| Background:Primary hepatic carcinoma(Primary hepatic carcinoma,PHC),is a kind of common malignant tumors,now the the global incidence rate is close to 626000/year,and the mortality is about 600000/year.It is the third on the rank order of causes of death,and more than half of new cases of liver cancer in the world’s are in China.According to statistics,annual new cases of liver cancer is 300,000,It is in the second place in the pathogenesis of cancer,the second on the rank order of causes of death.There is a highest incidence of liver cancer in the southeast coast of China.Compared with the country,liver cancer mortality rate in Guangdong Province,is higher than the national average of 50%,and compared with the global,is three to four times of the global average.3 large-scale inquest displayed that iver cancer Mortality was highest among malignant tumors in Shunde,Guangdong.Because of the occult onset,short duration,rapid disease progression of liver cancer,occurring in middle-aged men,the male to female ratio is 2:1-5:1,80%of patients were already in an advanced stage of cancer,they lost the best time for treatment.So it has ineffective treatment and high mortality,five year survival rate is only about 5%,It is seriously affecting the life and health of our residents.To sum,it is having become a local major public health problem to search and establish effective prevention and screening methods to reduce the incidence and mortality of liver cancer.Currently,it is certain that the incidence of liver cancer is a very complex process,it is the results of multiple factors from environment,genetic.There are many risk factors involved in Pathogenic start of hepatocellular carcinoma,tumor promotion and progression.So far,It has been confirmed that the infection of hepatitis B(HBV)and hepatitis C virus(HCV),schistosomiasis,liver cirrhosis,alcoholism and intake of aflatoxin are the important causes of liver cancer,.And in China,most of liver cancer patients have a history of HBV infection.But related research also shows that there are great individual differences in immunity against HBV virus.It means after HBV virus infection,the progression and deterioration of the disease depends on the polymorphisms of susceptibility gene in the patient’s own immune.And currently the data about the polymorphisms of susceptibility gene related to liver cancer in Guangdong Province is very few.In the past decades,research on the relationship between human genes and cancer was basically focused on the structure of genes and their associated expression control sequence,but in recent years,studies have shown that non-coding sequence of the human genes play a crucial role in tumor biology behavior.Recently long non-coding RNA(lncRNA)which once be considered as transcribed "noise" has been confirmed to play a key regulatory role in chromatin remodeling,histone modifications,transcription interference,proto-oncogene activation and other biological process.Compared to the protein-coding genes,lncRNA in tumors is very precise and specific expression,which makes it promise to be the new tumor marker.lncRNA has become a research hotspot in malignant etiology such as liver cancer.It has been confirmed the abnormal expression of lncRNA having a closely related to hepatic carcinoma’s appear,metastasis,prognosis or diagnosis.Recent studies have found that some lncRNA transcript nearby MYC,and be involved in the regulation of MYC multidimensionally.8q24 was being considered as"gene desert" which lacked of protein-coding genes.The IncRNA which locates in upstream region of MYC proto-oncogene of 8q24 chromosomal region was identified possible cancer sites by GWAS.And it is considered to be high-risk areas of a variety of cancers(prostate,ovarian and colon cancer).CCAT1、CCAT2、PVT1、CASC8、PRNCR1 gene locates in the area has been considered to be related to the incidence of many types of cancer.And some of them have been very effective\particular cancer biomarkers.But the report about the gene and liver cancer is very few.There are only a few of articles involving CCAT1 highly expressed in liver cancer.And these researches had limited efficacy because of small sample size and less comprehensive SNP locus.In summary,it’s has an important clinical significance to carry out epidemiological investigation and study to clarify evoking factors of liver cancer,and to study on the relationship between IncRNA genetic polymorphism and cancer susceptibility by using research methods based on single nucleotide polymorphisms and haplotype、expanding the sample size、applying case-control and cohort study.It can be helpful for predicting liver cancer risk of individuals and groups,finally,it brings benefit to raise the level of early prevention,early diagnosis and early treatment of liver cancer.So our study will be developed.In this paper,epidemiology,molecular biology,organic combination of bioinformatics and statistical method would be combined,and potential functional variant sites of CCAT1,CCAT2,PVT1,CASC8 and PRNCR1 would be chose.The purpose was to identify potential functional variant