| Part 1 Correlation analysis between candidate gene single nucleotide development of HBV chronic hepatic diseaseObjective:This section has 3 research purposes:1.The relationship between the SNPs of interferon and its receptor genes,the SNPs of oxidative stress genes,and rs2275959 with the occurrence and development of HBV chronic liver disease was analyzed by three genetic models.2.The study analyzes the relationship between haplotypes formed of SNPs with close physical position on the same chromosome and the occurrence and development of HBV chronic liver disease.3.The study analyzes the correlation between the interaction of SNPs and the occurrence and development of HBV chronic liver disease by three statistical methods.Methods:1. Selection of candidate genes.The study Used Pubmed database to retrieve functional SNPs or regions,these SNPs or regions may affect the transcription of genes or protein expressions related to HBV infection,and then affect the occurrence and development of HBV infection.Then the specific physical location and MAF of selected SNPs in the Han population in northern China were retrieved through UCSC database and Ensemble database,using Hapmap database to query haplotype information,using SNPinfo Web Server to predict the function of SNPs.A total of 21 SNPs?IFNA1-rs1332190?rs1831583,IFNA2-rs649053,IFNA5-rs7031048?rs3758236,IFNAR2-rs1051393?rs12233338,IFNLR1-rs10903035?rs11249006?rs7525481?rs4649203,IFNL4-rs12971396?rs8113007?rs7248668,CYBA-rs4673,NCF4-rs1883112,NOX4-rs1836882?rs3017887,SOD2-rs4880,GCLM-rs41303970,RP11-150O12.3-rs2275959?were included in this study.2.Sample selection.3128 subjects who met the inclusion criteria were selected from the Fifth Hospital of Shijiazhuang and the First,Second and Fourth Affiliated Hospitals of Hebei Medical University,including:840Negative Control?Ne C?individuals,496 natural clearances,691 patients with CHB,680 patients with LC,421 patients with HBV-HCC.The CHB group,LC group and HCC group are collectively referred to as HBV-induced liver disease group?HLD?.Fasting vein anticoagulant blood of the study subjects was extracted for backup.At the same time,the baseline data,examination results and disease status of the study subjects were collected in the form of questionnaire.3.Genotyping and data analysis.DNA was extracted from anticoagulant whole blood.21 SNPs were tested by MALDI-TOF MS platform.4.Statistical analysis.Comparison of multiple sets of measurement data using one-way analysis of variance?normal distribution with homogeneous variances?or K-W H rank sum test?non normal distribution and or heterogeneity of variance?.The chi-square test was used to compare two or more sample rates or constituent ratios.Unconditional logistic regression analysis was used to analyse the relationship between SNPs and the occurrence and development of diseases and calculate odds ratio?OR?and95%confidence intervals?95%CI?.The relationship between the baseline data and the occurrence and development of HBV chronic hepatic disease was analyzed.According to the research purpose,it was divided into different subgroups to study the association of 21 SNP loci with HBV susceptibility?HLD vs.Ne C individuals?,HBV natural clearance?HLD vs.natural clearances?and HCC susceptibility?HCC vs.LC+CHB,HCC vs.Ne C individuals?.P?0.05 was considered statistically significant,and the test level was bilateral.SPSS 21.0 statistical software was used for data processing and analysis.Haplo View software was used to analyze the association between haplotype and disease.The SNPStats software was used to perform Hardy-Weinberg equilibrium?HWE?on the genotype frequencies of 21 SNPs in Negative Control individuals.Gene-gene interaction analysis was performed by Generalized Multifactor Dimensionality Reduction?GMDR?,Logistic Regression Analysis?multiplication model?,and four-by-two table?addition model?.Results:1 The allele frequencies of 21 SNPs were consistent with the HWE