Molecular Mechanism Of AFP Affecting Carcinogenesis Of Hepatitis B-related Hepatocellular Carcinoma

Objective Study the relationship between the HBsAg and AFP, observe expression level of P21RAS, PIK3CA and P-AKT in HCC tissues and peficancerous tissues. Detect mutations of the exon 9 and 20 of PIK3CA gene in HCC tissues. To clarify relativity between HBV infection and AFP level, clarify relationship of AFP level and protein expression of RAS\PIK3CA\AKT signal pathway. To elucidate the relationship between AFP level and RAS\PIK3CA\ AKT signaling pathway and it's molecular mechanism roles in the liver cancer; by testing the PIK3CA gene mutations, further defined the relationship of PIK3CA\AKT signaling pathway abnormalities and HCC occurrence and prognosis.Methods Collected 80 HCC specimens which they are from the First Hospital of Lanzhou universityin the second department of general surgery from July, 2006 to October,2009. The specimens consisted of 50 paraffin imbedded and 30 fresh cancer tissue and corresponding cancer adjacent tissue, among the specimen,53 cases were HBV positive. All specimens were confirmed by pathological examination, and have not received radiotherapy and chemotherapy before surgery. All specimens are from typical cancerous focus, and fresh tissues were stored in nitrogen canister immediately. We used immunohistochemisty of Elivison to examin the expression of P21RAS, PIK3CA and P-AKT in HCC cancer tissue and cancer adjacent tissues. We extracted DNA in paraffin imbedded tissue by using DNA extraction kits, extracted DNA in fresh tissue by using Phenol chloroform method. The PCR premier of exon 9 and 20 of PIK3CA were designed with software Premier 5. PCR products were directly sequenced. Check the mutation point compared to standard sequence from Genebank. Using Statistical analysis software SPSS 16.0 to analyze the data from our study. (P<0.05) Results (1) There is certain relationship between the HBsAg and AFP level. The positive rate of AFP is higer in HBsAg positive cases than in HBsAg negative cases. (2)The expressions of P21RAS,PIK3CA,P-AKT are differentially in HCC tissue and peficancerous tissues. The average grey scale value of P21RAS were 137.16±2.75 in cancer tissre and 146.32±2.91 in cancer adjacent tissue (P<0.01). The average grey scale value of PIK3CA were 138.62±3.94 in cancer tissre and 151.42±1.78 in cancer adjacent tissue (P<0.01). The average grey scale value of P-AKTwere 141.38±3.19 in cancer tissre and 153.47±2.78 in cancer adjacent tissue (P<0.01). (3) 72 cases were successfully sequenced, result shows that 8 cases have gene mutation of PIK3CA,2 cases are E542K(G1624A),3 cases are E545K(G1633A) and 3 cases are H1047R(A3140G).Conclusion The positive rate of AFP is higer in HBsAg positive cases than in HBsAg negative cases. The expression level of P21RAS, PIK3CA and P-AKT in HCC cancer tissue were higer than in cancer adjacent tissue respectively. Furthermore, we found that AFP level is positively related to expression level of P21RAS, PIK3CA and P-AKT in HCC cancer tissue. Gene mutation of exon 9 (E542K,E545K) and 20 (H1047R) of PIK3CA were examined. The results showed that HBV infection promotes increase of serum AFP level, APF advances carcinogenesis of HCC though RAS\PIK3CA\AKT singal pathway. Hotpot mutaiton of PIK3CA was related to caricnogenesis and prognosis of HCC.