Expression Of CHDH In Colorectal Cancer And Its Impact On Prognosis

Objective:Colorectal cancer(CRC)is the malignant tumor with the second incidence rate and the fourth mortality in China,and the incidence is gradually younger.Meanwhile,there is still a lack of more obvious and effective targets for treatment.At present,with the rapid development of bioinformatics and high-throughput sequencing,it provides a foundation for the clinical application of genomic data.CHDH is a protein coding gene.The variation of this gene will affect the susceptibility to choline deficiency.Previous studies have shown that the expression of CHDH is associated with an increased risk of breast cancer.However,the impact of CHDH on the prognosis of CRC patients is still unclear.This study explored the expression of CHDH in CRC tissues and cells and its correlation with prognosis through public databases and clinical experiments.To provide individualized prognosis assessment for CRC patients,we further constructed nomograms.Methods:1.Bioinformatics analysis:The TIMER database was used to analyze the differential expression of CHDH in pan-cancer.The Kaplan-Meier survival analysis was performed in CRC.Furthermore,the multivariate COX regression analysis was carried out in combination with clinical pathological parameters.At the same time,the database was used to explore the correlation between the expression of CHDH in CRC and immune cells.Through Bio Portal database,we further study the type,frequency and location of CHDH gene mutation in CRC and conducted gene signaling pathway analysis providing theoretical basis for its research in CRC.2.Basic experimental verification:We investigated the expression of CHDH at the m RNA level in different CRC cell lines and normal colon cell lines by RT-qPCR,and further detected the expression of CHDH m RNA in CRC tissues and their matched paracancerous tissues of 106 patients.We explored whether there was any difference in the expression of CHDH in tumor tissues and paracancerous tissues.The relative expression was further verified at the protein level by Western Blotting(WB)and Immunohistochemistry staining(IHC).3.Clinical analysis and validation:Basic patients’information and clinico-pathological parameters were collected through the medical record system of Wujin Hospital affiliated to Jiangsu University.Patients’follow-up was conducted by telephone,outpatient follow-up or hospitalization.The patients were divided into CHDH high-and low-expression groups by plotting the receiver operating characteristic curve(ROC)and its calculated cut-off values.And,the correlation between CHDH high and low expression and clinicopathological parameters was investigated byX~2 testing.The survival analysis curves of CHDH high-and low-expression groups were drawn by Kaplan-Meier method.Meanwhile,to explore the independent prognostic factors affecting CRC,COX risk regression models with single and multiple factors were used.Patients were randomly divided into training and validation sets and we explored whether there were group differences between the training and validation sets.The components of the nomogram were independent prognostic factors for CRC patients.The discrimination and calibration of the model were assessed by ROC curves and calibration curves.The effect of CHDH on the migration ability of colon cancer cells was explored through scratch experiments.Results:1.The results of TIMER public database showed that the expression level of CHDH in colorectal adenocarcinoma,bladder cancer,hepatocellular carcinoma,prostate cancer,gastric adenocarcinoma,breast cancer,head and neck squamous cell carcinoma,kidney cancer and other cancers was different between adjacent tissues and cancer tissues(P<0.05).According to the results of Kaplan-Meier survival analysis,CRC patients’overall survival(OS)were not significantly different between the high and low expression groups of CHDH(P>0.05).The results of COX regression analysis demonstrated that age and TNM stage were independent prognostic factors in colon cancer.And age was an independent prognostic factor in rectal cancer.The results of immune infiltration analysis showed that the relative expression of CHDH in colon adenocarcinoma(COAD)was positively correlated with the estimated infiltration of immune cells such as central memory CD4+T cells(XCELL algorithm)and CD8+T cells(XCELL algorithm),and negatively correlated with the estimated infiltration of immune cells such as granulocyte monocyte progenitor cells(XCELL algorithm)and mast cells(CIBERSORT algorithm).In rectum adenocarcinoma(READ),there was a negative correlation with the estimated value of immune cell infiltration of naive CD4+T cells(CIBERSORT algorithm)and myeloid progenitor cells(XCELL algorithm).Gene mutation analysis showed that the amino acid mutation site of CHDH was mainly GMC oxidoreductase.The signal pathway analysis results in CRC showed that the signal pathways of CHDH positively correlated genes were mainly related to the Wnt signaling pathway,Erb B signaling pathway,Notch signaling pathway,etc.2.In basic experiments,RT-qPCR results showed that the expression level of CHDH in NCM460 cell lines was higher than that in SW480,SW620,and HCT116 cell lines(P<0.05).There was a statistically significant difference between paired CRC tissues and paracancerous tissues,and the expression level of paracancerous tissues was higher than that of tumor tissues(P<0.05).The results of WB and IHC were consistent with those of RT-qPCR.3.In the clinical analysis,the CHDH cut-off value obtained from the ROC curve was 0.969.The area under the curve(AUC)was 0.625 and its 95%confidence interval(CI)was 0.504-0.746,P=0.04.According to the cut-off value,enrolled patients were divided into two groups.CHDH≤0.969 and>0.969 respectively represented the low and high expression groups.Demographic characteristics and baseline clinicopathologic parameters such as sex,age,stage,etc.of the patients enrolled in the study were not significantly correlated with the high and low expression of CHDH(P>0.05).Kaplan-Meier survival analysis illustrated that the prognosis of patients with low expression of CHDH was better than that of patients with high expression of CHDH.COX regression analysis demonstrated that CHDH,CA199 and differentiation were independent risk factors for the prognosis of CRC patients.The results of relevant nomograms showed that there was no difference between the training and validation sets.The expression level of CHDH had the greatest impact on the prognosis evaluation,followed by CA199,and the smallest factor was the degree of tumor differentiation.The AUC of the ROC curves of the training and validation sets were 0.813 and 0.774,respectively.The calibration curve and the ideal curve fitted well.The distinguishability and calibration of the nomogram model were both acceptable.The scratch test results showed that overexpression of CHDH promoted the migration ability of colon cancer cells.Conclusions:1.Based on the results of previous basic experimental studies,there was a statistically significant difference in the relative expression of CHDH in m RNA and protein in tumor tissues and adjacent tissues(P<0.05).2.There was no significant correlation between CHDH and clinicopathological parameters such as sex,age,TNM stage,etc(P>0.05).3.For CRC patients,the expression level of CHDH,the degree of differentiation and CA199 were independent prognostic factors.4.The nomogram of prognosis prediction of tumor patients based on clinico-pathologic parameters and CHDH expression had good discrimination and calibration.5.The scratch test results showed that CHDH promoted the migration of colon cancer cells,which may be related to the occurrence and development of CRC.