expectations for controls?P>0.05?,which proved that the subjects were representative of the population.2 Association of interferons and their receptors gene polymorphisms with the occurrence and development of HBV chronic liver disease.2.1 SNPs related to the susceptibility of HBV chronic liver diseaseA case-control study was conducted with HLD as case group and Ne C individuals as control group.The 14 sites of interferon and its receptor series,together with smoking,drinking,gender and age,were introduced into the unconditional logistic stepwise regression model.The results showed that there were 5 loci related to the susceptibility of HBV chronic liver disease in the co-dominant model,of which three were risk SNPs:IFNAR2-rs1051393GT/TT vs.GG?P=0.001,OR=1.430;P=0.006,OR=1.471?,IFNLR1-rs4649203GG vs.AA?P=0.002,OR=1.676?,IFNLR1-rs7525481CT vs.CC?P=0.002,OR=5.907?;2 were protective SNPs:IFNLR1-rs11249006AG/GG vs.AA?P=0.000,OR=0.228;P=0.00234,OR=0.130?,IFNAR2-rs122333338CC vs.TT?P=0.003,OR=0.115?.In the dominant model,4 SNPs entered the equation,of which 3 were risk factors:rs1051393?GT+TT?,rs4649203?AG+GG?,rs7525481?CT+TT?,their OR values were 1.372,1.222and 3.975 respectively;one protective factor:rs11249006?AG+GG?,OR=0.239.In the recessive model,there are two sites entering the equation:compared with AA+AG,rs4649203GG is a risk factor:OR=1.478;compared with TT+TC,rs122333338CC is a protective factor:OR=0.185.Three SNPs near the IFNLR1 gene,rs7525481?T,rs10903035?G and rs11249006?G,constitute a haplotype block,and haplotype TAA was a risk factor for HBV chronic liver disease?P=0.0038,OR=1.208?,the haplotype composed of CAG was a protective factor(P=3.428×10-32,OR=0.116).The 3-factor model consisting of rs7525481,rs11249006,and rs10903035 was the best interaction model associated with HBV chronic liver disease.According to the interaction combination of three factors,the subjects were divided into"high-risk"group and"low-risk"group.The risk of HBV chronic liver disease in the"high-risk"population was 2.186?1.672,2.858?times higher than that in the"low-risk"population.The multiplication interaction results suggested that the risk of the main effects of rs7525481?T and rs11249006?A leading to disease were OR=2.258 and OR=1,respectively.The risk of disease due to their interaction was OR=1.240,so the two are positively multiplicative interaction.The risk of the main effects of rs1051393?T and rs649053?G leading to disease were OR=1.513 and OR=1,respectively.The risk of disease due to their interaction was OR=1.364,so the two are positively multiplicative interaction.There was a negative additive interaction between rs7525481?T and rs10903035?G,the RERI of disease due to the interaction of two loci was-2.67,when the two factors were present at the same time?CT+TT,AG+GG?,the risk of disease was 0.389 times that of the sum of them when they were present separately.2.2 SNPs affecting the ability of the body to clear HBVA case-control study was conducted with HLD as case group and natural clearance group as control group.The 14 sites of interferon and its receptor series,together with smoking,drinking,gender and age,were introduced into the unconditional logistic stepwise regression model.The results showed that:in the dominant model,IFNLR1-rs4649203AG+GG was a risk factor compared with wild-type homozygous AA?P=0.008,OR=1.344?,and individuals carrying AG and GG genotypes were less likely to clear HBV.No haplotypes associated with HBV clearance ability were found.The GMDR method did not find the optimal interaction model.The multiplication interaction results suggested that the risk of the main effects of rs7525481?T and rs11249006?A leading to disease were both OR=1.The risk of disease due to their interaction was OR=1.658,so the two are positively multiplicative interaction.The 10 pairs of SNPs showed no interaction based on the additive model.2.3 SNPs related to the progression of HBV-associated hepatocellular