The Experimental Research On The MRNA Expression Of ASPP Gene Family And The CpG Island Methylation Of ASPP Gene Promoters In The Tumor Cell Lines

Objective:The p53 protein is one of the best-known tumour suppressors. Recently, a new family of protein, the ASPP (for apoptosis-stimulating protein of p53) was discovered. This family consists of three members: ASPPi, ASPP2 and iASPP. ASPP1 and ASPP2 act as potent activators of p53. iASPP acts as an inhibitor of p53. The apoptotic function of p53 is stimulated by ASPPi and ASPP2 and inhibited by iASPP. To understand how important the ASPP proteins are in human cancers, we investigated the mRNA expression levels of ASPP members in eight kinds of cancer cell lines. To further understand the cause of abnormal ASPP expression in cancer cells. We detected the methylation status of ASPPi and ASPP2 gene promoter in tumor cell by methylation specific polymerase chain reaction.Methods:1.Cells were grown in Dulbecco modified Eagle medium supplement with 10% fetal calf serum and l00U/ml benzylpenicillin and 100 U/ml streptomycin sulfate in a humidified atmosphere containing 5% of CO2 at 37℃.2. mRNA expression levels of ASPP gene family were measured by RT-PCR.3. The CpG island status of ASPPi and ASPP2 gene promoter were analyzed and MSP primers were designed by Internet free primer design software. An analysis method of ASPPi and ASPP2 gene methylation status was established.4. Using this MSP method to detect the CpG island methylation status of ASPPi and ASPP2 gene promoter in three p53 wild-type tumor cell lines (Hepg, MCF-7, A549) and fibroblast cell.Results:1.ASPP gene family mRNA was expression in all cell lines, but the expression is different in each cell line.2. ASPPi gene mRNA expression in all tumor cell lines is prominent lower than theexpression in normal cells (fibroblast cell and blood monocyte), p<0.01. ASPP: gene mRNA expression is different in each tumor cell line. The expression level of the tumor cell lines show descendent trend comparing with that of the normal cells, P<0.05.3. iASPP gene mRNA expression is different in different cells. In A549 and HT-29, the expression is no difference, p>0.05. In the other 6 tumor cell lines the expression is higher than the normal cell expression, P<0.01.4. ASPPi and ASPP2 gene CpG island analysis using Internet free CpG island analysis software show that ASPPi gene has 10 CpG islands and there is one CG sequence average 3.4 bases in the first CpG island; ASPP2 gene has 3 CpG island and there is one CG sequence average 3.83 bases in the first CpG island.5. MSP analysis showed that CpG islands of ASPPi and ASPP2 gene promoters were methylated in the three p53 wild-type tumor cell lines Hepg-2, MCF-7, A549. Whereas in fibroblast cell, unmethylated bands was observed.Conclusions:1. The mRNA expression of ASPP gene family was measured by RT-PCR for the first time. The result indicated that ASPP gene family mRNA was expressed in all tumor cell lines researched.2. The electrophoresis photos were semiquantified analysis by Gel-Pro 3.1 software. The results showed that ASPPi and ASPP2 gene mRNA expression in tumor cell lines was lower than the expression of normal cells. iASPP gene mRNA expression in tumor cell lines was higher than the expression of normal cells. The result indicated that ASPPi and ASPP2 could enhance the apoptotic function of p53 and they could suppress tumor growth even in tumors expressing mutant p53; iASPP can restrain the apoptotic function of p53.3. The methylation status of ASPPi and ASP?2 gene was firstly analyzed by us and the rich CG sequence content exists in their first CpG island. This condition indicates that they were affected easily by methylation in this position.4. Using internet free primer design software MSP3.4 designed MSP primer of ASPPiand ASPP2 and established an analysis method-MSP to detect ASPPi and ASPP2 genemethylation status.5. ASPPi and ASP?2 gene CpG island methylation status was detected first time in three p53 wild-type tumor cell lines (Hepg-2 MCF-7 A549) and fibroblast cell. The resultsshowed that ASPPi and ASPP2 gene CpG islands were methylated in three p5...