Research Of Gene Expression And Promoter Hypermethylation Of Death Associated Protein Kinase In Bladder Urothelial Cell Carcinoma

Bladder cancer (BC) is an important question in public health area. It's the sixth most common cancer in the world, and the first common carcinoma of urinary system in China. About90%of bladder cancer is comprised of urothelial cell carcinoma (UCC)(also known as transitional cell carcinoma, TCC). One of the distinctive features of BUCC is that about75-85%of newly diagnosed cases are non-muscle-invasive superficial lesions (Tis, Ta, T1), with more than50%of them will recur. In addition, up to40%of patients with BUCC will suffer from tumour progression, and30%of the patients will die from initially non-musle-invasive bladder cancers. High recurrence, progression, adjuvant therapy as well as life-long follow-up undoubtedly add the medical expenses and suffering in BUCC patients, which also put great financial and social burden on our country.At present, detection and observation of bladder cancer is mainly performed by cystoscopy and urine cytology. As we all know, cystoscopy is an time-consuming, invasive procedure with exceeding discomfort for the patient, and the sensitivity of urine cytology is very low, especially for low-grade BUCC. Furthermore, treatment protocol with better therapeutic effects is urgently required, since traditional surgical intervention and adjuvant therapy perform worse than expected. Thus, it is a great challenge for us to identify molecular, genetic and epigenetic mechanisms which is associated with the development, recurrence and progression in BUCC, with the aim of exploring both non-invasive, more sensitive methods for cancer detection and prognosis evaluation, and potential programs for gene-targeting therapy.DNA methylation, an important part of epigenetics, has been a research focus in the past few years. Hypermethylation of the gene promoter regions represses DNA transcription in tumor suppressor genes (TSGs), thus leads to gene silencing, which is commonly observed in a variety of carcinomas. The change involving methylation in CpG island has been recognized as an vital part of Knudson's two-hits hypothesis.Death associated protein kinase (DAPK) gene, a TSG, is located in chromosome9q34.1. The protein product of DAPK gene is a160kD calcium/calmodulin dependent serine/threonine kinase with a unique domain structure. Interferone-gamma (IFN-y), tumour necrosis factor-alpha (TNF-α), Fas-ligand, other cytokines, or intracellular death signals can induce and activate apoptosis pathways which DAPK plays a vital role in. Researches indicated that expression of DAPK mRNA and protein were frequently at low levels in human cancer tissues and cell lines, probably as a result of gene silencing by DNA hypermethylation.Recently, researches from foreign scholars revealed that promoter methylation levels were significantly higher for DAPK gene in BC tissue samples and cell lines comparing with normal tissue samples and cell lines, while the expression of DAPK, DAPK mRNA were distinctly lower, what's more, detection of DAPK gene methylation in voided urine seemed to be more sensitive than conventional urine cytology. Hu and Zhao etc. ever studied the correlation between DAPK methylation and BC in China respectively, yet their research results were completely different. Study of Hu showed a significant association between gene methylation of DAPK gene and development of bladder cancer, as a result of promoter hypermethylation. In addition, the overall frequency of hypermethylation increased with tumor stage and recurrence, and detection of promoter methylation of DAPK gene may act as a biomarker for prognosis evalution in BC. On the contrary, Zhao etc. found that promoter methylation level of DAPK gene in BC was rather low, therefore, detection of promoter methylation of DAPK gene didn't seem to be a suitable biomarker for BC.In this study, we aimed to explore the correlation between DAPK methylation and BUCC, a non-invasive and sensitive method for cancer detection and prognosis evaluation, and a potential programs for gene-targeting therapy by analysing the CpG island hypermethylation of DAPK in clinical BUCC tissue samples and bladder cancers cell lines in this experiment. The current sudy was divided into3sections. In Section1, Western-blot, RT-PCR, and MS-PCR were separately performed to detect the expression of DAPK, DAPK mRNA and methylation status of DAPK in BUCC tissue and normal urothelial samples respectively, with the aim to evaluate the association between DNA methylation status and the clinical features of BUCC. In Section2, Western-blot, RT-PCR, and MS-PCR were performed respectively to detect the expression of DAPK, DAPK mRNA and methylation status of DAPK both in urothelial cell line5637(control group) and urothelial cell line5637with5-Aza-CdR (observed group). The purpose of this section of study was to investigate the correlation between DNA methylation and BUCC cell. In Section3, flow cytometry and tumour invasive assay detected apoptosis and migration activity both in urothelial cell line5637(control group) and urothelial cell line5637with5-Aza-CdR (observed group), which was to identify the correlation between DAPK gene and cell biological function in BUCC. Section One Analysis of gene expression and promoter hypermethylation of DAPK in tissue samples of human BUCCObjectives:To clarify the implication of gene expression and promoter methylation status of DAPK in human BUCC tissue.Methods:In this study,21fresh BUCC tissue samples as well as20cases of normal urothelial tissue (as control) were collected. Western-blot, RT-PCR, and MS-PCR were separately performed to detect the expression of DAPK, DAPK mRNA and methylation status of DAPK in BUCC tissue and normal urothelial samples respectively.Results:Western blotting confirmed that the protein expression of DAPK in BUCC samples was apparently lower than normal tissue, RT-PCR showed that the DAPK mRNA in BUCC samples was also apparently lower than normal tissue, in accordance with DAPK protein expression. Aberrant methylation was shown in16of21(76.2%) cases of BUCC tissues, and in4of20(40%) cases of control. No significant correlation between the hypermethylation of DAPK and the tumour grade, stage, or tumour occurrence was indicated. However, a correlation between gross hematuria and hypermethylation of DAPK was observed.Conclusions:According to the results above, we concluded that there was an important relationship between hypermethylation of DAPK gene and the occurence of BUCC as well as gross hematuria, implicating that BUCC patients with hematuria likely got a worse prognosis. Section Two Study of gene expression and aberrant promoter methylation of DAPK in human BUCC cell lineObjectives:In order to investigate the expression of DAPK, DAPK mRNA and detect the aberrant promoter methylation status in human BUCC cell line, as well as the effects of5-Aza-CdR on promoter methylation status in DAPK.Methods:Western-blot, RT-PCR, and MS-PCR were performed to detect the expression of DAPK, DAPK mRNA and methylation status of DAPK in BUCC cell line5637(control group) and urothelial cell line5637with5-Aza-CdR (observed group) resprctively.Results:Our results demonstrated extremely low expression of DAPK protein and mRNA in control group, and obviously increased expression of protein and mRNA in observed group, in which aberrant methylation of DAPK promoter was also detected.Conclusions:In summary, our results demostrated that there was an close correlation between the abnormal gene expression of DAPK and the development of BUCC, furthermore, the methylation status of CpG island palys a major role in the expression of DAPK. In addition,5-Aza-CdR can up-regulate the DAPK expression by reverse its promoter methylation. Section Three Analysis of relationship between promoter methylation status of DAPK and biological function in BUCCObjectives:Analyse the relationship between promoter methylation status of DAPK and biological function in BUCC.Methods:In this study, flow cytometry and tumour invasive assay detected apoptosis and migration activity both in urothelial cell line5637(control group) and urothelial cell line5637with5-Aza-CdR (observed group).Results:Flow cytometry indicated that cells in observed group had an apoptosis trend comparing with control group, and tumour invasive assay showed a slower migration of cells in observed group comparing with control group.Conclusions:In conclusion, DAPK can both promote the apoptosis and inhibit the migration ability in BUCC cells.