A Study On Clinical Significance And Assumed Regulatory Mechanism Of Abnormal CYP24A1 Expression In Colorectal Cancer

ObjectivesColorectal cancer?CRC?is the third most common type of malignancy worldwide,with an estimated incidence of 1.36 million newly diagnosed cases and 690 000 cancer deaths every year.It is the fourth leading cause of cancer-related mortality in both males and females.In recent years,the incidence of CRC is rapidly increasing in China and become a major disease affecting public health.Epidemiological and experimental studies have shown that Vitamin D closely related with the occurrence and development of colorectal cancer.Vitamin D can play a role in anti-colorectal cancer by promoting proliferation,promoting differentiation,promoting apoptosis,inhibiting angiogenesis and immunomodulation effects,Vitamin D and its metabolic pathways in colorectal cancer prevention and treatment has become a focus in recent years.Vitamin D₃ is the most important form of vitamin D,Its physiological role is played through the active metabolite 1,25?OH?₂D₃,1,25?OH?₂D₃ regulates the expression of multiple genes through binding to receptor VDR and plays anti-tumor biological activities in vivo,24-Hydroxylase,encoded by the CYP24A1 gene,is the rate-limiting enzyme that catabolizes 25?OH?D₃ and 1,25?OH?₂D₃ to the less active calcitriol acid,24,25?OH?₂D₃ and 1?,24,25?OH?₃D₃ respectively,plays an important regulatory role in the anti-tumor effect of 1,25?OH?₂D₃.Studies have shown that CYP24A1 is a potential oncogene,abnormal expression of CYP24A1 in breast cancer,lung cancer,esophageal cancer is closely related to tumor malignancy and poor prognosis,CYP24A1 expression is regulated by DNA methylation in certain types of tumors.So far,there has been few studies on the expression of CYP24A1 and VDR in colorectal cancer tissues.The relationship between the expression of CYP24A1 and VDR and the clinicopathological features and prognosis of patients with colorectal cancer is not clear.Whether DNA methylation can regulate the expression of CYP24A1 in colorectal cancer cells needs to be further elucidated.Based on this,this study intends to reveal the correlation between VDR and CYP24A1 expression with clinical pathology information and prognosis in the pathogenesis and progression of CRC process by the experiment,and study the impact on 1,25?OH?₂D₃ anti-colorectal cancer function due to the change of expression.At the same time,to explore the regulatory mechanism of CYP24A1 expression in colorectal cancer,to elucidate the role of DNA methylation in the regulation of CYP24A1 expression in colorectal cancer cells.Methods?1?According to the natural evolution of colorectal cancer,the tissues of various stages of development were collected,including 57 cases of normal intestinal mucosa,12 cases of intestinal adenoma and 99 cases of colorectal cancer tissues,to build tissue microarray,the changes of VDR and CYP24A1 expression were detected by immunohistochemistry,to explore the association between VDR and CYP24A1 and the clinical information of patients including age,sex,tumor size,tumor location,differentiation,depth of invasion,lymph node metastasis,venous permeation,perineural invasion and prognostic information.The levels of serum 25?OH?D₃ in 40 healthy subjects and 12 patients with adenoma and 46 patients with colorectal cancer were examined by ELISA.?2?In the colorectal cancer cell lines SW480 and Caco2,the relative survival rates of the tumor cells were measured by WST-1 kit at 24,48 h and 96 h after treatment with 1,10,100,200 n M doses of 1,25?OH?₂D₃,the changes in the level of expression of VDR and CYP24A1 mRNA in SW480 and Caco2 cell lines were detected by RT-PCR after 100 n M 1,25?OH?₂D₃ treatment.CYP24A1 gene expression was interfered by RNAi technique in Caco2 cell lines,to detect the inhibitory effect of 1,25?OH?₂D₃ on the proliferation of Caco2 cells after the intervention of CYP24A1.?3?The difference of methylation level of CYP24A1 promoter region in 5 cases of colorectal cancer tissues and matched normal tissues was detected by BSP experiment,the colorectal cancer cell lines SW480 and Caco2 were subjected to demethylation by 5-aza-2-deoxycytidine,then the change of expression of CYP24A1 mRNA was detected by RT-PCR.In addition,the Caco2 cells were subjected to demethylation treatment,the change of CYP24A1 expression induced by 1,25?OH?