| Background:Epigenetics, which refers to inherited changes in gene expression but do not alter DNA sequences, plays a vital role in embryonic development and diseases. Through DNA methylation, histone acetylation, chromatin looping, and small noncoding RNAs, epigenetics could activate or suppress gene expression. Epigenetics changes mainly affect chromatin structure stability, gene expression, tissue-specific modulation, genome integrity, genomic imprinting, embryonic development, and X-chromosome inactivation in female. Aberrant DNA methylation is one of the critical events in epigenetics and also as an essential participant during tumorigenesis. DNA methylated modification includes de novo methylation and maintenance methylation, which could transfer a methyl donor to cytosine under the catalysis of DNA methyltransferases (DNMT1 and DNMT3a, DNMT3b) and produce 5-methycytosine (5-mC). DNA methylated modification is a long-term and potentially heritable mark. Recent studies showed DNA methylated pattern was also dynamic in mammals. DNA demethylation has been observed in different biological processes, which includes passive demethylation and active demethylation. "Passive demethyltion" is dependent on replication to dilute the level of genomic 5-mC dependent, while "active demethylation" means removal or modification of methyl group from 5-mC. Passive demethylated pathway has been widely discussed but the enzymes involved in active demethylated pathway still unclear. It was not until 2009 that was unambiguously identified TET1, as methylcytosine dioxygenases, could catalyze 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC). TET (Ten-Eleven translocation) proteins, members of family Fe2+/α-ketoglutarate (a-KG)-dependent oxygenase family of enzymes, could iterative oxidate 5-mC to 5-hmC, then 5-formylcytosine (5fC) and 5-carboxylcytosine (5CaC), and finally complete DNA demethylated cycle. During tumorigenesis, decreased levels of TETs and 5-mC,5-hmC are widely reported. Hence, our research explored the level changes of TETs and 5-mC,5-hmC in colorectal cancer (CRC) and their roles in tumorigenesis. The thesis is organized into 3 parts including:the expression levels of TETs protein family in CRC and the relationship with clinical pathological characteristics and patients’prognosis; the genomic levels of 5-mC and 5-hmC and the association with clinical pathological characteristics and patients’prognosis; the biological functions of TET 1 in colorectal cancer cell lines.Part 1 The expression levels of TETs protein family in CRC and the relationship with clinical pathological characteristics and patients’prognosisMaterials and Methods:Cross-sectional study was used in our research. From 2006 to 2012,109 subjects with a diagnosis of colorectal cancer (CRC) who received surgical treatment at Taizhou hospital of Zhejiang province (Taizhou, China) were recruited. Primary colocrectal cancerous tissues (C) and corresponding adjacent normal tissues (N) from the surgical margin were obtained after surgery. QuantiGenelex assay was used to detect the expression levels of TET protein family. Then the different expression levels of TETs in colorectal cancers and corresponding adjacent normal tissues were discussed. The associations of expression levels of TETs and clinical pathological characteristics in CRC were analyzed. At the last, we discussed the relationship between TETs expression levels and patients prognosis.Results:the expression levels of TET1 and TET2 were significantly decreased in cancerous tissues than in corresponding normal tissues (TET1,0.02 vs 0.06; TET2,0.22 vs.0.31; all P<0.05), while the level TET3 was increased in tumor tissues (0.36 vs.0.22, P<0.05). Similar results were observed in different subgroup analysis by age, gender, colorectal cancer location, TNM stages and depth of tumor invasion, lymph node metastasis and distant metastasis (all P<0.05). Survival analysis showed that the ratio of TET1 in cancerous tissues to adjacent normal tissues (C/N-TET1) was an independent overall survival factor to predict patients prognosis (HR = 2.5,95% CI= 1.34-4.66; P< 0.01).Part 2 The genomic levels of 5-mC and 5-hmC and the association with clinical pathological characteristics and patients’prognosisMaterials and Methods:In our study, we have DNA samples of 71 patients which included cancerous tissues and corresponding normal tissues. So ELISA was used to detect the genomic level of 5-mC and 5-hmC. Then we analyzed the relationship between TETs and 5-mC,5-hmC. We also discussed the associations between the level of 5-mC,5-hmC and clinical pathological features, as well as the prognosis of CRC patients.Results:The genomic levels of 5-mC and 5-hmC were reduced in tumor tissues than that of corresponding normal tissues (5-mC,4.46% vs.6.15%; 5-hmC,0.05% vs.0.07%). In subgroup analysis, the similar tendency was also observed. However, there’s no statistically significant relationship between TETs and 5-mC or 5-hmC (all P >0.05). In clinical pathological studies, C/N-5-mC ratios decreased in patients with over 60 years old and T4 stage (all P values <0.05). Higher 5-mC and 5-hmC levels were found in rectal than in colon tissues (all P values <0.05). survival analysis showed that C/N-5-mC could act as an independent protect factor to predict patients survival outcome (HR=0.36,95% CI:0.13-0.96). Combined analyze of C/N-5-hmC ratio level and C/N-5-mC ratio level could be a potential model to predict patient survival outcome.Part 3 The biological functions of TET1 in colorectal cancer cell linesMaterials and Methods:According to survival analysis, the expression level of TET1 could affect patients’ prognosis. Then we further discussed the biological function of TET1 in vitro. Firstly we detected the expression level of TET1 in 7 colon cancer cell lines. Secondly high expression level of TET1 was chosen to build deficient-TET1 cell lines through lentiviral transfection and low expression level of TET1 was chosen to produce overexpression-TET1 cell lines by lipofection transfection. Thirdly ELISA assay was used to detect the genomic level of 5-mC and 5-hmC in constructed cell lines. Meanwhile cell cycle was examined by FACScan flow cytometry. CCK8 was used to detect cell proliferation and transwell assay was performed to measure cell migration and invasion.Results:Except SW620 cells, the expression level of TET1 was very low in other colon cancer cell lines. Difference from the result of analysis in tissue, reduced TET1 expression level showed corresponding decreased genomic5-hmC level and increased genomic 5-mC level. TET1-deficient could also induce G1/M transition to promote cell proliferation and facilitate cell metastasis and invasion. While overexpression of TET1 showed the opposite results except no significant difference in proliferation assay. These results implicated that besides acting as tumor suppressor gene, TET1 may be involved in tumor progression through acting as an oncogene in CRC.Above all, the changes of TETs and 5-mC,5-hmC levels in colorectal cancer played important roles in patients’ survival outcomes. This study uncovered the biological functions of TET1 may play vital roles in colorectal carcinogenesis. All of these suggested novel potential biomarkers and therapeutic opportunities for the diagnosis and treatment of CRC... |
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