Deletion, Mutation And Methylation Of P16 Gene And Its Protein Expression In Bladder Transition Cell Carcinoma

Objective To investigate the inactivation mechanism of p16 gene in bladder cancer and the relationship between p16 protein expression and bladder cancer. The homozygous deletion of p16 gene exon 1 and 2, the point mutation of exon2 and the methylation in exon 1 5' CpG islands of p16 gene and p16 protein expression in 25 bladder transitional cell carcinoma(bladder cancer) patients were examined. Methods To get tissue samples from 25 bladder cancer patients and extract DNA from tissue samples using phenol/chloroform method, design the primers for detecting the exonl and exon2 of p16 gene. To analyse the deletions of exonl and exon2 and point mutations of exon2 and methylation variations in exon 1 5' CpG islands of p16 gene by PCR(polymerase chain reaction), PCR-SSCP(PCR-single strand conformational polymorphism) and methylation-sensitive restriction enzyme hydrolysis PCR methods. The expression of p16 gene was determined by S-P immunohistochemical method in 25 specimens of bladder cancer and 6 cases of normal bladder mucosa tissue.Results The p16 gene deletions were found in six samples,four cases had deletion in exon2,one case had deletion in exonl and one in both exonl and exon2 in 25 bladder cancer samples. The homozygous deletion rate of p16 exon2 was 24% (6/25) ; no point mutation of p16 exon2 was found; the methylation rate of the exon 1 5' CpG islands of p16 gene was 69.6%(16/23). The positive rate of p16 gene expression was 52% (13 of 25) in bladder cancer and 100% (6/6) in all 6 normal bladder mucosae,significantly lower in bladder cancer than in normal tissue (P<0.01). The positive expression rate of p16 gene showed a tendency to descend with the progression of the pathological grade,clinical stage and poorer prognosis,but there was not a statistically significantdifference(P>0.05). Conclusions There are three ways of inactivation mechanism of p16 gene in bladder cancer: homozygous deletion, point mutation and 5' CpG island methylation. The deletion of p1 6 exon 2 was not a frequent event in Anhui bladder cancer patiens; a statistically significant association was also noticed between p16 gene exon 1 5' CpG island methylation and the inactivation of p1 6 gene in bladder cancer. Although no point mutation of p16 gene is found in these 25 bladder cancer patients, we can't conclude that it is not one of the inactivation mechanism and will make further study of the point mutation of p16 by enlarging the samples. There may be a correlation between p16 protein expression and the occurrence and development and prognosis of bladder cancer, p1 6 protein expression will be made further study by enlarging the samples,too.p16 expression protein may be one of the indicators of diagnosis of bladder cancer.