carcinoma?HBV-HCC?HBV-HCC was used as the case group,and CHB and LC were used as the control group for case-control study.The 14 sites of interferon and its receptor series,together with smoking,drinking,gender and age,were introduced into the unconditional logistic stepwise regression model.In the co-dominant model,there were two SNPs entering the equation:individuals with rs1051393TT were more likely to develop from HBV chronic liver disease to HCC than those with GG genotype?P=0.021,OR=1.497?;rs7248668GA was a dangerous genotype compared with GG?P=0.002,OR=1.876?.In the dominant model,only one locus entered the equation:compared with GG,rs7248668GA+AA was a risk factor,which was more likely to develop into HCC.In the recessive model,there were two SNPs entering the equation:compared with GG+TG,rs1051393TT was a risk factor?P=0.006,OR=1.512?;compared with AA+AG,rs649053GG was a risk factor?P=0.019,OR=1.421?.Three SNPs near the IFNL4 gene,rs12971396?G,rs8113007?T and rs7248668A,constituted a haplotype block,and haplotype GTA was a risk factor for HBV-HCC?P=0.0362,OR=1.440?,the haplotype composed of CAG was a protective factor?P=0.0423,OR=0.721?.The GMDR method did not find the optimal interaction model.The multiplication interaction results suggested that the risk of the main effects of rs1051393?T and rs7031048?G leading to HBV-HCC were both OR=1.The risk of disease due to their interaction was OR=2.325,so the two are positively multiplicative interaction.The risk of the main effects of rs4649203?A and rs7248668?A leading to HBV-HCC were OR=1 and OR=1.774,respectively.The risk of disease due to their interaction was OR=1.995,so the two are positively multiplicative interaction.3 The relationship between oxidative stress gene polymorphisms and the occurrence and development of HBV chronic liver disease.3.1 SNPs related to the susceptibility of HBV chronic liver diseaseA case-control study was conducted with HLD as case group and Ne C individuals as control group.The 6 SNPs of oxidative stress series,together with smoking,drinking,gender and age,were introduced into the unconditional logistic stepwise regression model.There were 3 SNPs in the codominant model:compared with wild type homozygous GG,mutant heterozygous rs4673AG was a risk factor?P=0.01,OR=1.412?;individuals carrying rs1883112AG/GG were less susceptible to disease than individuals carrying AA genotype?P=0.011,OR=0.783;P=0.007,OR=0.672?;mutant homozygote rs41303970AA was a risk factor compared to wild type homozygous GG?P=0.027,OR=1.951?.In the dominant model,3 SNPs entered the equation,of which there were 2 protective factors:the OR values of rs1883112?Ag+GG?and rs1836882?TC+CC?were 0.755 and 0.831,respectively;there was one risk factor:rs4673?Ag+AA?,whose OR value is1.358.In the recessive model,there were three sites entered the equation:rs1883112GG was a protective factor compared to AA+AG:OR=0.755;rs4880GG was a protective factor compared to AA+AG:OR=0.507;individuals carrying rs41303970AA are more likely to develop disease than individuals carrying AG+GG:OR=1.991.No haplotypes associated with susceptibility to HBV chronic hepatic disease were found.The 4-factor model consisting of rs1883112,rs1836882,rs4880 and rs3017887 was the best interaction model associated with HLD.According to the interaction combination of four factors,the subjects were divided into"high-risk"group and"low-risk"group,among them.The risk of HLD in the"high-risk"population was 2.029?1.479,2.782?times higher than that in the"low-risk"population.There was a positive multiplication interaction trend between rs1836882?A and rs1883112?T?P=0.061,OR=1.200?.The 15 pairs of SNPs showed no interaction based on the additive model.3.2 SNPs affecting the ability of the body to clear HBVA case-control study was conducted with HLD as case group and natural clearance group as control group.The 6 SNPs of oxidative stress series,together with smoking,drinking,gender and age,were introduced into