associated with HCC,reveal an important influence on liver cancer gene-gene and gene-environment interaction patterns by analyzing the interaction between the candidate gene’s functional variation and liver cancer’s environmental risk factors.Above all,a further research of liver cancer’s genetic susceptibility mechanism can be done,it will provide genetic markers for constructing liver cancer prediction system to promote early prevention、early detection of liver cancer;and provide some theoretical bases for the control of liver cancer ’s epidemic,and reduce the burden of liver cancer.Objective:To screen and predict the CCAT1,CCAT2,PVT1,CASC8 and PRNCR1 located on chromosome 8q24,research the susceptibility between the candidate gene SNPs and primary hepatic carcinoma;reveal the gene-gene and gene-environment interaction model which have a major impact on liver cancer,provide scientific basis for further study on liver cancer genetic susceptibility mechanism.Methods:(1)Based on control study strategy,choose the crowd in Shunde area,Guangdong in 619 cases of liver cancer and 619 healthy controls,do epidemiological surveys to understand the environmental and genetic factors of liver cancer information.The risk factors of liver cancer were analyzed by logistic model;(2)By using the Haploview software,R2>0.8,MAF>0.05 standard,and bioinformatics technology,filtering and prediction CCAT1,CCAT2,PVT1,CASC8 and PRNCR1 potential functional genes of genetic variation;(3)Extract the subject’s DNA by using silicone membrane assay,perform SNP genotyping with the DNA by using iPLEX GOLD technology systems which is belong MassArray molecular array platform in flux that operate by Bio Miao Biological Technology(Beijing)Co.,Ltd,calculate the distribution of genotype and allele frequencies,do Hardy-Weinberg equilibrium test in case group and control group,research the distribution of genetic variation haplotype and frequency analysis by using PHASE2.1 software,analysis the correlation between candidate gene SNPs and the susceptibility of Primary hepatic carcinoma.(4)Reveal candidate genes CCATl,CCAT2,PVT1,CASC8 and PRNCR1’s gene-gene and gene-environment interactive mode that have a major impact on primary hepatic carcinoma based on logistic regression model,crossover analysis,multifactor dimensionality reduction,multi factor variance analysis method,to research the influences that the interaction among gene polymorphism to the genetic susceptibility of liver cancer,finally for the screening of high risk human primary hepatic carcinoma group to provide scientific theoretical bases.Results:1.subjects a total of 1238 cases,include 619 people in cases group,619 in control group.Male to female ratio was 6.5:1,the male mean age(60±12),the female mean age(57±16).The average first diagnosed liver cancer patients in the age of 55±12 among them,mostly in the age of 50-70,accounting for 57.19%,72.05%of patients with a history of hepatitis B infection,68.5%of patients had a history of chronic hepatitis,43.13%of patients have a history of liver cirrhosis,11.47%of patients have liver fluke infection,68.98%of patients are smokers,57.19%of patients drinking,24.88%in patients eating raw fish,58.48%of patients had drinking ditch pond,25.52%of patients have a family history of hepatitis B,14.54%had a family history of liver cancer,19.22%patients have a family history of cancer.2.According to Logistic regression model:Viral hepatitis,hepatitis B virus infection,liver fluke infection,smoking,alcohol consumption,with or without a history of eating raw fish,whether ditch pond drinking,family history of hepatitis,liver cancer family history,family history of cancer a total of 10 factors may be the incidence associated risk factors of primary hepatic carcinoma(P<0.05).The OR and 95%confidence intervals were:18.78(13.48-25.98),18.88(13.53-25.61),3.15(1.46-4.77),1.56(1.37-2.23),1.62(1.22-2.06),1.48(1.12-1.85),1.68(1.34-2.16),2.53(1.84-3.43),3.81(2.27-5.95),1.76(1.23-1.97).3.The 23 candidate genes which selected Mass ARRAY technology typing, according to the HWE law of genetic equilibrium test:except for the rs 10808556 locus in CCAT2 gene,the rs4733813 and rs77290463 loci in PVT1 gene,the rs13281615 locus in CASC8 gene,the rest of loci differences were not statistically significant(P>0.05),so the survey has a good group representation.According to analysis the distribution of 20 SNP loci’s genotype and allele frequencies which is conform HWE law of genetic equilibrium in the case and control group:the rs6989575 site in CCAT1 gene,the rs4733813 and rs77290463 loci in PVT1 gene,their genotype and allele frequencies distribution was statistically significant difference between the two groups(χ2=11.5172,P=0.041,χ2=6.262,P=0.044,χ2=24.654,P=0.000),the others’ differences were not statistically significant(P>0.05).4.According to genotype correlation analysis to 20 SNP loci by using conditional Logistic regression model,the results suggest:the genotype of rs13257657,rs13281615,rs6989575 locus were statistically associated with the risk of liver cancer(P<0.05,OR values>1),it suggested that these