| Esophageal cancer is one of the most common malignancies worldwide, with the post-operation five-year survival rate less than 30%. Squamous cell carcinoma accounts for the major histopathologic type of esophageal cancer. In spite of the improvements in ESCC treatment during last decades, the prognosis of ESCC is still terrible. It’s of great importance to make early diagnosis and interventions for ESCC patients to improve the therapies.The carcinogenesis of ESCC is determined by both environmental and genetic factors, in previous studies, researchers have found that the tumor susceptibility is closely correlated with genetic variations. For example, it’s reported that the carcinogenesis is associated with single nucleotide polymorphisms from the growth or metastasis-asoociated genes. As another major type of genetic variation, copy number variation attracted much less attention than SNP, recent studies revealed that copy number variations which occur in cancer-related genes play important roles in predicting the cancer risk, which should be explained by gene dosage or site effects. The well-known caner risk-associated CNVs include those presented on genes related to tumor growth, metastasis and metabolism, almost all of which are located on m RNA coding regions. However, as the emergence of noncoding RNA, researchers realized that single nucleotide polymorphisms presented on noncoding RNA coding regions may also influence the susceptibility of cancers, but it’s still unclear weather the copy number variation of noncoding RNA could change the cancer rik.In this study, we evaluated the association of five CNV sites(including those presented on oncogenes MALAT, PRKCI and so on) with ESCC risk in a case-control study based on candidate genes strategy. All the five sites are from the fragile chromosome regions in ESCC tissues, which are represented by 3q26,8q24 and 11q13. These five genes are reported to be closely associated with tumor growth and metastasis, which included m RNAs and long noncoding RNA MALAT1.Long noncoding RNA(lnc RNA) is defined as a group of RNAs(>200 nt) with limited or no coding capacity due to a lack of an intact open reading frame, but participates in most vital biological activities. Lnc RNAs regulate gene expression by various ways, such as by chromatin reprogramming through binding of polycomb protein complex, playing as micro RNA sponge, mediating inactivation of chromosome X and so on. Lots of reports have demonstrated the association between dysregulation of lnc RNAs and human malignancies, such as the well-known tumor associated HOTAIR, ANRIL, XIST, HULC, and MALAT1.Metastasis Associated Lung Adencarcinoma Transcript 1(MALAT1), located on 11q13 of human chromosome, was originally found in NSCLC and treated as a prognostic marker for patients’ survival. Previous studies have indicated that MALAT1 was over-expressed in diverse tumor tissues and promoted proliferative and metastatic phenotypes of tumor cells. It could accelerate tumor growth and metastasis by multiple mechanisms, such as by mediating phosphorylation of SR family proteins, directly binding to chromosomes, and regulating alternative splicing of oncogenic m RNAs. However,it’s still unclear how it functions in human ESCC.Furthermore, the factors which contribute to MALAT1 up-regulation in malignant tissues remain poorly understood, previous studies have suggested that the expression of MALAT1 was regulated by methylation of histone H3, transcriptional factors, and micro RNAs; but it’s still insufficient to explain its deregulation in diverse tumor tissues. Role of the epigenetic mechanism in regulation of cancer-associated genes expression has been confirmed repeatedly, and it’s well known that beside the modification on histone, methalytion of the Cp G island is another main way in gene expression regulation, but no previous study reported whether the methylation of Cp G island contributes to MALAT1 regulation yet.To characterize its roles in ESCC and the elements associated with its deregulation, we examined the copy number alterations in tumor samples and tested the clinical significance of MALAT1 in esophageal cancer. Then we evaluated its oncogenic functions by a series of experiments. We have also checked the changes of ATM-CHK2 pathway upon MALAT1 knockdown, to uncover the underlying mechanisms by which MALAT1 controls cell cycle and facilitates tumor growth. To find out the epigenetic factors associated with its up-regulation in ESCC tissues, we investigated the methylation status of the Cp G island at the promoter region.The results indicated that: 1. No association of the germline CNVs in the five genes with ESCC susceptibility was identified in the case-control study; 2. MALAT1 was amplified in ESCC somatic tissues obviously, and is mainly over-expressed in tumor tissues from late-stage patients, suggesting that it should play a role in advancing of ESCC but not in its initialization phase. 