Studies Of CDK6 Expression And Its Molecular Basis Function In Nasopharyngeal Carcinoma

Background and objectivesNasopharyngeal carcinoma is one of the most common malignant in southern China and Southeast Asia.It is a multi-gene,multi-stage,multi-channel process,the new gene related to nasopharyngeal carcinoma will continue to help us better investigate the pathogenesis of nasopharyngeal carcinoma.Based on the previous study,the cell cycle-dependent kinase CDK6 was selected as the object of this study,and the abnormality of its cell cycle signal pathway was observed by gene chip technique and the results of previous experiments.Based on the above study,this study in order to study the role of CDK6 in nasopharyngeal carcinoma,to understand which miRNA expression and regulation of the pathways related in.Contents and methods1.Detect the expression level of CDK6 in nasopharyngeal carcinoma tissues and cell lines by immunohistochemistry and quantitative real-time PCR(qPCR).The clinical data were analyzed by statistical analysis.2.Construct the interference carrier targeting CDK6,To observe the change of CDK6 expression after inhibition in the ability of cells growth,migration and invasion,through MTT,Transwell chamber to define the function of CDK6 genes in NPC cells.3.Analysis miRNA array expression before and after CDK6 knockdown and Function,the mechanism of authentication by MTT,tranwell,boyden,drug susceptibility test and other methods.Result1.Immunohistochemical results showed that CDK6 were high expression in nasopharyngeal carcinoma and cell lines.The difference was statistically significant(P<0.001)compared with normal nasopharyngeal tissues and cell lines.The results after PCR validation are the same.At the same time,the overall survival rate of patients with CDK6 expression was worse than that of patients without CDK6 expression.2.Silence CDK6 genes influence biology behavior of NPC cellTransient transfection method was used to infect HONE1 and 5-8F cells.Western blot was used to verify the infection efficiency.1)MTT assay was used to detect the proliferation of CDK6 gene in vitro.The results showed that the proliferation of the experimental group was significantly lower than that of the blank control group.The results were statistically significant.2)Transwell migration assay was used to detect the migration ability of nasopharyngeal carcinoma cells after CDK6 knockdown.The results showed that the cell movement ability after CDK6 knockdown was significantly decreased compared with the blank control group.3)The Boyden test was used to detect the invasive ability of nasopharyngeal carcinoma cells after CDK6 gene knockdown.The results showed that the invasion ability of nasopharyngeal carcinoma cells was significantly decreased after CDK6 interference.3.Analysis miRNA array expression before and after CDK6 knockdown and Function,the mechanism of authenticationAfter knockdown the expression of CDK6,the expression of Let-7d,which was the most significant difference,was screened by miRNA expression microarray.1)The proliferation of Let-7d mimics group was significantly lower than that of the control group(P<0.001),and the cell growth of Let-7d inhibitor group was significantly higher than that of the control group(P<0.001),the results were statistically significant.2)Transwell migration assay,compared with the control,Let-7d mimics group to reduce the number of cells through the membrane,Let-7d inhibitor group increased the number of cells through the membrane,the difference was statistically significant(Mimics t = 7.572,P<0.001,Inhibitor t-5.494,P<0.001).3)Boyden invasion assay,compared to the control group,Let-7d mimics group to reduce the number of cells through the membrane,Let-7d inhibitor group increased the number of cells through the membrane,the difference was statistically significant.4)Western blot,cell cycle gene c-myc expression regulated down when Let-7d mimics transfected cells.On the contrary,c-myc expression regulated up.5)The effect of CDK6 and let-7d on the sensitivity of nasopharyngeal carcinoma cells to(5-fu)was detected by MTT assay.It was observed that let-7d was able to increase the sensitivity of NPC cells to 5-Fu.Conclusion:1.CDK6 expression in nasopharyngeal carcinoma was significantly higher than that in normal nasopharyngeal carcinoma tissues,and its positive expression was significantly correlated with patient survival rate.2.CDK6 gene can enhance the ability of nasopharyngeal carcinoma cells in vitro and promote its migration and invasion.3.CDK6 may be negatively correlated with let-7d in miRNA and regulate the expression of let-7d through the c-myc in NPC cells.Results suggested that Let-7d suppressed cell growth by CDK6/c-myc signal in NPC.