three genes may be risk factors for liver cancer,after adjustment for age,sex,history of hepatitis B virus infection,hepatitis B family history and other factors,three SNPs genotypes and the risk of primary hepatic carcinoma remained statistically association(P<0.05,OR values>1).Further analysis of showed that the dominant model,recessive model and additive model showed:rs13281615,rs6989575 were statistically significant in the dominant model(P<0.05,OR values>1),rs13257657 were statistically significant in the dominant model,recessive model and additive model(P<0.05,OR values>1),while the other SNPs genotypes was not statistically associated with primary liver cancer risk(P>0.05).5.The interaction of each genes SNP loci by using the analysis table,based on conditional Logistic regression model,used multiplicative and additive interactions model,it results:there exists effect of PVT1 rs13281615 and CCAT1 rs77628730,CCAT1 rs6989575 and CASC8 rs1562430 gene,the model difference had statistical significance(P<0.05),and the others model difference had no statistical significance(P>0.05).6.CCAT1 rs6989575,rs77628730 and rs7816475,CCAT2 rs6983267,PVT1 rs13281615 and rs13257657,CASC8 rs1562430,PRNCR1 rs1016343,a total of eight genes analysis with drinking history,if had consumed ditch pond,hepatitis B virus infection,history of liver fluke infection,family history of liver cancer,family history of cancer a total of five environmental factors,it resulted:CCAT1rs7816475 Genetic Variation had multiplicative interaction effect with if had consumed ditch pond(P=0.020),HBV(P=0.032),carry rs7816475 GG genotype ditch the drinking pond water was an increased risk of developing liver cancer,OR a value of 1.513(95%CI=1.052 2.176);Carrying rs7816475 GG genotype,AA+AG genotypes of hepatitis b virus infection was significantly increased the risk of liver cancer,the ORvalues were 22.288(95%CI=14.155 35.095);29.163(95%CI=15.503 54.858);CCAT2rs6983267 had multiplicative interaction effect with family history of cancer(P=0.047),Carrying rs6983267 TT genotype,GG+GT genotype of family history was an increased risk of developing liver cancer,liver cancer OR values were 2.981(95%CI=1.792 4.960),8.616(95%Cl=3.013 24.635);PVT1rs13281615Genetic Variation had multiplicative and addition interaction effect with drinking history(P=0.011,P=0.008),Carrying rs13281615 GG genotype,AA+AG genotype drinking history was an increased risk of developing liver cancer,OR a value of 2.625(95%CI=1.884 3.658),1.548(95%CI=1.154 2.078),carrying rs13281615 GG genotype,AA+AG genotypes of hepatitis b virus infection was significantly increased the risk of liver cancer,the OR values were 26.637(95%CI=16.90641.969),17.032(95%CI=11.512 25.199);PRNCR1rs1016343 Genetic Variation had multiplicative interaction effect with drinking history(P=0.048),Carry rs1016343 TT+CT genotypes of alcohol was an increased risk of developing liver cancer,OR a value of 1.416(95%CI=1.047 1.916).7.By analysis of CCAT1 rs6989575,rs77628730 and rs7816475,CCAT2 rs6983267,PVT1 rs13281615 and rs13257657,CASC8 rs1562430,PRNCR1 rs1016343 a total of eight genes,MDR resulted:the best two-factor model were CCAT1 rs6989575 and CASC8 rs1562430 polymorphism,these two-factor model could calculate the highest prediction accuracy(TA=59.25%).And cross-validation consistency reached 10/10.But after 1000 times of permutation test model there was no statistically significant(P>0.05).8.CCAT1 rs6989575,rs77628730 and rs7816475,CCAT2 rs6983267,PVT1 rs13281615 and rsl3257657,CASC8 rs1562430,PRNCR1 rs1016343,a total of eight genes analysis with drinking history,if had consumed ditch pond,hepatitis B virus infection,history of liver fluke infection,family history of liver cancer,family history of cancer a total of five environmental factors,MDR resulted:the best three-factor model were PVT1 rs13281615 polymorphism,hepatitis B virus infection and family history of liver cancer,these three-factor model could calculate the highest prediction accuracy(TA=78.92%).And cross-validation consistency reached 10 times,that means the best three-factor model could operated 10/10.After 1000 times of permutation test model was statistically significant(P<0.001).Conclusions:1.liver cancer incidence is high in medium-elderly in Shunde area,Guangdong province.The relevant risk factors of HCC:Hepatitis b virus infection,liver fluke infection,smoking,drinking,eating fish or not,whether drinking pond water,family history,family history,family history of cancer,cancer of the liver of hepatitis b.2.PVT1 rs13257657,PVT1rsl3281615,CCAT1 rs6989575 may be associated with liver cancer risk,but need further cohort study was confirmed.3.CCAT1,CCAT2,PVT1,CASC8 and PRNCR1 gene-gene model difference had not statistical significance(P>0.05).4.PVT1 rs13281615 and history of hepatitis b virus(HBV)infection,alcohol had multiplicative interaction effect in gene-environment model,But the further study is necessary in the future on a large scale,in other regions and ethnic groups and research in cell and animal level function. |
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