Polymorphisms At The Microrna Binding Site In The 3′untranslated Region Of M RNA Are Associated With Outcome In Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC) is the fifth most common cancer, and it is responsible for more than half a million deaths each year, making it the third leading cause of cancer-related death worldwide, and seriously threatens the health of people. The data about epidemiological and experimental research indicate that this disease is strongly associated with several risk factors,including chronic hepatitis B virus, chronic hepatitis C virus, alcohol abuse,nitrosamines, aflatoxin, liver cirrhosis, water pollution, trace elements, and sex hormones. The molecular mechanism of HCC is not completely understood.Despite improved clinical detection methods and therapies, the prognosis of postoperative HCC patients is still poor due to a high recurrence rate. While the molecular mechanism of HCC carcinogenesis is still not fully understood,there are many prognostic factors and predictors of recurrence associated with the disease, including tumor size, tumor quantity, cell differentiation, venous invasion, and degree of inflammation.Tumor is a kind of new biological formation which is formed by the interaction of multiple risk factors, leads to abnormal regulation of cell division, proliferation and apoptosis. The instability of genomic expression is one of the basic characteristics of tumor. The deletion or amplification of genes often occurs in the morbidity and development of tumor. These alterations can cause antioncogene to deactivation or activation, and cells to a major change. About 50% genes of micro RNA(mi RNA) are located in the fragile sites or gene regions of tumor. The expression of mi RNA is significantly different between tumor and normal human tissues, and the regulation of mi RNA is significantly different in tumor cell lines with among different tissues. Mi RNAs are ~22 nucleotide RNA molecules that act asposttranscriptional regulators of messenger RNA(mRNA) expression by base pairing to the 3′untranslated region(3′UTR) of m RNAs to repress translation.Specifically, mi RNAs target nucleotides at the 5′end, which is known as the seed region of an m RNA’s 3′UTR. Combination perfectly between mi RNA and its target m RNA sequence result in reduced protein levels due to RNA silencing. It can affect cell growth, differentiation, proliferation, apoptosis and other biological processes. Mi RNA is a program valve for function and the regulation of cells. It can not only regulate the expression of multiple proteins,but also the signal transduction of multiple signaling pathways.Recently, more and more scholars pay attention to the research of miRNA single nucleotide polymorphisms(SNPs). SNPs are polymorphisms for sequence of genes that are caused by mutations in a single nucleotide at the chromosomal level, including nucleotide substitution, deletion, insertion, and so on. SNPs widely exist in the human genome, with an average of per500~1000 bases, which accounts for more than 90% types of all known genetic polymorphisms. It is one of the most common genetic variants in human being. With the large-scale scanning of the human genome,the results show that we have about 20000 bases of the SNPs at mi RNA binding site.It not only has the function of genetic markers, but also has the regulating effect of gene expression to cause the difference of disease susceptibility.Increasing evidence suggests that SNPs in the 3′UTR targeted by mi RNAs alter the expression of target gene, thus increasing an individual’s risk for cancer.Polymorphisms at the miRNA binding site can affect significantly to susceptibility, tumor cell metastasis, surgery and natural disease outcome after prognosis in HCC, and some of the SNPs can inhibit the proliferation of tumor cells to protect body. The analysis about mi RNA binding SNPs can help us to estimate the risk with HCC and treatment prognosis.In this study, we genotyped four miRNA binding site SNPs located in the3′UTR of RYR3(rs1044129), C14orf101(rs4901706), KIAA0423(rs1053667)and GOLGA7(rs11337) in HCC patients to assess their relationships withcancer risk and outcomes, and evaluate its role in pathogenesis and development of HCC.Part one Selection and identification of single nucleotide polymorphismsPart one Selection and identification of single nucleotide polymorphisms at the micro RNA binding site in hepatocellular carcinomaObjective: In this study, we selected sites in the 3′UTR of mRNA thainfluences the morbidity and prognosis of HCC, combined with literature anthe NCBI SNP database. We could lay foundation firmly for precisiotreatment of HCC.Methods: 1) Blood samples were collected from 95 HCC patients whundergo HCC resection at the Department of General Surgery of the SeconHospital of Hebei Medical University from January 2008 to December 2010 Blood samples were also collected from 90 healthy controls without a historof any cancer. All procedures were supervised and approved by the hospital’Human Tissue Research Committee, informed consent was obtained from althe patients. 