3. Knockdown of MALAT1 by si RNAs inhibited growth and metastasis of esophageal cancer cells, and lead to G2/M arrest with increased cell apoptosis. MALAT1 depletion also activated the ATM-CHK2 pathway, which should be responsible for G2/M arrest. A negative correlation of MALAT1 expression level with phosphorylation status of ATM-CHK2 pathway was found in tumor tissues, suggesting that over-expression of MALAT1 may accelerate ESCC growth by dephosphorylation of the ATM-CHK2 pathway, which may release cells from cycle arrest; 4. No impact of the methylation status of the Cp G island on MALAT1 expression was identified by our research.The major contents are presented as follwing:1. Analysis of the association between the candidate CNVs and ESCC susceptabilityWe made bioinformatic analysis and dual-luciferase reporter assays to evaluate the properties of the candidate CNVs located on the fragile chromosme regions 3q26,8q24 and 11q13, which contain the oncogenes of PRKCI, MALAT1, SKIL and so on. Then we genotyped the selected CNVs in a case-control study by a newly developed method called Accucopy, a total of 201 ESCC patients and 193 matched healthy controls were included in this study. The results showed that no association between the germline CNVs and ESCC risk was identified.2.Amplification of MALAT1 in ESCC tissues contributes to its upregualtion and its expression level was correlated with clinical parametresTo make the prediction of the functional role of MALAT1 in ESCC, we first detected its expression level in 54 pairs of tissues and five cell lines(four ESCC cell lines and one normal control), and analysed the association between the expression level of MALAT1 with the clinical pathological patterns, such as the tumor size and metastasis pattern. We also detected its copy number in the 54 ESCC tissues and tested the association between its copy number and expression level. Our results showed that MALAT1 was over-expressed mostly in late-stage ESCC tissues, its expression level correlated tightly with the tumor size, and lymph node metastasis, indicating that MALAT1 may promote tumor proliferation and metastasis. We also found that MALAT1 was amplified in 22.2% of the ESCC tissues, and its amplification contributes to its up-regualtion in ESCC.3.The role and mechanisms of MALAT1 in ESCC proliferation and meatastasisThen we knocked down MALAT1 in ESCC cell lines EC109, EC9706, KYSE150 and KYSE450 by si RNAs, and tested the proliferation, colony formation, migration and invasion ability by a series of assays. The cell biological experiments confirmed our speculation from ESCC tissues, and we found that knockdown of MALAT induced G2/M cell cycle arrest, which may explain the growth inhibition caused by MALAT1 knockdown. According to previous reports, MALAT1 participated in genome maintanence and cell cycle regulation, so we speculated that MALAT1 may be involved in regulation of DNA damage response pathay, which may contribute to cell cycle arrest. We tested our hypothesis by western-blotting and found that the ATM-CHK2 pathway was phosphorylated upon MALAT1 depletion, indicating that the over-expression of MALAT1 was correlated with dephosphorylation of the ATM-CHK2 pathway, which may release cells from cell cycle arrest. Then we tested the association between MALAT1 expression and ATM-CHK2 pathway in ESCC tissues, the results suggested negative association between MALAT1 expression and phosphorylation of ATM-CHK2 pathway.4.No impact of the methylation status of MALAT1 promoter on its expression was identified in this studyPrevious studies suggested that expression of MALAT1 was controlled by methylation of histone H3, mi RNA and transcriptional factor. The epigenetics mechanism in MALAT1 expression regulation remains largely unclear. In this study, we sequenced the Cp G Island located on the promoter of MALAT1 coding region in esophageal tissues fro the first time, and analyzed the association between the methylation stasus of Cp G island and MALAT1 expression level. We found no difference on the methylation rate between cancerous and normal tissues, indicating that the methylation of the Cp G island didn’t influence MALAT1 expression. |
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