG132处理的细胞中,RNF216过表达显著增加TLR4多泛素化(P＜0.05).与RNF216组相比,RNF216+TLR4组GC-1细胞活力、克隆数和迁移数均显著降低(P＜0.05).结论 RNF216通过K48-连接的多泛素化降解TLR4来促进GC-1细胞迁移和增殖.
Abstract
Objective To explore the molecular mechanisms underlying the expression and function of ring finger protein 216(RNF216)in spermatogonia.Methods siRNA targeting RNF216 and pcDNA3.1-Flag-RNF216(RNF216 overexpression plasmid)were transfected into mouse spermatogonial cell line(GC-1 cell)to knock down or up-regulate RNF216 expression in cells.Cell proliferation was determined by cell counting kit 8(CCK-8)and crystal violet cell proliferation test,and cell migration ability was determined by Transwell test.The expression of RNF216 and toll-like receptor 4(TLR4)was detected by protein blot.Results Compared with si-NC group,the number of cell clones and migration in si-RNF216#l group and si-RNF216#2 group decreased significantly(P＜0.001).Compared with NC group,the number of clones and migration of GC-1 cells in RNF216 group increased significantly(P＜0.001).In cells treated with proteasome inhibitor MG132,the overexpression of RNF216 significantly increased the ubiquitination of TLR4(P＜0.05).Compared with RNF216 group,RNF216+TLR4 group GC-1 cells significantly decreased in cell viability,clone number and migration number(P＜0.05).Conclusions RNF216 can promote the migration and proliferation of GC-1 cells by degrading TLR4 through K48-linked ubiquitination.
关键词
环指蛋白216/精原细胞/泛素化/细胞运动/细胞增殖Key words
ring finger protein 216/spermatogonia/ubiquitination/cell movement/cell proliferation引用本文复制引用
基金项目
湖北省卫生健康委项目(W2021F084)
随州市卫生健康委科研项目(2018SZ32008)
出版年
2024
NETL
NSTL
万方数据