₂D₃ was detected by RT-PCR.Regulation mechanism of DNA methylation in CYP24A1 gene expression was investigated at the tissue and cell level.Results?1?The colorectal cancer tissue microarray was successfully constructed and the expression of CYP24A1 and VDR was detected by immunohistochemistry.The high expression rates of CYP24A1 in colorectal cancer,adenoma and normal tissues were 69.7%,66.7% and 21.1% respectively.The expression of CYP24A1 in colorectal cancer tissues and adenoma tissues was significantly higher than that in normal tissues,and there was no difference between colorectal cancer and adenoma.The high expression rates of VDR in colorectal cancer,adenoma and normal tissues were 27.3%,58.3% and 64.9% respectively.The high expression rate of VDR in colorectal cancer was significantly lower than that in adenoma tissue and normal tissues,there was no difference between adenoma tissues and normal tissues.Furthermore,the expression of CYP24A1 was closely related to the depth of tumor invasion,lymph node metastasis and venous permeation,and the expression of VDR was correlated with the depth of tumor invasion.?2?The CRC patients with high CYP24A1 expression showed significantly reduced overall survival and disease-free survival compared to the patients with low expression.A multivariate Cox regression analysis showed that CYP24A1 expression was an independent prognostic factor for CRC recurrence?HR=2.829,95%CI=?1.092-7.330?,P=0.032?.However,no differences were found between CRC patients with high VDR expression and CRC patients with low VDR expression.?3?The serum 25?OH?D₃ level in the patients with colorectal cancer was non-normal distribution,with a median of 10.80 ng / ml,a quartile of?4.58,28.21?,the serum 25?OH?D₃ level in adenoma patients was 63.29 ± 32.95 ng / ml,the level of serum 25?OH?D₃ in healthy subjects was 67.91 ± 33.26 ng / ml.The level of serum 25?OH?D₃ in colorectal cancer patients was significantly lower than that in healthy group and adenoma group,no difference was found between adenoma group and healthy group.In addition,the level of serum 25?OH?D₃ was negatively correlated with CYP24A1 in adenoma patients?r=-0.614,P= 0.034?,but there was no correlation in colorectal cancer.There was no correlation between serum 25?OH?D₃ levels and VDR expression in colorectal cancer and adenoma patients.?4?Different doses of 1,25?OH?₂D₃ were applied to SW480 cell line and Caco2 cell line,and the inhibitory effect of cell proliferation was observed at different time points.The results showed that 100 n M 1,25?OH?₂D₃ was applied to SW480 cell line,and the inhibitory effect was observed at 48 h.The inhibitory effect was increased with the prolongation of the time and the increase in dose,but no inhibition effect was observed in Caco2 cell lines at all time points.The expression of CYP24A1 in Caco2 cell line was significantly higher than that in SW480 cell line,The inhibitory effect was observed when 100 n M 1,25?OH?₂D₃ acts on Caco2 cell lines at 48 h time point after CYP24A1 expression was inhibited by si RNA,the relative survival rate of the cells was 76.23%,which was statistically different from that of the control group.?5?The expression of CYP24A1 mRNA was not correlated with the promoter methylation level in normal intestinal mucosa and colorectal cancer?r =-0.483,P = 0.226?,Caco2 cell lines were treated with different doses of DAC for demethylation,CYP24A1 mRNA expression were increased compared with the control group after the treatment and the difference was statistically significant,however,this effect was not observed in the SW480 cell line.In the Caco2 cell line,the expression of CYP24A1 induced by 1,25?OH?₂D₃ was significantly higher in cells treated with DAC demethylation than in the untreated group,and the difference was statistically significant.Conclusions?1?CYP24A1 expression was increased in colorectal cancer and adenoma tissues,while VDR expression in colorectal cancer was lower than adenoma tissue and normal tissues;The high expression rate of CYP24A1 in T3-T4 stage was higher than that in Tis-T2 stage,and in lymph node metastasis group was higher than that in the untreated group,and the infiltration group was higher than that in the non-invasive group.The high expression rate of VDR was higher in Tis-T2 stage than that in T3-T4 stage.?2?The high expression of CYP24A1 was associated with poor prognosis in patients with colorectal cancer,and the overall survival and disease-free survival of patients with high expression of CYP24A1 were lower than those with low expression.Cox proportional hazards regression model showed that CYP24A1 was an independent factor for disease-free survival.?3?The serum levels of 25?OH?D₃ in colorectal cancer patients were lower than those in healthy and adenomas.The expression of CYP24A1 was negatively correlated with serum 25?OH?D₃ levels in adenoma patients.?4?The up-regulation of CYP24A1 expression in colon cancer cells affects the antitumor activity of 1,25?OH?₂D₃.In Caco2 cell lines,inhibition of CYP24A1 expression will enhance the antitumor activity of 1,25?OH?₂D₃.?5?The expression of CYP24A1 mRNA in colorectal cancer tissues was not correlated with the methylation level of CYP24A1 promoter region.However,in specific cell lines,DNA methylation was involved in the regulation of CYP24A1 expression.