the unconditional logistic stepwise regression model.The results showed that:the mutant heterozygote rs4673AG in the co-dominance model was a risk factor compared with wild homozygote GG?P=0.045,OR=1.398?.The other 5 SNPs in this series?NCF4-rs1883112,NOX4-rs1836882?rs3017887,SOD2-rs4880,GCLM-rs41303970?were not related to the spontaneous clearance of HBV.3.3 SNPs associated with HBV-HCC progressionHBV-HCC was used as the case group,and CHB and LC were used as the control group for case-control study,no SNPs related to HBV-HCC were found.4 Relationship between RP11-150O12.3-rs2275959T and the occurrence and development of HBV chronic liver disease.Rs2275959T was significantly associated with the occurrence of HBV-HCC.HBV-HCC was used as the case group,and CHB and LC were used as the control group for case-control study.The results showed:compared with CC genotype,CT and TT were risk factors of HCC susceptibility in co-dominant model?P=0.016,OR=1.427;P=0.006,OR=1.561?;the CT+TT genotype in the dominance model was a risk factor compared with wild homozygote CC?P=0.005,OR=1.477?.Taking the Ne C individuals as the control group and HCC as the case group,compared with CC genotype,TT was risk factor of HCC susceptibility in co-dominant model?P=0.003,OR=1.748?;the CT+TT genotype in the dominance model was a risk factor compared with wild homozygote CC?P=0.016,OR=1.463?;the TT genotype in the recessive model was a risk factor compared with CC+CT genotype?P=0.014,OR=1.459?.Conclusions:1.Five SNPs of interferon and its receptor series are associated with the susceptibility of HBV chronic liver disease,of which three are risk SNPs:IFNAR2-rs1051393T,IFNLR1-rs4649203G,IFNLR1-rs7525481T;two are protective SNPs:IFNLR1-rs11249006G,IFNAR2-rs122333338C.Rs4649203G is also associated with the natural clearance of HBV.IFNAR2-rs1051393T,IFNL4-rs7248668G,IFNA2-rs649053G are risk factors for HBV-HCC.2.Three SNPs near the IFNLR1 gene,rs7525481?T,rs10903035?G and rs11249006?G,constitute a haplotype block,and haplotype TAA is a risk factor for HBV chronic liver disease.Three SNPs near the IFNL4 gene,rs12971396?G,rs8113007?T and rs7248668A,constitute a haplotype block,and haplotype GTA is a risk factor for HBV-HCC.3.The interaction of SNPs of interferon and its receptor genes may play an important role in the occurrence and development of HBV infection.4. Five SNPs of oxidative stress genes are associated with the susceptibility of HBV chronic liver disease,including two risk SNPs:CYBA-rs4673A,GCLM-rs41303970A and three protective SNPs:NCF4-rs1883112G?NOX4-rs1836882C?SOD2-rs4880G.Rs4673A is also associated with the natural clearance of HBV.5. Rs2275959T is associated with the occurrence of HBV-HCC.Part 2 The effect of lnc-RP11-150O12.3 polymorphic site rs2275959 C of hepatocellular carcinomaObjective:The study explores the molecular mechanism of the effect of lnc-RP11-150O12.3 polymorphism rs2275959T on the occurrence and development of HCC.Methods:1.Bioinformatics Analysis.RNASNP database was used to predict the secondary structure of RP11-150O12.3.The Lnc RNASNP2 database was used to predict the mi RNAs bound to RP11-150O12.3 and the expression of RP11-150O12.3 in liver cancer and adjacent tissues.We used UCSC database to download RP11-150O12.3 expression data in liver cancer and adjacent tissues.2.Cell and tissue experiments were used to verify the interaction between RP11-150O12.3 and mi R-6739-3p.QRT-PCR was used to detect the expression of RP11-150O12.3 in five cell lines,and the suitable hepatoma cell line was selected for follow-up study.RP11-150O12.3 was localized in four cell lines by FISH assay of cell slide.We used q RT-PCR to detect the expression of RP11-150O12.3 and mi R-6739-3p in liver cancer and adjacent tissues.Whether the expression of RP11-150O12.3 and mi R-6739-3p in liver cancer tissues was changed due to different rs2275959 genotypes was analyzed.Besides,the correlation between the expression of RP11-150O12.3 