The Effects Of P16 Gene Alteration On The Carcinogenesis And Progression Of Hepatitis B Virus Related Hepatocellular Carcinoma

Objective To initially approach the effects of pl6 gene abnormal alteration on the carcinogenesis and progression of hepatitis B virus (HBV) related hepatocellular carcinoma (HCC) and provide some experimental data for the further researching for the molecular biologic mechanism of the development and progression of HBV related HCC , and for the prevention, diagnosis and treatment of HCC using pl6 gene by examining the infection status of HBV, the alteration of pl6 gene, the expression of p16 protein and Rb (Retinoblastoma susceptibility gene) protein and the transcription activity of nuclear factor-KappaB (NF-kB) in heptocellular carcinoma, peritumoral areas and liver cirrhosis (LC) tissues, then analyzing the relationship among HBV DNA integration, pl6 gene alteration, pl6 protein and Rb protein expression and NF-kB transcription activity and the relationship between these factors and HCC.Methods The specimens involved in this study were divided into three groups: HCC, peritumoral areas tissue and LC. HCC tissue and the liver tissue in peritumoral areas were obtained from 35 cases with HCC by hepatectomy and LC tissue was obtained from 35 cases with LC in the corresponding period by biopsy during other operation (4 of them was out for the lack of specimen during the late period of this study). Histopathologic examination of the three kinds of tissues was done by histopathologist. Polymerase chain reaction (PCR) was applied to examine HBV DNA in the tissues of HCC, peritumoral area and LC. The integration of HBV DNA in the specimens was detected by the re-amplification of reclaimed larger size DNA segments by PCR and was confirmed by Southern blot hybridization. The homozygous deletion of pl6 gene exon1 α , exon2 and exon3 was detected by PCR. The point mutation of pl6 gene exons was examined by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and the methylation status of p16 gene was detected with CpGenome^TM DNA Modification Kit and CpGWIZ? pl6 Amplification Kit. The expression of pl6 mRNA in the three kinds of tissues was detected by reverse transcriptase PCR (RT-PCR). pl6 protein and Rb protein in HCC, peritumoral area and LC tissue were detected by Western blot analysis. Alpha-fetoprotein (AFP) in serum was examined by enzyme-linkedimmunoad sorbent assay (ELISA). The transcription activity of NF-kB in these tissues was examined by Electrophoretic Mobility Shift Assays (EMSA), Competition Assays and Super-Shift Assays.Results HBV DNA in the tissues of HCC, peritumoral area and LC was still replicating actively. The positivity rates of HBV DNA in HCC, peritumoral area and LC were 94.3%, 91.4% and 94.3% respectively and the integration rates of HBV DNA in the three kinds of tissues were 71.4%(25/35), 65.7%(23/35), 60.0% (21/35) respectively, there was no significant difference among them CP>0.05) . But the integration of HBVX gene was dominating in HCC and the integration of HBVS gene was dominating in LC, the integration rates of HBVX gene in the three kinds of tissues integrated by HBV with DNA were 100.0% (25/25), 95.7% (22/23) and 66.7% (14/21) respectively, while that of HBV S gene were 50.0% (12/24), 52.2% (12/23) and 81.0% (17/21) respectively, the disparity among the three kinds of tissues was statistically significant (P<0.05).There was high deletion rates of pl6 gene exons in HCC, peritumoral area and LC. The deletion rates of the exonl a of pi6 gene in the three kinds of tissues had no significant difference (P>0.05) , neither had the deletion rates of exon2 and exon3. The mutation rates of exon2 of pl6 gene in HCC and LC were 20.0%(7/35) and 3.2%(1/31), there was significant difference ( x 2=4.3422, i><0.05). The methylation rates of pl6 gene in the three kinds of tissues had no statistical difference (P>0.05), but the sum of the homozygous deletion rate of exonl a and the methylation rate of pl6 gene in LC, peritumoral area and HCC took on ascending trend and there was significant difference among the three kinds of tissues ( x 2=7.4111, P<0.05, v =2).The expression loss rates of pl6 mRNA in HCC, peritumoral area and LC tissues were 51.4% (18/35) , 28.6% (10/35) and 22.6% (7/31) respectively, there was significant difference (P<0.05) .The expression loss rates of pl6 protein in the three kinds of tissues were 62.9% (22/35) , 51.4% (18/35) and 29.0% (9/31) respectively, with significant difference (F<0.05) . There was positive correlation between the expression loss rate of pl6 mRNA and the low differentiation degree of HCC and the integration of HBV DNA (coefficient of correlation 0.202 and 0.185 respectively, P<0.05).The expression rates of Rb protein in HCC, peritumoral area and LC tissues were 34.3% (12/35), 74.3% (26/35 ) and 83.9% (26/31) respectively, there were significant difference between HCC and peritumoral area (x2=11.2829, P<0.001) and between...