Study On The Relationship Between The Upper Digestive Tract Cancer And The Infection Of Human Papillomavirus & Epstein-barr Virus

Objective1. Human papillomavirus ( HPV ) infection rates and typs were examined in tissues of esophageal carcinoma( EC ) patients from different areas of China to understand HPV prevalence in EC tissues; The ratios of E2 / E6 genes were detected to analyze integration status of HPV16 in EC; HPV location in the chromosome were detected; Meta-analysis of relevant literature for the published articles in Chinese related to HPV and EC, and further investigate the relationship between HPV infection and etiology of EC in China.2. The present study pooled the data from the papers in PubMed database concerning EBV-related gastric cancers and performed a meta-analysis of 22 research papers to figure out the relationship between Epstein-Barr virus (EBV) infection and clinicopathological features of gastric carcinoma; Construct eukaryotic expression vector pcDNA3.1(+)/BARF1-his with recombinant of BARF1 gene encoded by EBV and pcDNA3.1(+)-his for establishment in vitro model of EBV infection in the future to further explore the relationship between EBV infection and the etiology of gastric cancer.Methods1. HPV positive rates were determined in EC samples from different areas of China by nested polymerase chain reaction ( NPCR ) with the general primer sets of PGMY09/11 and GP5+/6+ for HPV L1 gene, and type HPV16 and HPV18 were detected with the nested multiplex PCR ( NMPCR ) assay, which composed of degenerate primers of E6-E7 conservative region and type-specific primers, and analysis HPV infection of the EC samples in different regions.2. The HPV16 positive specimens were subjected to real-time quantitative PCR for determination of the ratios of E2 to E6, analysis physical status of HPV16;Detection ofβ-actin content using fluorescent PCR in each sample,and calculation viral load of HPV16 in every cell of these specimens by HPV16E6 andβ-actin ratio.3. Amplification of first strand cDNA by RT-PCR with Oligo (dT) 17-P3 primer, and amplification the first round with HPV E7-specific P1 forward primer and the P3 reverse primer, and amplification the second round with HPV E7-specific P2 forward primer and Oligo (dT) 17-P3 reverse primer, the second round amplified products were cloned into pMD-18T vector and sequenced to analyze HPV integration in the human chromosome location.4. We searched and collected the published articles in Chinese related to HPV and EC, and selected the articles for detection HPV in the esophageal cancer specimens and meta-analysis were performed for the papers concerning HPV-related EC of China. according to Cochrane system, in accordance with the Mantel-Haenszel method, the literature heterogeneity test data to the application immediately effect model to calculate the research literature in esophageal cancer cases and normal control group, the odds ratio (odds ratio, OR), weight (weight), 95% confidence interval (95 % CI) and the combined weight and 95% CI. Statistical analysis was performed by using the Review Manager 4.2 for meta-analysis software version; P value less than 0.05 was considered statistically significant. Further verify the etiology role in occurrence of esophageal cancer in China.5. Articles were searched from the PubMed database (http:// www.ncbi.nih.gov/pubmed) using the keywords"Epstein-Barr virus and gastric carcinoma". Data was summarized from the eligible articles and a meta-analysis was performed to explore the association between EBV and the clinicopathological characteristics of gastric cancer. According to Cochrane system, in accordance with the Mantel-Haenszel method, the literature heterogeneity test data to the application immediately effect model to calculate the research literature in esophageal cancer cases and normal control group, the odds ratio (odds ratio, OR), weight (weight), 95% confidence interval (95 % CI) and the combined weight and 95% CI. Statistical analysis was performed by using the Review Manager 4.2 for meta-analysis software version; P value less than 0.05 was considered statistically significant. Further verify the etiology role in occurrence of gastric carcinoma.6. The specific primers were designed according to the BARF1 gene cDNA sequence in Genbank provided and restriction sites added at both ends; BARF1 construct eukaryotic expression vector of BARF1.Results1. HPV was