and mi R-6739-3p was analyzed.Luciferase reporter gene detection technology verified the sponge adsorption of RP11-150O12.3 and mi R-6739-3p.Finally,we used q RT-PCR to detect whether the binding of RP11-150O12.3 and mi R-6739-3p affected the expression of RP11-150O12.3.3.Cell and animal experiments were used to study the effect of RP11-150O12.3 on the biological function of QGY7703.We constructed QGY7703 HCC cell lines with stable overexpression and knockdown of RP11-150O12.3 using lentivirus packaging and infection techniques.The effects of RP11-150O12.3 on the proliferation of QGY7703hepatoma cells were detected in vitro assays by MTT,clonogenic experiment,and in vivo assays by subcutaneous tumorigenesis in nude mice.Changes in migration and invasion ability of QGY7703 cells after overexpression or knockdown of RP11-150O12.3 were detected by Transwell chamber assay.Finally,we used flow cytometry to make clear whether RP11-150O12.3 can regulate QGY7703 cell cycle and apoptosis.Finally,flow cytometry was used to analyze whether RP11-150O12.3 affected the cell cycle and apoptosis.Results:1.Bioinformatics Analysis.The local secondary structure of RP11-150O12.3 predicted by the RNASNP database showed that the wild-type base C of rs2275959 is more easily complementary combine to other nucleic acids,while the mutated base U is located at the stem-loop junction and continuous A-U/C-G pairs can form stable hydrogen bonds,increase intermolecular force and make molecules more stable.Subsequently,the Lnc RNASNP2 database presents that RP11-150O12.3 and mi R-6739-3p have a sponge adsorption effect when rs2275959 is wild-type C.When rs2275959 is mutated to U,the adsorption may disappear.The Lnc RNASNP2and UCSC databases showed that the expression of RP11-150O12.3 in liver cancer tissues is higher than that in adjacent tissues,and the original data downloaded by TCGA supports this conclusion.2.Cell and tissue experiments were used to verify the interaction between RP11-150O12.3 and mi R-6739-3p.The expression of RP11-150O12.3 in BEL7402,QGY7703 and Hep G2=0.002),the expression of RP11-150O12.3 in hep3B cells was not significantly different from that in LO2 cells.The localization of RP11-150O12.3 in three hepatoma cell lines BEL7402,QGY7703,Hep G2and normal hepatocyte LO2 was mainly located in the cytoplasm.The expression of mi R-6739-3p in BEL7402,QGY7703,Hep G2 and Hep3B cells was lower than that in LO2 cells,the difference was statistically significant?P=0.000?.RP11-150O12.3 was detected in 30 pairs of fresh liver cancer tissues and their adjacent tissues.The expression of RP11-150O12.3 in liver cancer tissues was higher than that in adjacent tissues(Z=-3.898,P=9.7×10-5).The expression of RP11-150O12.3 in the liver cancer tissue increased gradually from individuals carrying the rs2275959CC genotype?Median=1.765?to individuals carrying the CT genotype?Median=4.496?=0.040).The expression level of mi R-6739-3p in liver cancer tissues is lower than in adjacent tissues?Z=-3.363,P=0.001?.The correlation between RP11-150O12.3 and mi R-6739-3p expression levels was analyzed in liver cancer tissues,and there was a negative correlation between the two(rs=-0.693,P=2.2×10-5).Further analysis of the correlation between the expression levels of RP11-150O12.3 and mi R-6739-3p in liver cancer tissues of individuals with different rs2275959 genotypes,it was found that the expression levels of the two RNA were negatively correlated in the cancer tissues of individuals with CC and CT genotypes(rs=-0.734,P=3.5×10-4),there was no correlation between the expression levels of the two RNA in the cancer tissues of individuals with TT genotype?rs=0.009,P=0.979?.The results of the dual luciferase reporter gene showed that rs2275959-C mediated sponge adsorption of RP11-150O12.3 and mi R-6739-3p.q RT-PCR further demonstrated that this adsorption can result in a decrease in the expression of RP11-150O12.3.3.Cell and animal experiments were used to