The Significance Of NMNAT2 Protein Expression And The Research Of Its Regulatory Factors In Colorectal Cancer

ContentThe whole project includes two parts:Part I Expression and clinical significance of NMNAT2 in colorectal cancerObjectivesTo explore the expressions of NMNAT2 and its clinical significance in human colorectal cancerMethods:The CRC tissues and adjacent normal tissues were obtained from 95 colorectal cancer patients which met the inclusion criterias.All the tissues were fixed in formalin,embedded in paraffin and then were processed to sections.Both the CRC and adjacent normal tissues were sections stained with immunohistochemistry(IHC)for the location and expression of nicotinamide mononucleotide adenylyl transferase 2(NMNAT2).Collecting,recording,then analyzing the clinical information of the patients for exploring the relationship between NMNAT2 expression and CRC progression.The follow-up information and survival rate was analyzed by the Kaplan-Meier method and the log-rank test.The multivariate Cox proportional hazard regression analysis was conducted as well to estimate if NMNAT2 could influence prognosis and survival rate of the CRC patients.Results:1?NMNAT2 protein expression was localized primarily in cytoplasm.Compared to adjacent normal tissues,the NMNAT2 was significantly upregulated in CRC tissues.2?The expression level of NMNAT2 protein was increasing with degree of tumor invasion,TNM staging progress expression,but it had nothing to do with the other clinical parameters such as gender,tumor size and so on.3.The expression level of NMNAT2 was not a statistically significant influence factor for postoperative survival rate.According to the univariate analysis,the independent risk factors for survival time were age,average diameter,morphology,differentiation,depth of invasion and TNM stages.Moreover,average diameter,morphology and TNM stages were demonstrated to be the combined factors by multivariate analysis.However,the expression of NMNAT2 had no significant effect on survival time.Conclusion:1.The specificity expression of NMNAT2 protein was involved in the development of CRC.The NMNAT2 might be a new oncogene for CRC,and it could be a promising therapeutic and diagnostic target point.2.The expression level of NMNAT2 was neither a statistically significant influence factor for postoperative survival rate nor a independent risk factor for survival time.NMNAT2 could not help to predict the prognosis or survival time of the CRC patients.Part ? The correlation of NMNAT2 and SIRT6 expression in colorectal cancer and its clinical significanceObjectives:Explore expression level of SIRT6 in colorectal cancer,and whether SIRT6 protein affected the progress of the colorectal by regulating the expression of NMNAT2.Methods:The fresh CRC tissues and adjacent normal tissues were obtained from 29 colorectal cancer patients which met the inclusion criterias.Detect the expression of Suituins 6(SIRT6)protein and nicotinamide mononucleotide adenylyl transferase 2(NMNAT2)protein in the tissues by Western blotting.The expression of SIRT6 and NMNAT2 mRNA was tested by RT-qPCR.Analyze specific expression of SIRT6 protein and mRNA in colorectal cancer tissues and its relevance to NMNAT2.Another 113(including the former 29 cases)CRC tissues and adjacent normal tissues were enrolled to the trial as well.All the tissues were fixed in formalin,embedded in paraffin and then were processed to sections.Both the CRC and adjacent normal tissues were sections stained with immunohistochemistry(IHC)for the location and expression of SIRT6 and NMNAT2.Then the association of the expression level between NMNAT2 and p53 was assessed by Spearman nonparametric correlation.Collecting,recording,then analyzing the clinical information of the patients for exploring the relationship among SIRT6 expression,NMNAT2 expression and CRC progression.Explore whether SIRT6 protein affected the progress of the colorectal by regulating the expression of NMNAT2 and its clinical significances.Results:1.According to the Western blotting results,the expression levels of SIRT6 protein were significantly reduced in CRC tissues compared to the adjacent normal tissues;whereas NMNAT2 protein were significantly increased in CRC tissues.2.Immunohistochemical results indicated that SIRT6 protein expression was localized primarily in CRC nuclear,and the expression of it in CRC tissues were significantly lower than the adjacent normal tissues?3.Compared to the adjacent normal tissues,SIRT6 mRNA were reduced in CRC tissues.On the contrary,the expression of NMNAT2 mRNA in CRC tissues were more than the adjacent normal tissues.4.Both the histograms of SIRT6,NMNAT2 protein expression and mRNA expression showed an obvious opposite trend.And the immunohistochemistry confirmed a negative correlation between the expression of SIRT6(reduced)and NMNAT2(increased)in CRC tissue samples(r=-0.246).5.Expression of SIRT6 protein in CRC tissues was positively correlated with the depth of tumor invasion,tumor differentiation(grade),lymph node metastasis,and TNM stage.but it had nothing to do with the other clinical parameters such as gender,tumor size and so on.Conclusion:1.The absence of SIRT6 gene expression was involved in the process of Colorectal cancer multiple genetic changes.The expression level of SIRT6 protein could be a reference index of the CRC progression stage and prognosis of the patients.2.A negative correlation between the expression of SIRT6(reduced)and NMNAT2(increased)in CRC tissue samples,and both of them are proved to involve in the progression of CRC.SIRT6 may be one of the control factors of NMNAT2 in CRC progression.It could be a new pathway to the study NMNAT2 role in occurrence and development of CRC.