High Throughput Analysis Of Differentially Methylated Genes In Human Esophageal Squamous Cell Carcinoma

Esophageal cancer is the eighth most common cancer and sixth most common cause of cancer relative deaths worldwide. It is one of the most lethal diseases in China, and 70% of esophageal squamous cell carcinoma (ESCC) having been diagnosed occur ed in China. Despite there is a decline in its morbidity, ESCC continues to have a poor prognosis, with 5-year survival rate ranging from 15% to 25%. The alterations in DNA methylation is one of the common mechanisms which can lead to the accurance and development of cancer, promoter hyper-methylation of suppressor genes and global DNA hypo-methylation of oncogene are commonly observed in cancer. Present studys show that the abnormal methylation of genes are responsible for the initiation and progression of ESCC, and identifying these abnormal methylation biomarkers, the further studies are warranted to take these biomarkers as the important prognostic factors and drug targets for clinical practice.In the first part of the dissertation, Genome-scaled DNA methylation and mRNA expression profiles between 7 ESCCs and their adjacent normal tissues are analyzed by reduced representation bisulfite sequencing (RRBS) and digital gene expression profiling. After analysis the annotation and pathways of these differential methylation genes, we find they enrichmented at molecular binding and regulation, cellular component and activity, metabolic regulation and catalytic activity. Moreover, there is a change in cell movement, material transportation and cell-cell interaction. Besides, combining with the result of gene expression profiling, we screen the candidate genes located in promoter region and there is an inverse correlation between gene expression level and DNA methylation. According to the degree of methylation differences in top 10 and document retrieval, we screen our candidate gene Cyclin Y-like 1 (CCNYL1), with hyper-methylation in its promoter region and decreased expression in carcinoma gene expression profiling. Prodicted on TCGA, CCNYL1 has no mutation, deletion and amplification in ESCC, so we choose it to do the follow-up study.CCNYL1 gene is characterized by using Bisulfite sequencing PCR (BSP) in 17 pairs of tissues from ESCC, and another 10 pairs of tissues were detected by RT-PCR, the consequences show that CCNYL1 gene is hyper-methylation in promoter and decreased expression in ESCCs, which are consistent with RRBS analysis and gene expression profiling results. According to statistical analysis, in differential methylated region (DMR), CpG site 13, CpG site 16, CpG site 17, CpG site 18 and CpG site 19 were hyper-methylation in ESCC patients. Compared to esophageal epithelial immortalized cell lines, CCNYL1 is hyper-methylation and decreased expression in ESCC cell lines. Then, we evaluate the alteration of cell growth by growth curve assay in CCNYL1-transfected cells, we find the CCNYL1-transfected clones inhibited cell growth when compared with vector alone. Finally, we adopt epigenetic therapy to treatment ESCC cell lines with 5-Aza-2-deoxyctidine(5-aza-dC), the DNA methylation level of CCNYL1 in promoter is decreased and gene expression level of it is increased. In addition, the methylation level of the CpG site 13, CpG site 16, CpG site 17 and CpG site 19 in DMR are remarkable decreased, and it provides a molecular basis for tumor treatment. Therefore, CCNYL1 promoter hyper-methylation may play a role in ESCC initiation and its DMR maybe a new target for tumor therapy.In the second part of the dissertation, we screen the negative correlation genes which are different in lymphatic metastasis or not by high through analysis. There are 5 differential genes in promoter, besides,ODC1 have been studied in ESCC as well as PHB2, PCMT1, ARHGEF4 in many tumors, except HSD11B1L. We detailedly study the role of HSD11B1L in the initiation and progression of ESCC. According to the experiment, the DNA methylation level of HSD11B1L in promoter is decreased (P< 0.001) and there is a negative correlation with gene expression. Moreover, its methylation level is deeper in ESCC tissues with lymph node metastasis than that without lymph node metastasis (P<0.001). Furthermore, the study demonstrates that knockdown of HSD11B1L remarkably attenuated the malignances of ESCC cells in vitro, which inhibits tumor cells growth and migration. After that, the HSD11B1L methylation is examined in 20 paraffin-embedded ESCC tissues with lymph node metastasis and 20 without lymph node metastasis by BSP. Consistently, we confirm that HSD11B1L is hypo-methylation in ESCC tissues with lymph node metastasis (P< 0.001), indicating that patients with HSD11B1L hypo-methylation are vulnerable to lymph node metastasis. Additionally, statistical analysis reveals that methylation of HSD11B1L is significantly correlated with differentiation degree (P=0.039) and lymph node metastasis (P=0.004). The result of Kaplan-Meier analysis indicates that patients with hypo-methylation of HSD11B1L is correlated with shorter overall survival (P=0.003). Combined with multivariate Cox regression survival analysis, our results comprehensively demonstrate that HSD11B1L plays a critical role in the initiation and progression of ESCC and its methylation is an independent prognostic factor in ESCC.