Fus依赖ac4C修饰调控促进宫颈癌细胞增殖  

Fus promotes cell proliferation by ac~4C-modification in cervical cancer cells

作　　者：金泽源 李咏梅 JIN Ze-yuan;LI Yong-mei(Department of Pathogenic Biology,School of Basic Medical Sciences,Tianjin Medical University,Tianjin 300070,China)

机构地区：[1]天津医科大学基础医学院病原生物学系,天津300070

出　　处：《天津医科大学学报》2023年第1期15-20,共6页Journal of Tianjin Medical University

摘　　要：目的:探讨Fus RNA结合蛋白(Fus)影响宫颈癌细胞增殖的机制。方法:通过MTT实验观察Fus对宫颈癌细胞HeLa增殖的影响;采用RT-qPCR和Western印迹观察人N-乙酰转移酶10(NAT10)对Fus表达的影响;利用RNA免疫沉淀实验(RIP)检测Fus mRNA的富集情况;通过RNA稳定性实验观察NAT10对Fus mRNA稳定性的影响;基于生物信息学软件PACES预测Fus mRNA上的ac4C修饰位点,构建含有Fus mRNA上ac4C修饰位点的野生型和突变型的EGFP报告质粒,利用EGFP-reporter实验和乙酰化RIP(ac4C-RIP)确定Fus mRNA上发生ac4C修饰位点;最后通过挽救实验观察NAT10介导Fus对宫颈癌细胞增殖的影响。结果:Fus促进宫颈癌HeLa细胞增殖(tFus=14.06,tsh R-Fus=5.74,均P<0.05);Fus蛋白和mRNA的表达水平随NAT10表达增加而增加(tNAT10=10.71,tsh R-NAT10-1=13.17,tsh R-NAT10-2=7.65,均P<0.05);RIP实验结果提示Fus mRNA与NAT10蛋白间存在相互作用(t=4.20,P<0.05),且NAT10过表达细胞中Fus mRNA具有较高ac4C修饰水平(t=30.62,P<0.05);另外,相对于载体对照细胞,NAT10过表达细胞中Fus mRNA的稳定性增高(P<0.01);发现Fus mRNA的925~939序列中的胞嘧啶为ac4C修饰位点;挽救实验结果显示NAT10介导Fus促进宫颈癌细胞的增殖(t=9.67、6.71,P<0.05)。结论:Fus mRNA被NAT10催化发生ac4C修饰,Fus表达水平被上调,继而促进宫颈癌细胞的增殖。Objective:To explore the mechanism of Fus RNA binding protein(Fus)in the promotion of cervical cancer cell proliferation.Methods:The effects of Fus on cervical cancer cell proliferation were observed using MTT assays.Western blotting analysis and RT-q PCR were used to detect the influence of human N-acetyltransferase 10(NAT10) on expression levels of Fus.RNA immunoprecipitation assay(RIP)was detected the enrichment of Fus mRNA.RNA half-life assay was conducted to determine the effect of NAT10 on the stability of Fus mRNA.Based on the searching results of the possible ac~4C-modification sites on Fus mRNA through PACES bioinformatics software,the EGFP reporter plasmids containing the possible wild-type or mutant ac~4C-modification sites were constructed.EGFP-reporter assay and ac~4C-RIP assay were used for the identification of ac~4C modification sites on Fus mRNA.Lastly,the effect of NAT10 mediated Fus on the proliferation of cervical cancer cells was observed by rescue experiment.Results:Fus promoted cell proliferation of HeLa cells (tFus=14.06,tsh R-Fus=5.74,all P<0.05).The expression level of Fus protein and mRNA was increased with NAT10 expression(tNAT10=10.71,tsh R-NAT10-1=13.17,tsh R-NAT10-2=7.65,all P<0.05).The results of RIP experiment indicated that there was an interaction between Fus mRNA and NAT10 protein(t=4.20,P<0.05),and Fus mRNA in NAT10 overexpression cells had a higher ac~4C modification level(t=30.62,P<0.05).In addition,the stability of Fus mRNA in NAT10 overexpression cells was higher than that in vector control cells(P<0.01)Furthermore,cytosine in 925-939 sequence of Fus mRNA was ac~4C modified site.The rescue experiment results showed that NAT10 mediated Fus to promote the proliferation of cervical cancer cells (t=9.67,6.71,both P<0.05).Conclusion:Fus mRNA can be ac~4C modificated by NAT10,which resulting in the enhancement of Fus expression and the promotion of cell proliferation in cervical cancer cells.

关 键 词：宫颈癌 ac4C FUS NAT10 

分 类 号：R737.33[医药卫生—肿瘤]