重组多杀性巴氏杆菌毒素介导FGFRL1诱导Hela细胞增殖研究
Study of Recombinant Pasteurella multocida Toxin-mediated FGFRL1-induced Proliferation in Hela Cells
林忠森 1赵勤 1袁建林 1伍锐 1颜其贵 1文翼平 1黄小波 1杜森焱 1曹三杰1
作者信息
- 1. 四川农业大学动物医学院/猪病研究中心/国家级动物类实验教学示范中心/农业农村部兽用药物与兽医诊断技术四川科学观测站,成都 611130
- 折叠
摘要
成纤维细胞生长因子受体样因子1(fibroblast growth factor receptor-like 1,FGFRL1)作为成纤维细胞生长因子受体家族成员,在调控细胞增殖、分化上起作用.多杀性巴氏杆菌毒素(Pasteurella multocida toxin,PMT)是多杀性巴氏杆菌(P.multocida,Pm)产生的重要毒力因子,属于一种促有丝分裂原蛋白,在多种细胞上引起细胞增殖,导致组织增生、占位,使其他正常组织细胞生存受限而死亡,但目前PMT引起促增殖作用的机制以及介导的因子均不清晰.前期CRISPR/Cas9全基因文库筛选发现FGFRL1提高了重组PMT(recombinant PMT,rPMT)蛋白染毒处理的Hela细胞存活率.为探究PMT在Hela细胞上的促增殖作用是否通过FGFRL1介导,本研究分别使用0、0.01、0.1、1.0 μg/mL的rPMT蛋白处理正常及FGFRL1基因缺失的Hela细胞各48 h,CCK-8(Cell Counting Kit-8)法检测细胞增殖率、EdU-488标记法检测细胞DNA复制情况、划痕试验法检测增殖细胞的迁移率.结果显示,在rPMT浓度为0.1 μg/mL处理48h时,缺失FGFRL1的Hela细胞增殖显著下降(P＜0.05),而划痕恢复率与正常Hela细胞相比下降接近70%(P＜0.05).说明FGFRL1的缺失减弱了rPMT对Hela的促增殖作用.利用实时荧光定量PCR检测rPMT处理后Hela细胞磷脂酰肌醇3激酶/丝氨酸-苏氨酸蛋白激酶(phosphatidylinositol 3 kinase/serine-threonine protein kinase,PI3K-AKT)通路上游因子变化水平、Western blot检测该通路关键激活因子AKT的磷酸化水平进行初步验证.结果显示:rPMT处理后PI3K-AKT通路上游因子转录水平显著升高(P＜0.05),缺失FGFRL1的Hela细胞AKT与磷酸化AKT水平极显著低于正常Hela细胞(P＜0.01),说明PI3K-AKT通路激活受到抑制.由此可以初步确认PMT可能通过FGFRL1靶向PI3K-AKT通路引起Hela细胞增殖.本研究丰富了PMT促增殖作用的理论基础,初步探索了PMT引起细胞增殖的通路激活方式,为进一步揭示PMT的促增殖作用提供参考.
Abstract
Fibroblast growth factor receptor-like 1(FGFRL1),as a member of the fibroblast growth factor receptor family,plays a role in the regulation of cell proliferation and differentiation.Pasteurella multocida toxin(PMT)is an important virulence factor produced by P.multocida(Pm),which belongs to mitogenic protein,and causes cell proliferation on a variety of cells,leading to tissue proliferation,occupancy,and causing impair growth and death of other normal tissue cells.However,the mechanism and the factor that PMT induce a pro-proliferative effect,are currently unclear.Previous CRISPR/Cas9 whole gene library screen revealed that FGFRL1 increased the survival rate of recombinant PMT protein(rPMT)treated Hela cells.In order to investigate whether PMT affects the proliferation of Hela cells through FGFRL1 in the PI3K-AKT pathway,in the present study,normal and FGFRL1-knockout Hela cells were treated with rPMT at 0,0.01,0.1,and 1.0 μg/ml for 48 h.The cell proliferation rate was detected by Cell Counting Kit-8(CCK-8)assay,cell DNA replication by EdU-488 labeling assay,and the cell migration rate by scratch assay,The results showed that cell proliferation in the FGFRL1 knockout Hela cell was significantly decreased(P＜0.05)when treated with rPMT at a concentration of 0.1 μg/mL for 48 h,while the rate of scratch recovery was decreased by nearly 70%compared with that of normal Hela cell(P＜0.05).It indicated that the absence of FGFRL1 diminished the pro-proliferative effect of rPMT on Hela cell.Initial validation of the pathway was performed using qPCR to detect changes in the transcriptional levels of factors upstream of the phosphatidylinositol 3 kinase/serine-threonine protein kinase(PI3K-AKT)pathway in Hela cells after rPMT treatment,and phosphorylation levels of AKT,a key activator of the pathway,were detected by western blot..The results showed that the upstream factors of PI3K-AKT pathway were extremely significantly elevated after rPMT treatment(P＜0.05),and the levels of AKT and phosphorylated AKT in the FGFRL1 knockout Hela cell were significantly lower than those in the normal Hela cells(P＜0.01),which indicated that the activation of the PI3K-AKT pathway was inhibited.Thus,it could be preliminarily confirmed that PMT might cause Hela cell proliferation by targeting the PI3K-AKT pathway through FGFRL1.This study enriches the theoretical basis of the PMT's proliferation-promoting effect,preliminarily explores the pathway activation mode of PMT-induced cell proliferation,and provides information for further revealing the PMT's proliferation-promoting effect.
关键词
成纤维细胞生长因子受体样因子1(FGFRL1)/重组多杀性巴氏杆菌毒素(rPMT)/Hela细胞/细胞增殖/PI3K-AKT通路Key words
Fibroblast growth factor receptor-like 1(FGFRL1)/Recombinant Pasteurella multocida toxin(rPMT)/Hela cells/Cell proliferation/PI3K-AKT pathway引用本文复制引用
基金项目
四川省"十四五"川猪重大科技专项(2021ZDZX0010)
中央引导地方科技发展专项(2021ZYD0086)
出版年
2024
国家科技期刊平台
NSTL
万方数据