Investigation Of SNP Within The APOBEC3A/B Promoter Relevant To The Risk Of Cholangiocarcinoma

Research background and purpose:Bile tract cancer?BTC?are tumors formed in bile duct epithelium,which including cholangiocarcinoma?CCA?and gallbladder cancer?GBC?.CCA accounts for about 3%of digestive system tumors and could be further divided into intrahepatic cholangiocarcinoma?ICC?,perihilar cholangiocarcinoma?p CC?and distal cholangiocarcinoma?d CC?by the anatomical difference,while the later two were already categorized into extrahepatic cholangiocarcinoma?ECC?according to the third edition of“International classification of tumors”^[1].GBC is the sixth most common cancer in digestive system^[6].All the tumors mentioned above have significant differences in etiology,susceptible population,incidence and mortality rates,prognosis characteristics.In terms of etiology,parasite infection?mainly hepatic paragonimus?was the main inducement in coastal areas.Cholelithiasis which characterized by chronic inflammation was the main inducement in China.In Europe and America,primary sclerosing cholangitis?PSC?is the main risk factor^[2-6].CCA has a significantly higher incidence rate in Southeast Asia and a relatively lower rate in Europe and America.GBC has the highest incidence rate in Latin America and the secondary in Asia,while the lowest in Europe and America.From the perspective of the susceptible population,the Asians usually got more risks of CCA.Even in America,the incidence of CCA in Asian-American and Hispanic-American population was higher than that in pure American and African-American population.At the same time,the mortality rate in African-American population was the lowest.It could be seen that the occurrence of BTC was not only affected by the differences in anatomy and tissue type,but also by the geographical,ethnic and gender^[8-11].The prognosis of BTC was poor for the difficulty in early diagnosis so that the primary prevention showed a particularly importance.At present,most of the researches on tumorigenesis focus on the influence of gene mutation which may conduct tumor occurrence and progression.However,it can not be ignored that there is a complex and close internal relationship between tumorigenesis and hereditary background.Single nucleotide polymorphism?SNP?is the variation of DNA sequence caused by single nucleotide change.It is the most common one of mammalian's heritable gene variation which mainly manifested as single base conversion?C>T,Complementary G>A?or transversion?C>A,complementary G>T?.They are widely existed in human genome with good stability.When SNP falls near or inside pathogenic genes,the variation will impact the functions of genes than which may conduct the occurrence and development of diseases.These characteristics makes SNP not only a breakthrough point for cancer research,but also a good combination point for hereditary background and key tumorigenesis gene's function.Apolipoprotein B m RNA editing enzyme catalytic polypeptide?APOBEC?is a kind of protease.In human beings,there are 11 members in APOBEC family whose coding gene was located on chromosome 22,including APOBEC1,APOBEC2,APOBEC3?with 7subfamily member:APOBEC3A,APOBEC3B,APOBEC3C,APOBEC3DE,APOBEC3H,APOBEC3F,APOBEC3G?and activation induced cytosine deaminase?AID?^[40].The main physiological function of APOBEC3 subfamily is introducing high-frequency mutation in the process of reverse transcription with high-efficiency of nucleic acid editing,which plays an important role in the innate and acquired immune response,retrovirus resistant,endogenous reverse transcription inhibition and the breakdown of exogenous DNA^[39,41].However,when the high-frequency mutation caused by it occurs wrongly in vivo,the role of APOBEC3 will change to the main source of mutagenicity.Many research already showed that APOBEC3 family plays some key roles in the occurrence of breast cancer,bladder cancer or colon cancer,especially when its mutagenicity occurs in the oncogenes or anti-oncogenes^[46].At present,there are few studies on APOBEC3 in BTC.To sum up,this research focus on exploring the connections between the risk of BTC and the function of APOBEC3A/3B with the hereditary background in Chinese and the internal mechanism of these connections.This research aimed to add a new way for the early screening of BTC in China and provide new evidence for the tumorigenesis mechanism of BTC.Research methodIn this research,we first used gene database in Ensembl and haploview-4.2 to analyze the promoter region of APOBEC3A/3B.Finally three SNP sites were selected with their characteristics and relationships clarified.Then 3231?1315 female and 1916 male?clinical data and peripheral blood samples were collected,including 1240 healthy people,735patients with CCA,453 patients with GBC,803 patients with cholangitis.The collected clinical information includes:age,gender,ethnic,clinical diagnosis,hepatitis B infection.The level of AFP,CEA and CA19-9 were collected in patients with biliary system diseases.After plasma were separated from blood samples,SNP detection and analysis were carried out step by