Study Of Correlation Between HPV16 E6 And P53Arg72Pro Polymorphisms

Objective: To observe the change of P53 protein and P53 associated protein in the Arg homozygous cells and Pro homozygous cells after being effected by HPV16 E6. And to discuss correlation between HPV16 E6 and p53Arg72Pro polymorphisms, it provide academic evidence for the mechanism of cervical cancer.Methods: We constructed eukaryotic expression vector pcDNA3.1/myc-His (-) A-HPV16 E6. Then we transfected pcDNA3.1/myc-His (-) A-HPV16 E6 into TP53 codon 72 genotype cell lines, arginine (Arg) homozygous (SK-BR-3) and proline (Pro) homozygous (MIA PaCa-2). Immunocytochemistry was also used to detected the expression of protein of P53,P21,Bax,Ki67 proteins in these cells which were transfected by HPV16 E6.Results: (1) It was very successful for constructing of recombinant pcDNA3.1/myc-His (-) A-HPV 16 E6 plasmid. (2) The eukaryotic expression vector pcDNA3.1/myc-His (-) A-HPV16 E6 was successfully transfected into the Arg homozygous or Pro homozygous cells. (3) In the Arg homozygous or Pro homozygous cells, the expression of P53 protein was respectively 0.899±0.040 OD and 1.074±0.015 OD. After being transfected by pcDNA3.1/myc-His (-) A, the expression of P53 protein was respectively 0.821±0.019 OD and 1.052±0.010 OD. There were no significance between those (P=0.185 and 0.229). While after being transfected by HPV16 E6, the expression of P53 protein in two cells was lower than that of before being transfected by HPV16 E6, respectively 0.604±0.043 OD and 1.006±0.008 OD. There were significance between those (P=0.000 and 0.001). And P53 protein was obviously degraded in the Arg homozygous cells. (4) The expression of P21 protein was very low or even no any in the Arg homozygous and Pro homozygous cells. And those cells were transfected by HPV16 E6, there was no any change of the expression of P21 protein. (5) In the Arg homozygous and Pro homozygous cells, the positive rate of Ki-67 protein was respectively 80.26% and 79.66%. After being transfected by pcDNA3.1/myc-His (-) A, the positive rate of Ki-67 protein was respectively 81.48 % and 81.14%. There were no significance between those (P=0.624 and 0.462). While after being transfected by HPV16 E6, the positive rate of Ki-67 protein in two cells was higher than that of before being transfected by HPV16 E6, respectively 86.73% and 87.89%. There were significance between those (P=0.047 and 0.016). And the positive rate of Ki-67 protein was higher in the Pro homozygous cells than in the Arg homozygous cells. (6) In the Arg homozygous and Pro homozygous cells, the expression of Bax protein was respectively 0.297±0.013 OD and 0.416±0.025OD. After being transfected by pcDNA3.1/myc-His (-) A, the expression of Bax protein was respectively 0.292±0.004 OD and 0.412±0.031 OD. There was no significance between those. After being transfected by HPV16 E6, the expression of Bax protein in Arg homozygous cells was 0.296±0.009 OD. There was significance between those (P=0.958). But the expression of Bax protein, 0.332±0.013, in the Pro homozygous cells after being transfected by HPV16 E6 was obviously decreased. And there was significance between those (P=0.023).Conclusion: (1) It was very successful for constructing of recombinant pcDNA3.1/myc-His (-) A-HPV 16 E6 plasmid. (2) After the eukaryotic expression vector pcDNA3.1/myc-His (-) A-HPV16 E6 was transfected into the Arg homozygous and Pro homozygous cells, the P53Arg and P53Pro protein can be degraded by HPV16 E6. And the P53Arg protein was obviously degraded. (3) After the P53Arg and P53Pro protein was degraded by HPV16 E6, the ability of proliferation and apoptosis were depressed by different degree. Therefore, the P53Arg and P53Pro protein can cause the cervical cancer by different approaches.①The expression of Ki-67 protein was improved. It suggested the proliferation ability of cells was increased. And the proliferation ability of Pro cells was stronger than that of Arg homozygous cells after the P53 protein was degraded.②The expression of Bax protein was decreased. It suggested the apoptosis ability of cell was decreased. And the apoptosis ability of Pro cells was lower than that of Arg homozygous cells after the P53Pro protein was degraded.③The expression of P21 protein was no any change. It suggested the p21 gene was not transcribed by P53Arg and P53Pro protein.