| Backgroud:Nasopharyngeal carcinoma(NPC)is mainly originated from nasopharyngeal epithelium.It mainly occurs in Southeast Asia and China’s Guangdong and Guangxi regions.The occurrence and development of nasopharyngeal carcinoma involves the activation of several oncogenes and the inactivation of tumor suppressor genes.The imbalance of their interaction is the molecular basis of the occurrence and development of nasopharyngeal carcinoma.MiRNAs participate in a variety of signaling pathways and regulate important biological processes by regulating target genes,such as cell proliferation,metastasis,apoptosis,aging,differentiation,autophagy,immune response,etc.In the last few years,plenty of studies have found that miR-22 as a tumor suppressor is involved in the occurrence and development of a variety of tumors,such as lung cancer,breast cancer,gastric cancer and other malignant tumors.However,the expression of miR-22 in nasopharyngeal carcinoma and its effect on nasopharyngeal carcinoma cells have not been reported,Therefore,this study will detect the expression level of miR-22 in nasopharyngeal carcinoma and its effect on the cell multiplication,migration and apoptosis of nasopharyngeal carcinoma cells,and use bioinformatics to analyze the target genes of miR-22 and the signal pathways it involved,and preliminarily explore its possible mechanism in nasopharyngeal carcinoma,so as to provide valuable molecular markers for nasopharyngeal carcinoma.MethodsDetection and analysis of miR-22 expression in different nasopharyngeal carcinoma cells and normal nasopharyngeal epithelial cells by q RT-PCR;the micro RNA expression dataset GSE32960 of nasopharyngeal carcinoma was screened from the public database,and the differential expression of miR-22 was analyzed by R language limma package.LV-miR-22-up was transfected into nasopharyngeal carcinoma cell line HK-1.QRT-PCR was used to detect the effect of exogenous up-regulation of miR-22 expression.Transwell migration assay,wound healing assay,CCK-8 proliferation assay and flow cytometry were used to observe the changes of migration,multiplication and apoptosis of nasopharyngeal carcinoma cells after upregulation of miR-22;LV-miR-22-down was transfected into nasopharyngeal carcinoma cell line HK-1.The effect of exogenous down-regulation of miR-22 expression was detected by q RT-PCR.Transwell migration assay,wound healing assay,CCK-8proliferation assay and flow cytometry were used to observe the changes of migration,proliferation and apoptosis of nasopharyngeal carcinoma cells after down-regulation of miR-22;Mir DB,targetscan and micro T-CDs databases were used to predict the possible target genes of miR-22.The target genes predicted by the three databases were selected as follow-up research objects.The target genes were annotated by David website for GO function annotation and KEGG signal pathway enrichment analysis,and then the protein interaction network diagram of target genes was constructed in STRING database.The top 50 of the key target proteins with node centrality were screened out.ResultCompared with NP69,the relative expression levels of miR-22 in NPC cell lines were S26(1.688 ± 0.623),5-8F(0.544 ± 0.131),6-10B(0.213 ± 0.200)and HK-1(0.020 ± 0.008),respectively.The expression levels of miR-22 in 5-8F,6-10 B and HK-1 were significantly lower(P<0.05).In the GSE32960 data set,miR-22 expression was decreased in nasopharyngeal carcinoma tissues(P<0.01).Over-expression lentivirus of miR-22(LV-miR-22-up)and its negative control(LV-NC)were transfected into HK-1 cell line.The proportion of fluorescent cells was more than 90% under microscope.Compared with LV-NC group and blank group,the relative expression level of miR-22 in LV-miR-22-up group was significantly increased(P<0.05).Transwell migration assay and wound healing assay signified that up-regulation of miR-22 could significantly inhibit the migration ability of nasopharyngeal carcinoma cells(P<0.01).CCK-8 test showed that up-regulation of miR-22 could significantly inhibit the proliferation ability of nasopharyngeal carcinoma cells(P<0.01).Flow cytometry showed that upregulation of miR-22 could promote the apoptosis of nasopharyngeal carcinoma cells(P<0.01).Low-expression lentivirus of miR-22(LV-miR-22-up)and LV-NC were transfected into HK-1 cell line.The proportion of fluorescent cells was more than90% under microscope.Compared with LV-NC group and blank group,the relative expression level of miR-22 in LV-miR-22-down group was significantly decreased(P<0.05).Transwell migration assay and wound healing assay signified that down-regulation of miR-22 could significantly inhibit the migration ability of nasopharyngeal carcinoma cells(P<0.01).CCK-8 test showed that downregulation of miR-22 could significantly promote the proliferation ability of nasopharyngeal carcinoma cells(P<0.01).Flow cytometry showed that downregulation of miR-22 could inhibit the apoptosis of nasopharyngeal carcinoma cells(P<0.01).A total of 210 target genes were obtained from the intersection of the predicted results of the three databases.The go function annotation results of the target genes showed that the target genes of miR-22 were mainly involved in the regulation of cellular process,regulation of metabolic process and regulation of biosynthetic process;they were mainly located in organelles and intracellular organelle;they played the functions of protein binding,DNA binding and kinase activity.KEGG pathway analysis showed that the target genes involved in the signal pathway mainly included endocytosis pathway and cancer pathway.The target genes which included in cancer pathway were MAX,NRAS,TGFBR1,CBL,LAMC1,MECOM,PTEN,AKT3,CSF1 R and TPM3.String database analysis showed that proteins encoded by key target genes such as PTEN,SIRT1 and CSF1R played a key role.Conclusion1.MiR-22 was low expressed in nasopharyngeal carcinoma.2.MiR-22 plays a tumor suppressor role in nasopharyngeal carcinoma.3.MiR-22 may be involved in the occurrence and development of tumor by targeting PTEN,SIRT1,CSF1 R and other target genes. |
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