step.Finally,a population study database was established based on clinical information and SNP typing results,then the relationship between SNPs in the promoter region of APOBEC3A/3B and the risk of BTC could be analyzed.Then,APOBEC3A/3B promoter plasmids were constructed with different alleles,transfection assays were conducted in normal bile duct epithelial cells?HIBEpic?,bile duct cancer cells?RBE?and gallbladder cancer cells?GBC-SD?,respectively.The transcriptional activity of APOBEC3A/3B promoter in different cells were observed by double luciferase assays.The apoptosis assays using overexpression plasmids of APOBEC3A/3B were conducted by flow cytometry in 293T cells with Western blot was used to verify the effect of over expression.TCGA database was explored to find out the expression levels of APOBEC3A/3B in CCA tissue and to analyze the correlation between APOBEC3A/3B and cell mutation,which can confirm their ability to affect cell status.Using the online prediction website of PROMO,we predicted the transcription factor which can be combined to APOBEC3B promoter region.Then si RNA?small interference RNA?was used to knockout the transcription factor and luciferase assay was used to screen the key transcription factors that may play a regulatory role.Finally,si RNA and promoter plasmids were co-transfected to observe the change of promoter activity in APOBEC3B promoters,while RT-q PCR was used to quantify the expression levels of key transcription factor in different cells.The main statistical methods are t-test or one-way ANOVA test?when the data conform to the normal distribution?,rank sum test?when the data do not conform to the normal distribution?,?² test?classification variable?,and logistic regression.In the study,age and gender were considered to have distribution differences between the biliary disease group and healthy control group,so they were included in the analysis for a correction,then AORs?adjusted odds ratio?and 95%confidence interval?CI?were obtained.All the statistical tests were double tail tests,and the difference was statistically significant when p<0.05.The statistical software used is SPSS 23.0.Research results1.SNP site determination:3 SNP sites finally selected:?1?APOBEC3A rs12157810?-525bp,promoter region,A>C?;?2?APOBEC3B rs2267401?-338bp,promoter region,T>G?;?3?APOBEC3A rs12628403?+4340bp,intron 4 region,A>C?.Among them,APOBEC3A rs12628403 is the deletion type"proxy"site of APOBEC3B rs2267401,and the"proxy"compliance rate is 97.5%in 200 random verification samples.2.Analysis of the relationship between the SNPs of APOBEC3A/B promoter and the occurrence risk of BTC:the impact of APOBEC3A/3B on the risk of BTC in the context of SNP reflects many characteristics with anatomy,tissue type and gender differences.In conclusion,the C allele of APOBEC3A rs12157810 plays the key role in reducing the risk of BTC,while APOBEC3B rs2267401's G allele showed an opposite effect between CCA?reducing the risk?and GBC?increasing the risk?.3.The difference of the activity of APOBEC3A/3B promoter in cells:in HIBEpic,RBE and GBC-SD,the promoter transcription activity of APOBEC3A rs12157810 C-allele is stronger than that of A-allele.The promoter transcription activity of APOBEC3B rs2267401 G-allele is weaker than that of T-allele in HIBEpic and RBE,but is significantly stronger than that of T-allele in GBC-SD.4.The effects of overexpression with APOBEC3A/3B on the apoptosis of 293T cells:the overexpression of APOBEC3A significantly promoted the apoptosis of 293T cells?apoptosis rate:293T APOBEC3A 22.48%vs 293T vector 10.54%p<0.05?.The overexpression of APOBEC3B did not significantly promote apoptosis.5.The correlation between APOBEC3A/3B and mutation?TCGA?:In CCA,APOBEC3A was significantly correlated with mutation?r²=0.39?,while the expression level was significantly lower than that of APOBEC3B?P<0.05,3B?10 times 3A?,indicating that APOBEC3A has high efficiency of mutagenicity.6.Prediction,screening and regulation method studies of transcription factors related to APOBEC3B promoter:after screening,the transcription factor TFAP2A showed a significant inhibitory effect on the activity of APOBEC3B G allele promoter.Its expression level was high in CCA while low in GBC,resulting a regulatory difference between cell types.Research conclusion1.Under the context of SNP,the activity difference of APOBEC3A/3B promoters conducted different effects on the risk of BTC with anatomy,tissue type,gender and susceptible population characteristics.The SNP detection of APOBEC3A/3B promoters's style in peripheral blood can be an effective means to offer some clues for early screening in BTC.2.APOBEC3A which related to reduced risk of CCA,has efficient mutagenic effect.Over mutation may lead to tumor cells'apoptosis and reduce the final tumor formation,and ultimately reduce the risk of CCA.3.The different risk effects of APOBEC3B between CCA and GBC were related to the difference of promoter activity between tissues.4.The difference of APOBEC3B promoter activity among tissues maybe mainly due to the inhibitory regulation level of TFAP2A,which could be the main mechanism.