study the effect of RP11-150O12.3 on the biological function of QGY7703.We have successfully constructed stable cell line of QGY7703 that over expressed and knocked down of RP11-150O12.3.The results of MTT assay showed that the proliferation ability of QGY7703 cells over expressing RP11-150O12.3 was enhanced?P=0.04?after 48 hours of culture compared with the empty plasmid control cells?Control?.The cell proliferation ability decreased?P=0.042?compared with the unrelated sequence control group?shcontrol?when we knocked down RP11-150O12.3 in QGY7703.The clone formation rate of QGY7703 cells overexpressing RP11-150O12.3 was higher than that of the Control group?P=0.005?,and the clone formation ability of QGY7703 cells decreased after knocking down of RP11-150O12.3(P=4.26×10-4).Tumor forming experiment in athymic mice showed:the tumor growth rate and final weight of Balb/c mice subcutaneously injected with QGY7703 cells overexpressing RP11-150O12.3 are higher than those of mice injected subcutaneously with Control cells(Pweight=0.004);Balb/c mice subcutaneously injected with QGY7703 cells that interfered with RP11-150O12.3 had lower tumor growth rates and final weights than mice injected subcutaneously with shcontrol cells(Pweight=0.011).Changes in migration and invasion ability of QGY7703 cells after overexpression or knockdown of RP11-150O12.3 were detected by Transwell chamber assay.The results showed that the migration and invasion ability of RP11-150O12.3 overexpression group was higher than that of the Control group?P=0.028,P=0.010?,and the migration and invasion ability of RP11-150O12.3 knockdown group was lower than that of the shcontrol group?P=0.002,P=0.024?.The results of flow cytometry showed that:compared with the Control group,QGY7703 cells overexpressed RP11-150O12.3 decreased the proportion of G0/G1 phase?P=0.001?,significantly increased the proportion of S phase(P=1.9×10-4),and promoted the transformation of cell cycle from G0/G1 phase to S phase.In contrast,compared with the Control group,QGY7703 cells with RP11-150O12.3 knockdown increased the proportion of G0/G1 phase(P=1.13×10-4)and decreased the proportion of S phase?P=0.049?,which made the cells block at G0/G1 phase.The results of apoptosis experiment showed that:Compared with the Control cells,the apoptosis rate of QGY7703 overexpressed RP11-150O12.3T decreased?P=0.0011?,and the difference between the two groups was more obvious after treatment with5-Fu(P=1.12×10-4).In contrast,compared with the control group,the proportion of apoptosis of QGY7703 cells knocked down of RP11-150O12.3was increased?P=0.003?,and the difference between the two groups of cells treated with 5-Fu also increased?P=0.001?.Conclusion:1.The expression of RP11-150O12.3 in BEL7402,QGY7703,and Hep G2 cells is higher than that in LO2 cells,and it is localized in the cytoplasm.The expression of mi R-6739-3p in BEL7402,QGY7703,Hep G2and Hep3B cells is lower than that in LO2 cells.2.The expression of RP11-150O12.3 in liver cancer tissues is higher than in adjacent tissues.The expression of RP11-150O12.3 in the liver cancer tissue increases gradually from individuals carrying the rs2275959CC genotype to individuals carrying the CT genotype to individuals carrying the TT genotype,mi R-6739-3p expression has a tendency to gradually decrease.The expression level of mi R-6739-3p in HCC is lower than that in adjacent tissues.The expression levels of RP11-150O12.3 and mi R-6739-3p are negatively correlated in the cancer tissues of individuals with CC and CT genotypes.3.The interaction of RP11-150O12.3 with mi R-6739-3p depends on the rs2275959C and results in a down-regulation of the expression of RP11-150O12.3.4.As an oncogene,RP11-150O12.3 can promote the proliferation,migration and invasion of HCC cells,promote the progress of the cell cycle,and inhibit apoptosis in vitro;in vivo can promote tumor formation in nude mice. |
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