Research On Regulatory Mechanism Of DNA Hydroxymethylation Modification And Post-transcriptional Regulation In Colitis And Colorectal Cancer

?Backgrounds?Colon cancer(CRC)is one of the most common gastrointestinal malignancy,its morbidity and mortality rate was increasing year by year,a serious threat to human health.Colon cancer is closely related to colitis.Due to repeated outbreak and protracted course of disease,chronic non-specific ulcerative colitis with intraepithelial neoplasia change is the basis of the occurrence of colorectal cancer,sequence evolve from low-level intraepithelial neoplasia to high-level intraepithelial neoplasia,and become colorectal cancer in the end.Aberrant epigenetic alterations play an important role in the occurrence and development of malignant tumors.Abnormal DNA methylation and microRNA expression changes are the most important factors.In-depth study on these two factors is benefit to intervene the occurrence of colon cancer.It plays an important role in the CRC early dignosis and early treatment.It can reduce the morbidity and mortality rate of colon cancer.A variety of gene regulation ways were exist in organism,including transcriptional regulation,post-transcriptional regulation,pre-translational regulation,post-translational regulation and epigenetic regulation.The abnormal regulation of colorectal cancer-associated oncogene and tumor suppressor gene is closely related to the development of colorectal cancer.An epigenetic modification(DNA methylation and hydroxymethylation)and a post-transcriptional regulation(microRNA)play an important role in gene regulation,they are associated with the maintenance of multiple functions of cells and they are inseparable with the occurrence and development of colorectal cancer.5-hydroxymethylcytosine(5-hmC)is produced by the oxidation of 5-methylcytosine(5-mC)by the TET(ten-eleven translocation)family of enzymes.It not only participates in the process of DNA demethylation,but also has a dual regulation of gene expression.Recent reports showed that the level of 5-hmC as a useful epigenetic biomarker was decreased in malignant neoplasms.It has also been confirmed that the level of 5-hmC was decreased in solid tumors such as colorectal cancer and liver cancer.The mechanisms underlying may be the reduction in the expression level of TET in tumor tissue.However,the expression of TET family protein and 5-hmC in colitis and colorectal cancer is unclear,and its function and regulation mechanism has not been fully elucidated.In this study,we examined the expression of TET family protein and 5-hmC in colitis and colon cancer,and discussed the correlation between TET family protein and 5-hmC in the occurance of colon cancer,for providing a new theoretical basis of 5-hmC as a important epigenetic marker of CRC.miRNAs are a group of evolutional highly conserved small non-coding RNAs with a length of approximately 19-24 nucleotides.These miRNAs regulate tumor related genes expression by targeted combination with mRNAs at the 3'-untranslated region(3'-UTR)nucleotides,promptting mRNA degradation and translational suppression.They affected cell migration,proliferation,differentiation and apoptosis,acted as an oncogene or tumor suppressor gene.A high-throughput sequencing method for colorectal cancer and adjacent tissues was screened to obtain microRNA molecules that were significantly elevated in colon cancer,mi R-647 and miR-1914 are positioned very close to each other on chr20q13,with a distance only 1086 bp from each other.The study found that miR-647 and miR-1914 cooperate to promote cancer progression by down-regulating nuclear factor IX in colorectal cancer,the specific mechanism to be further clarified.?Objectives?1.To detect the expression of TET protein family and 5-hmC in colitis and colon cancer,and to clarify its relevance to the development of colitis and colorectal cancer.2.To detect the expression of miR-647 and miR-1914 in colorectal cancer tissues and cell lines,and to elucidate its function and regulatory mechanism in the occurrence of CRC.?Methods?The expression of 5-hmC in colitis and colorectal cancer was detected by immunohistochemistry and immunofluorescence assay;The expression of TET protein family in colitis and colon cancer was detected by qPCR;Bioinformatics high-throughput sequencing and RNA-seq analysis was used to detect the expression of TET protein family and microRNAs in CRC tissues and adjacent normal tissues.Screening out the most obvious changed microRNA molecules;The expression of miR-647 and miR-1914 in CRC tissues and adjacent normal tissues were detected by qPCR in 22 patients;The expression of miR-647 and miR-1914 in HIEC and HT-29 was detected by qPCR;miR-647 mimics and miR-1914 mimics or miR-647 antagomir and miR-1914 antagmoir were transfected into colon cancer cells or human intestinal epithelial cells;MTT assay was used to detect the proliferation of colon cancer cells;Cell wound scratch assay and transwell assay were used to detect the migration of colon cancer cells;To investigate the effect of miR-647 and mi R-1914 on the sensitivity of colon cancer cells to 5-FU.The target genes of miR-647 and miR-1914 were screened and identified by bioinformatics analysis and i-TRAQ;The target gene was verified by luciferase reporter assay and qPCR assay at last.To study and discuss the function of target gene in CRC by introducing small molecule regulators of target gene.?Results?1.Immunohistochemistry and immunofluorescence showed that the expression of 5-hmC was decreased in colon cancer and increased in colitis.2.The cell inflammation model was established by stimulating HIEC cells and colon cancer cells with IL-6.Immunohistochemical and immunofluorescence were used to detect the expression of 5-hmC.In the colitis,the expression of 5-hmC was upregulated,and its elevation was related to the increase of TET protein.TET1 and TET2 played an important role in it.3.The acute intestinal inflammation model in mice and chronic intestinal inflammation model in mice were established by stimulating with DSS.Immunohistochemical and immunofluorescence were used to detect the expression of 5-hmC.In the colitis,the expression of 5-hmC was upregulated,and its elevation was related to the increase of TET protein.TET1 and TET2 played an important role in it.4.The results of transcriptions showed that the expression of TET protein in colorectal cancer was lower than that in adjacent tissues,but there was no significant difference.5.The results of transcriptions also showed that miR-647 and miR-1914 were the most obvious changed microRNAs in colon cancer,which was closely related to the occurrence of colon cancer.The target gene NFIX was also abnormal in colon cancer.6.The expression of miR-647 and miR-1914 in colon cancer tissues and cells were increased.7.MTT assay showed that miR-647 and miR-1914 could promote colon cancer cell proliferation.8.Cell wound scratch assay and transwell assay showed that mi R-647 and miR-1914 could promote colon cancer cell migration.9.miR-647 and miR-1914 can both enhance the sensitivity of colon cancer cells to 5-FU.10.i-TRAQ and target gene prediction software predict that NFIX is the target gene for miR-647.Luciferase reporter assay and qPCR assay showed that NFIX is a common direct target of miR-647 and miR-1914.11.The introduction of small molecule modulators testified that miR-647 and miR-1914 cooperate to promote cancer progression by downregulating NFIX in colorectal cancer.?Conclusions?1.The expression of 5-hmC was increased in colitis,TET1 and TET2 play an important role.2.miR-647 and miR-1914 cooperate to promote the proliferation and migration of CRC cells by directly targeting NFIX.