has-miR-497-5p靶向CCNE1对类风湿性关节炎滑膜细胞增殖的影响

Effects of has-miR-497-5p targeting cyclin E1 on the proliferation of human fibroblast-like synoviocytes-rheumatoid arthritis cells

目的 评价has-miR-497-5p对类风湿性关节炎滑膜细胞(HFLS-RA)增殖的影响。方法 利用miRDB、TargetMiner、TargetScan和miRTarBase数据库预测has-miR-497-5p的作用靶基因,然后利用DAVID数据库对靶基因进行GO分析和KEGG信号通路富集,String数据库对靶基因进行互作分析;以HFLS-RA为研究对象,MTT检测has-miR-497-5p对细胞增殖的影响;流式细胞仪检测has-miR-497-5p对细胞周期的影响;利用qRT-PCR检测has-miR-497-5p对HFLS-RA细胞CCNE1、CCND1、CCND2和CDK6 mRNA的影响;MTT评价has-miR-497-5p在沉默CCNE1后对细胞增殖的影响。结果 生物信息学预测has-miR-497-5p的靶基因共计125个,生物学过程主要集中于胸腺发育、蛋白质磷酸化、细胞凋亡和细胞周期等方面;信号途径主要富集于癌症相关的microRNA、PI3K-Akt途径和细胞周期等;细胞周期中的富集蛋白互作分析显示,CCNE1、CCND1、CCND2和CDK6之间存在相互作用;has-miR-497-5p过表达可以显著抑制细胞增殖(P<0.05),明显抑制细胞周期的S期(P<0.05);qRT-PCR结果显示,has-miR-497-5p模拟剂可以显著降低CCNE1、CCND1、CCND2和CDK6 mRNA表达(P<0.05);沉默CCNE1显著抑制细胞增殖(P<0.05)。结论 has-miR-497-5p可能通过靶向CCNE1抑制HFLS-RA增殖。

Objective To evaluate the effects of has-miR-497-5p on the proliferation of human fibroblast-like synoviocytes-rheumatoid arthritis(HFLS-RA) cells.Methods Databases including miRDB, TargetMiner, TargetScan and miRTarBasewere used to predict the target genes of has-miR-497-5p, DAVID database was used to conduct GO analysis and KEGG signaling pathway enrichment for target genes, and String database was used to conduct interaction analysis for target genes.HFLS-RA cells were used as the research objects. MTT assay was used to explore the impact of has-miR-497-5p on the proliferation of HFLS-RA cells. Flow cytometry was used to detect the effects of has-miR-497-5p on cell cycles. The mRNAlevels of cyclin E1(CCNE1), cyclin D1(CCND1), cyclin D2(CCND2), and cyclin-dependent kinase 6(CDK6) were measured by real-time quantitative reverse transcription PCR(qRT-PCR). MTT assay was used to detect the effect of has-miR-497-5p on cell proliferation after siRNA-mediated CCNE1 silencing in HFLS-RA cells.Results The predicted target genes(total 125 target genes) of has-miR-497-5p were enriched in thymus development, protein phosphorylation, negative regulation of the apoptotic process, cell cycle, etc. The signal pathway was mainly enriched in microRNAs in cancer, PI3K-Akt pathway, cell cycle, etc. The PPI showed that CCNE1, CCND1, CCND2, and CDK6 played a key role in the interaction network. MTT assay showed that has-miR-497-5p could significantly inhibit the proliferation of HFLS-RA cells(P<0.05), and has-miR-497-5p significantly inhibited cell cycles at S phase(P<0.05). And qRT-PCR results showed that has-miR-497-5p significantly decreased the mRNA levels of CCNE1, CCND1, CCND2, and CDK6(P<0.05). CCNE1 knockdown affected the has-miR-497-5p induced inhibition of proliferation(P<0.05).Conclusion Over-expressed has-miR-497-5p in HFLS-RA cells could suppress HFLS-RA cells proliferation by targeting CCNE1.