miR-145-5p对口腔鳞癌细胞增殖的调控作用及其机制

Regulation of miR-145-5p on the Proliferation of Oral Squamous Cell Carcinoma Cells and Its Mechanism

目的 探究miR-145-5p对口腔鳞状细胞癌(OSCC)细胞增殖的调控作用及其机制。方法 利用GEO数据库构建OSCC的miRNA差异表达谱;通过生物信息学技术预测miR-145-5p靶基因并对其进行GO、KEGG及PPI分析;以人OSCC细胞株(HSC-3)为实验对象,将实验分为CN组(未做处理)、NC组(转染mimic NC)和miR-145-5p组(转染miR-145-5p mimic),通过CCK-8法检测不同剂量(50、100、200 nmol/L)miR-145-5p对HSC-3细胞增殖的影响;JC-1和Annexin V-FITC/PI染色结合流式细胞术检测miR-145-5p对HSC-3细胞的凋亡诱导情况;RT-qPCR检测miR-145-5p调控神经母细胞瘤鼠肉瘤同系物(NRAS)、C-JUN氨基端激酶(C-JUN)、信号转导和转录激活因子1(STAT1)的表达情况。结果 miRNA差异表达谱显示,miR-145-5p在OSCC中表达下调(P<0.05);GO和KEGG分析显示,miR-145-5p靶基因主要调控MAPK信号通路;PPI蛋白互作显示,NRAS是miR-145-5p的关键靶基因之一;CCK-8结果显示,50、100、200 nmol/L的miR-145-5p转染HSC-3细胞后,细胞活力较CN组降低(P<0.05);JC-1染色结果显示,miR-145-5p组HSC-3细胞线粒体膜电位低于CN组(P<0.05);Annexin V-FITC/PI染色结果显示,miR-145-5p组HSC-3细胞凋亡率较CN组明显增加(P<0.05);RT-qPCR结果显示,与CN组比较,miR-145-5p组NRAS表达降低(P<0.05),而MAPK信号通路关键转录因子C-JUN和STAT1表达升高(P<0.05)。结论 miR-145-5p可通过靶向NRAS,并经过MAPK信号通路诱导细胞凋亡,从而抑制OSCC细胞增殖。

Objective To explore the regulation of miR-145-5p on the cell proliferation in oral squamous cell carcinoma(OSCC)and its mechanism.Methods The miRNA differential expression profile of OSCC was constructed using Gene Expression Omnibus(GEO)database.The target genes of miR-145-5p were predicted by bioinformatics and analyzed by using Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG),and Protein-Protein Interaction(PPI)networks.Human OSCC cell line(HSC-3)was used as the experimental object.The HSC-3 cells were divided into CN group(untreated),NC group(transfected with mimic NC),and miR-145-5p group(transfected with miR-145-5p mimic).The proliferation inhibition of different doses of miR-145-5p(50,100,and 200 nmol/L)on HSC-3 cells was detected by CCK-8 assay.The apoptosis of HSC-3 cells induced by miR-145-5p was detected by JC-1 and Annexin V-FITC/PI staining combined with flow cytometry.RT-qPCR was used to detect the expressions of neuroblastoma ratsarcoma homolog(NRAS),C-JUN amino-terminal kinase(C-JUN),and signal transduction and activator of transcription 1(STAT1)regulated by miR-145-5p.Results The differential expression profile of miRNA showed that the expression of miR-145-5p was down-regulated in OSCC(P<0.05).GO and KEGG analysis showed that the target genes of miR-145-5p mainly regulated signaling pathways such as MAPK.PPI analysis showed that NRAS was one of the key target genes of miR-145-5p.CCK-8 results showed that the cell viability of HSC-3 cells transfected with 50,100,and 200 nmol/L of miR-145-5p decreased compared with that of CN group(P<0.05).The results of JC-1 staining showed that the mitochondrial membrane potential of HSC-3 cells in miR-145-5p group was lower than that in CN group(P<0.05).Annexin V-FITC/PI staining showed that the apoptosis ratio of HSC-3 cells in miR-145-5p group was significantly higher than that in CN group(P<0.05).RT-qPCR results showed that compared with CN group,miR-145-5p group exhibited decreased expression o f NRAS(P<0.05),but increased expressions of key transcription factors C-JUN and STAT1 in MAPK signaling pathway(P<0.05).Conclusion miR-145-5p may induce apoptosis through MAPK signaling pathway by targeting NRAS,thereby inhibiting OSCC cell proliferation.