The Experimental Research On The MRNA Expression Of TIP30 Gene And The CpG Island Methylation Of Its 5'-UTR In Liver Cancer Cell Lines

Backgroud and Objective:Hepatocellular carcinoma (HCC) is one of the most common malignant cancers with the highest death rate in China, and the death number of HCC, in China, accounts for as 50% of the total number of the world. Hepatocellular carcinoma is characterised by supreme malignancies and rapid development with poor prognosis and has poor excision rate, high recurrence and metastasis, which badly influence the therapeutic efficacy of hepatocellular carcinoma .The genesis,development and metastasis of HCC is a multistep and complex process involved by multiple genes and factors. In the progress, tumor progression in molecular level is based on loss of heterozygosity involving tumor suppressor genes on many chromosomes or gene amplification of selected oncogenes. To find the gene changed in malignant tumor, study their mechanism and illustrate their effect in the development of tumor may provide new markers for tumor staging, for assessment of the relative risk of tumor formation, and open new opportunities for therapeutic intervention.Tip30, weighing 30 kD, is a Tat-Interactive Protain, also called HTATIP2(HIV-1 Tat interactive protein 2), which is found by Xiao H and his collaborator, when they studied the transcription in vitro of human immunodeficiency virus. Tip30 may bind with Transactivator of transcription (Tat) as cofactor to specifically enhances HIV-1 Tat-activated transcription. Its expression has been detected in many human normal tissues such as heart, brain, lung, kidney, skeletal muscle and pancreas; however, in some tumor types, its expression was found to be decreased. Studies revealed that, on one hand, TIP30/CC3 was a transcriptional cofactor with Ser/Thr protein kinase activity, that could bind to the largest RNA polymeraseⅡsubunit and phosphorylate the heptapeptide repeats of the C-terminal domain of its, regulating expression of genes involved in apoptosis and angiogenesis to promote apoptosis and suppress tumor growth and metastasis on the specially physiological and pathological manner; on the other hand, TIP30/CC3 may bind directly to the members of Importinβfamily in a RanGTP-insensitive manner and associate with nucleoporins to suppress tumor growth through promoting apoptosis induced by the inhibition of nuclear transport. DNA methylation is epigenetic modification, which do not chang gene sequence, but may influence the structure of chromtin or modify the three-dimensional structure of DNA to regulate gene expression. The alteration of DNA methylation is a important factor inducing cell transformation and silencing suppressor gene. DNA methylation may result in aberrant expression of genes involving proliferation and differentiation by gene and epigenetic mechanism to make cell escape from the control of normal precess, further to induce malignant alteration of cell and development of the tumor. The role of DNA methylationin tumorigenesis has attracted conciderable attention recently. The detection of aberrant DNA methylation is helpful for the understanding of the fundamental mechanisms oncogenesis and present the useful information for the early diognosis of the cancer.There are no related reports which whether tip30 gene have aberrant methylation and its expression is influenced by the methylation. In this experiment, we examined the expression level of Tip30 gene mRNA to reveal the relationship between Tip30 gene and HCC. To further understand the mechanism of Tip30 gene downregulation, we detected the methylation status of Tip30 gene 5'-UTR in HCC cells by MSP and BSP.Methods:1. We detected the expression of TIP30/CC3 gene mRNA in multiple hepatocellular carcinoma cell lines by Real-Time PCR, and analized the difference of TIP30 gene mRNA expression between caner cells and normal cells.2. we investigated the funtion of Tip30 gene promoter in HL7702 using luciferase reporter gene vector PGL3-Enhancer. The 5'serial deletion of Tip30 gene promoter-pGL3 Enhancer luciferase reporter gene recombinants was constructed. 3. we analyzed the CpG island status of TIP30 gene 5'-UTR by PLOTCpG and designed MSP and BSP primers using Methyl Primer Express Software 1.0. An analysis method of TIP30 methylation status wes established.4. We detected the CpG island methylation status of TIP30 gene 5'-UTR using the MSP and BSP respectively in multiple hepatocellular carcinoma cell lines.5. We finally observed the influence of methylation inhibitor on mRNA expression of TIP30gene.Results:1. The mRNA expression of TIP30gene were detected in normal and malignant cell lines, but their expression level is different in esch cell line.2. The mRNA expression of TIP30 gene in most of the tumor cell lines is prominently lower than the expression in normal cell(p<0.01) . But in PLC and XJC, the expression is higher than the normal cell. This suggests that there were different mechanism of gene expression regulation in different cell line.3. In HL7702 cell line, the -135/-45 region is the major active transcription region of TIP30 gene promoter. The result of TESS analysis showed multiple SP1 binding sites in the region.4. The result of PLOTCpG analysis showed that TIP30 had two typical CpG islands, one covering the transcription start site and another covering the tranlation start site.5. MSP and BSP results showed that the CpG island of TIP30 gene 5'-UTR were methylated in some tumor cells with lower TIP30 mRNA expression, whereas some tumor cells with lower TIP30 mRNA expression and tumer cells with higher TIP30 mRNA expression and normal cell were not methylated. This suggests methyaltion influence the transcriptional regulation of TIP30 gene and result in the lower expression of TIP30 gene in tumor.6. The methylation inhibitor restored the expression of TIP30 gene in tumor cells with methylation.Conclusion: 1. The mRNA expression of TIP30 gene wes measured by Real-Time PCR for the first time in liver cancer cells and normal liver cell. The result indicated that the TIP30 expression is different in different cell lines, and to compared with normal liver cell, the TIP30 gene mRNA expression was prominently low in most liver cancer cells, revealing the relationship between TIP30 gene and HCC.2. we studied the TIP30 gene promoter function in liver cell line for the first time and discovered that the -135/-45 region is the major active transcription region of its promoter, and TESS analysis showed multiple SP1 binding sites in the region.3. The CpG island of TIP30 gene 5'-UTR was firstly analyzed by us using PLOTCpG and two typical CpG islands were found, the first one of which had richer CpG dinucleotide sequence content indicating that they were methylated easily in this region.4. The MSP and BSP primers for the methylation analysis of TIP30 gene were firstly designed and established the analysis method of TIP30 gene methylation status.5. The methylation status of TIP30 gene 5'-UTR CpG island was explored for the first time in different liver cancer cells and found out that the methylation of TIP30 gene 5'-UTR was an important factor regulating TIP30 gene expression.