miR-488-3p靶向调控NSUN2对喉癌细胞恶性生物学行为的影响实验研究OA

Objective:To explore the effects of microRNA-488-3p(miR-488-3p)on the malignant biological be-haviors of laryngeal carcinoma cells through targeting regulation of NOP2/Sun RNA methyltransferase 2(NSUN2).Methods:Human bronchial epithelial cell line BEAS-2B and human laryngeal carcinoma cell lines TU138,TU686 and AMC-HN-8 were selected.RT-qPCR was used to detect the expression levels of miR-488-3p and NSUN2 mRNA in these cells,and the cell line with the most significant changes was chosen for subsequent experiments.TU686 cells were randomly divided into Control group,miR-488-3p overexpression empty plasmid group(miR-NC group),miR-488-3p overexpression group(miR-488-3p mimics group),miR-488-3p mimics+NSUN2 overexpression empty plas-mid group(miR-488-3p mimics+pcDNA-NC group)and miR-488-3p mimics+NSUN2 overexpression group(miR-488-3p mimics+pcDNA-NSUN2 group).Except for the Control group,miR-NC,miR-488-3p mimics,pcDNA-NC and pcDNA-NSUN2 were transfected into TU686 cells of corresponding groups respectively.RT-qPCR was per-formed to detect the expression levels of miR-488-3p and NSUN2 mRNA in each group.CCK-8 assay and EdU stai-ning were adopted to evaluate cell proliferation ability.Transwell assay and wound healing assay were used to detect cell invasion and migration abilities.AO/EB staining and flow cytometry were applied to assess cell apoptosis.West-ern blot was used to measure the expression of proliferation related proteins,epithelial mesenchymal transition relat-ed proteins and NSUN2 protein.Dual-luciferase reporter gene assay was conducted to verify the targeting relationship between miR-488-3p and NSUN2.Thirty male nude mice were randomly assigned into five groups with 6 mice in each group:Model group,miR-NC group,miR-488-3p mimics group,miR-488-3p mimics+pcDNA-NC group,and miR-488-3p mimics+pcDNA-NSUN2 group.Except for the Model group,TU686 cells transfected with correspond-ing plasmids were inoculated into the axilla of nude mice to establish xenograft tumor models;TU686 cell suspension was injected into the axilla of mice in the Model group.After 35 days,the tumor weight and volume among groups were compared.Western blot was used to detect NSUN2 protein expression in tumor tissues.Results:Compared with human bronchial epithelial BEAS-2B cells,the expression of miR-488-3p was decreased while NSUN2 mRNA was in-creased in laryngeal carcinoma TU138,TU686 and AMC-HN-8 cells(all P＜0.05).TU686 cells presented the most significant changes and were selected for follow-up experiments.Compared with the Control group and miR-NC group,TU686 cells in the miR-488-3p mimics group had higher miR-488-3p expression,higher apoptosis rate,as well as lower NSUN2 mRNA level,cell viability,EdU-positive cell rate,number of invasive cells and wound healing rate.Meanwhile,the protein levels of NSUN2,cyclin-dependent kinase 1(CDK1),CyclinD1,zinc finger E-box binding ho-meobox 1(ZEB1)and Snail family transcriptional repressor 1(Snail1)were also decreased(all P＜0.05).In the miR-488-3p mimics+pcDNA-NSUN2 group,the expression of NSUN2 mRNA,cell viability,EdU-positive cell rate,number of invasive cells,wound healing rate and the protein levels of NSUN2,CDK1,Cyclin D1,ZEB1 and Snail1 were elevated,whereas cell apoptosis rate were reduced when compared with the miR-488-3p mimics group and miR-488-3p mimics+pcDNA-NC group(all P＜0.05).The luciferase activity of cells co-transfected with WT-NSUN2 and miR-488-3p mimics was lower than that of cells co-transfected with WT-NSUN2 and miR-NC(P＜0.05).In vi-vo experiments showed that tumor weight,tumor volume and NSUN2 protein expression in tumor tissues were markedly reduced in the miR-488-3p mimics group relative to the Model group and miR-NC group(all P＜0.05).These indicators were increased in the miR-488-3p mimics+pcDNA-NSUN2 group(all P＜0.05).Conclusion:MiR-488-3p may inhibit the proliferation,migration and invasion of laryngeal carcinoma cells as well as the growth of xen-ograft tumors in nude mice,and promote apoptosis by targeting NSUN2.