| Part 1Expression of miR-34a-5p in nasopharyngeal carcinoma and it relationship on nasopharyngeal carcinoma patientsObjective:To investigate and analyze the expression of miR-34a-5p in nasopharyngeal carcinoma and its relationship with patients with nasopharyngeal carcinoma.Methods:The pathological wax block of patients with nasopharyngeal carcinoma and nasopharyngeal inflammation diagnosed in the First Affiliated Hospital of Guangxi Medical University from March 2010 to May 2012 was collected.105 patients with nasopharyngeal carcinoma(nasopharyngeal carcinoma group)and 30 patients with nasopharynx inflammation(non-tumor group).The follow-up time of the nasopharyngeal carcinoma group was 13-60 months,the median follow-up time was 47 months.qRT-PCR was used to detect the expression of miR-34a-5p in nasopharyngeal carcinoma and non-tumor groups,ROC curves were performed on nasopharyngeal carcinoma and non-tumor groups,and Kaplan-Meier analysis was performed on survival data of nasopharyngeal carcinoma patients.Results:(1)The expression level of miR-34a-5p in the nasopharyngeal carcinoma group was significantly lower than that in the non-tumor group(P<0.001),and the difference was statistically significant.(2)The results of ROC curve of nasopharyngeal carcinoma group and non-tumor group were AUC=0.7894,95%CI=0.6968-0.8821,indicating that miR-34a-5p has good diagnostic efficiency.(3)Kaplan-Meier analysis showed that the survival time of patients with low expression of miR-34a-5p in nasopharyngeal carcinoma was lower than that of patients with high expression of miR-34a-5p(P<0.05).Conclusion:miR-34a-5p is under-expressed in patients with nasopharyngeal carcinoma.miR-34a-5p has the potential to be a tumor marker for the diagnosis and prediction of the prognosis of patients with nasopharyngeal carcinoma.Part 2Effect of miR-34a-5p on the biological function of nasopharyngeal carcinoma cells in vitroObjective:To investigate the effects of miR-34a-5p on the biological function of nasopharyngeal carcinoma cells in vitro.Methods:The nasopharyngeal carcinoma cell lines C666-1 and CNE1 were cultured to logarithmic growth phase.The expression of miR-34a-5p in C666-1 and CNE1 was detected by fluorescence quantitative qRT-PCR.The miR-34a-5p and PCDH were packaged with lentivirus.Transfected C666-1 and CNE1,the transfected cells were divided into CNE1 PCDH control group(group A),CNE1 miR-34a-5p transfection group(group B),C666-1 PCDH control group(group C),C666-1.miR-34a-5p transfection group(group D),qRT-PCR method to detect the expression of miR-34a-5p in the above cells;CCK8 method to detect the proliferation rate of each group,cell colony formation assay to detect the ability of each group of cells to clone;cell invasion test(Transwell)was used to detect the invasive ability of each group;the cell migration assay was used to detect the migration ability of each group;the apoptosis rate of each group was detected by flow cytometry.Results:(1)miR-34a-5p in nasopharyngeal carcinoma cell lines C666-1 and CNE1 was lowly expressed.(2)The expression of miR-34a-5p in miR-34a-5p transfection group(group B and group D)was higher than that in C666-1 and CNE1 cells,and the difference was statistically significant(P<0.05).(3)Cell proliferation rate,cell colony forming ability,cell invasion ability and cell migration ability of group B and group D were lower than Group A and group C,the difference was statistically significant.(4)The apoptosis rate of group B and group D was higher than that of group A and group C,and the difference was statistically significant.Conclusion:The increase of miR-34a-5p expression can promote the apoptosis of nasopharyngeal carcinoma cells and inhibit the proliferation,growth,clonal formation and invasion of nasopharyngeal carcinoma cells,suggesting that miR-34a-5p may play an important role of anti-oncogene in nasopharyngeal carcinoma cells.Part 3Effect of miR-34a-5p on the growth of nasopharyngeal carcinoma xenografts in nude mice in vivoObjective:To investigate the effect of miR-34a-5p on the growth of xenograft tumor in nude mice with nasopharyngeal