detected from collected specimens of esophageal cancer patients, and HPV-positive rates were: 93.2% (109/117) in Llinzhou city of Henan province; 88.1% (37/42) in Baoding city of Hebei province; 95% (19 / 20) in Yanting city of Sichuan province. HPV16 type was main type, second was HPV18 type. Among them, HPV16-positive rates was 59.6% (65/109), 51.4% (19/37) and 57.9% (11/19); HPV18-positive rate was 19.3% (21/109), 21.6% (8/37) and 21.1% (4/19).2. The real-time quantitative PCR results showed that only 6.3 % ( 2/32 ) of the HPV16 positive specimens harbored exclusively episomal form, with E2/E6 ratio > 1, the mixture of episomal and integrated forms 84.3 % ( 27/32 ), with 0< E2/E6 ratios<1, and exclusively integrated form 9.4 % ( 3/32 ) , with E2/E6 ratio =0, indicating a predominance of integration of viral DNA in the host genome; Viral load of HPV16 were 0.066-65.2 copyies/cell in 32 HPV16 E6 positive samples.3. Sequence analysis revealed that HPV18 integration in chromosome 5 from two HPV18-positive cancer patients.4. For 17 articles, HPV positive rates were 44.0% in EC specimens, and 16.7% in normal controls. HPV infection in esophageal carcinoma tissues compared with normal controls was statistically significant ( P<0.05 ); HPV16 type-specific were tested with 12 articles, the results showed the positivity of HPV16 were 38.6% in EC, and 15.9% in normal tissues, HPV16 positive rates were significantly difference for EC specimens contrast to normal esophageal tissues by statistical analysis ( P<0.05 ). HPV infection and geographical distribution of significant differences ( P <0.05 ) were found in Henan, Guangdong, Xinjiang, Sichuan, Hebei, Shanghai, Fujian and Hong Kong eight regions.5. A total 5475 cases from 22 papers with gastric cancer were enrolled, of whom 411 cases were found EBV-positive. Among the EBV-positive gastric cancer cases, the detection rate was 11.1% in males and 3.0% in females. Compared with EBV-negative gastric cancer, EBV-positive gastric cancer had less lymph node metastasis. EBV infection rate was 28.3% in residual gastric cancers; EBV infection rates were no statistically significant difference ( P <0.05 ) for histological type and the specimen type.6. 666 bp products of BARF1 were obtained by RT-PCRwith RNA of B95-8 cells. right referred to plasmid identified. BARF1 gene fragment was inserted pUM-T vector by identification of double-digestion. BARF1 gene has been properly connected to the pcDNA3.1 (+)-his eukaryotic expression vector through sequencing analysis.Conclusions1. Our study showed that HPV positive rates were 93.2 %, 88.1 %, 95.0 %, respectively, and average positive rate was 92.2 % in EC samples from Linzhou, Baoding, and Yanting City; Among them the average HPV16 E6 positive rate was 53.1 %, and HPV18 E6 positive rate was 18.4 %. it suggested that HPV plays an etiologic role in the development of esophageal cancer in China.2. Integration of viral DNA in the host genome of HPV16 positive EC samples suggests that integration HPV DNA is common, and implies that HPV16 infection may play an etiologic role in EC development.3. HPV18 may be integrated in one part of chromosome 5. Integration of HPV genome in the host cell chromosome suggests that persistent HPV infection is a key for malignant cell transformation and canceration.4. HPV infection in Chinese esophageal cancer compared with normal tissue was significant difference ( p<0.05 ), and high risk HPV infection prevalence in different geographical locations. Meta-analysis results suggested the HPV prevalence in the esophageal cancer samples of China and clued the possible etiological relationships between HPV infection and the esophageal cancer development.5. Meta-analysis showed that EBV infection occurred only in gastric cancer tissue cells and was significantly associated with the patients'gender, lymph node metastases, and the location where tumor tissue generated and geographical distribution( P<0.05 ), but was not significantly associated with the patients'histological types of tumor and the type of specimen ( P>0.05 ), and indicated the possible etiological relationships between EBV infection and gastric cancer development.6. The eukaryotic expression vector pcDNA3.1(+)/BARF1-his was constructed successfully, and for establishment in vitro model of EBV infection in the future to further explore the relationship between EBV infection and the etiology of gastric cancer.