carcinoma cells in vivo.Methods:CNE-1 was transfected with lentiviral-packed miR-34a-5p and PCDH.Nude mice were randomly divided into C666-1 miR-34a-5p transfection group and C666-1 PCDH control group.The transfected cell suspension was injected into nude.Under the skin of the left axilla,a nude mouse model of nasopharyngeal carcinoma was constructed and the gross morphology,weight and volume of the xenograft tumor were observed.Results:Compared with the C666-1 PCDH control group,the weight and volume of xenograft tumors in the C666-1 miR-34a-5p transfected group were decreased,and the difference was statistically significant.Conclusion:High expression of miR-34a-5p can inhibit the growth of xenograft tumors in nasopharyngeal carcinoma cells in nude mice,suggesting that miR-34a-5p may be an important tumor suppressor in nasopharyngeal carcinoma xenografts in nude mice.Part 4miR-34a-5p target gene prediction and bioinformatics analysisObjective:To predict miR-34a-5p potential target genes and possible signaling pathways via miRNA databases.Methods:miR-34a-5p target genes were predicted by miRanda,miRTarBase,miRDB and TargetScan sites,respectively,and the miR-34a-5p target genes were taken.GO and KEGG enrichment analysis was performed on the target genes in the intersection,and the possible cellular processes,cellular components,molecular functions and potential regulatory pathways were obtained.Results:In the GO analysis,Notch signaling pathway,cellular immunity,apoptosis,cell proliferation,mitosis,DNA replication were enriched.The KEGG analysis is also enriched in signaling pathways such as Notch signaling pathway and miRNAs in cancer.The target gene prediction site was found to have a binding site for miR-34a-5p and NOTCH1.Conclusion:The bioinformatics method was used to infer that the 3' UTR of the NOTCH1 gene contains the predicted binding site of miR-34a-5p.It is suggested that the target gene of miR-34a-5p is likely to be NOTCH1 and regulate cells through the NOTCH signaling pathway.Part 5Molecular mechanism of miR-34a-5p targeting and regulating the inhibitory effect of NOTCH1 on nasopharyngeal carcinomaObjective:To investigate the molecular mechanism of miR-34a-5p targeting the regulation of NOTCH1 on the tumor suppressor effect of nasopharyngeal carcinoma.Methods:The lentiviral-packed miR-34a-5p and PCDH were transfected into C666-1 and CNE1,and the transfected cells were divided into CNE1 PCDH control group(group A)and CNE1 miR-34a-5p transfection group(group B)and C666-1 PCDH control group(C group),C666-1 miR-34a-5p transfection group(D group),the expression of NOTCH1 gene was detected by qRT-PCR,the expression of NOTCH1 and Hes-1 protein was detected by western blot;miR-34a-5p and PCDH were transfected into C666-1.Nude mice were randomly divided into C666-1 miR-34a-5p transfection group and C666-1 PCDH control group.The transfected cell suspension was injected into the left lobe of nude mice to construct xenograft model of nasopharyngeal carcinoma in nude mice.The expression of NOTCH1 gene was detected by qRT-PCR.The expression of NOTCH1 and Hes-1 protein were detected by western blot.Results:(1)Compared with group A and group C(control group),the expression of NOTCH1 gene and the expression of NOTCH1 and Hes-1 protein in group B and group D(miR-34a-5p transfection group)were significantly decreased,and the difference was statistically significant.(2)Compared with the C666-1 PCDH nude mice control group,the Notch1 gene and NOTCH1 and Hes-1 protein levels of C666-1 miR-34a-5p nude mice with high expression of miR-34a-5p were significantly decreased,and the difference was statistically significant.(3)Luciferase reporter experiment results indicate that NOTCH1 is a target gene of miR-34a-5p.Moreover,when the expression level of miR-34a-5p increases,the expression of NOTCH1 will decrease accordingly.Conclusion:miR-34a-5p can inhibit the growth of nasopharyngeal carcinoma.The main molecular mechanism is to reduce the expression of target gene Notch1 and possibly affect the downstream gene of NOTCH pathway. |
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