2) The clinical data of patients with HCC were selected includinsex, age, Child-classification, portal vein thrombosis, number of tumor, size otumor, TNM classification. We followed up the patients with HCC btelephone in order to understand the survival of the patients, or their familmembers when they were serious or death. The patients were followed for years. 3) Statistical analysis of the comparisons between the 3 years survivarate of the patients after surgery, and find the factors that may affect thsurvival rate.Genomic DNA was extracted immediately with the WizarGenomic DNA extraction kit. We designed PCR amplification primers of thfour mi RNA SNPs binding site sequences according to the NCBI databasePolymerase chain reaction(PCR) was performed using a PCR Master Mix Kiaccording to the manufacturer’s instructions. DNA sequencing results werjudged by Snapshot technology. 4) The student t-test was used to compare thdifferent between the gender and age of the HCC and normal group. The χtest was used to analyze dichotomous values, such as the presence or absencof an individual SNP in patients and healthy controls. Survival curves wercalculated using the Kaplan-Meier method, and comparisons between thcurves were made using the log-rank test. Multivariate survival analysis was performed using a Cox proportional hazards model. All statistical analyses were performed using the SPSS 18.0 software package. A probability level less than 0.05 were used as the criterion for significance.Results: 1) The results showed that there were no significant differences between the age and gender of HCC group and control group(58.74±12.58 vs 56.83±9.80, P=0.853, and 84/11 vs 81/9, P=0.730). The clinical features of postoperative 3 years survival rates are as follows up: male vs female(31.6%vs 22.2%, P=0.530), ≤ 60 years vs >60(28.8% vs 34.5%, P=0.742),Child-Pugh classification A vs B(32.9% vs 0%, P=0.019), portal vein thrombosis formation vs no portal vein thrombosis formation(32.5% vs 12.5%,P=0.027), diameter of tumor(cm) ≤5 vs > 5(35.7% vs 28.3%, P=0.765),TNM staging 0-I vs II-III(46.7% vs 22.4%, P=0.017), single tumor vs multiple tumor(30.9% vs 30.0%, P=0.871). 2) None of these SNPs,rs1044129, rs11337, rs1053667 and rs4901706, was associated with HCC cancer risk by distribution frequency. 3) We compared the survival rates of the post-operational patients with HCC between genotypes for each SNP. The three-year survival rate for RYR3 is AA vs AG+GG(39.4 % vs 25.5 %,P=0.047), C14orf101 is GG vs AA+AG(27.1 % vs 35.0 %, P=0.573),KIAA0423 is TT vs CT+CC(32.8% vs 26.7%, P=0.717), GOLGA7 is GG vs GT+TT(30.8% vs 30.6%, P=0.594). We performed a multivariate analysis with the Cox proportional hazards model for these predictive factors. The relative risks of these factors are as follows: RYR3(rs1044129) is 1.812(95%confidence interval: 1.026~3.201, P=0.041), Child-Pugh classification is2.464(95% confidence interval: 1.020~5.951, P=0.045),TNM classification is1.922(95% confidence interval: 1.037~3.562, P=0.038), Portal vein thrombosis 1.571(95% confidence interval: 0.664~3.719, P=0.304).Part two Polymorphism at the microRNA binding site in the3′untranslated region of RYR3 is associated with outcome in the post-operational patients with hepatocellular carcinomaObjective: In this study, we were located on the miRNA 3’UTR SNPbinding site RYR3 to analyze the expression in tissue of patients with HCC. In addition to analyze the functional effect of RYR3 expression, we constructed a vector containing the genotype of rs1044129 in the 3′UTR region and transfected them in He La cells. RYR3 was genotyped to assess their relationships with the risks and outcomes of HCC.Methods: 1) Liver samples were collected from 88 HCC patients who underwent HCC resection at the Department of General Surgery of the Second Hospital of Hebei Medical University, and also collected from 45 healthy controls with the cavernous hemangioma of the liver, intrahepatic bile duct stones and other benign liver diseases. 2) Genomic DNA was extracted immediately with the Wizard Genomic DNA extraction kit,the experimental methods were same as part 1. 3) We measured RYR3 levels in HCC tissue by immunohistochemistry. The results for all receptors were semiquantified using the histochemistry score(HSCORE). 4) We constructed a vector containing the AA or GG genotype of rs1044129 in the Renilla luciferase gene and transfected them in He La cells. A dramatic reduction in Renilla luciferase activity was observed in the genotypes by psi CHECKTM-2 vector. 5) The χ2test was used to analyze dichotomous values, such as HCC group each genotype expression in HCC. Kaplan-Meier test was used to compare RYR3 low antibody expression in patients with HCC high expression of the survival rate after operation. The t-test was used to compare the different expression levels between genotypic groups with Renilla luciferase reporter assays. All statistical analyses were performed using the SPSS 18.0 software package. A probability level less than 0.05 were used as the criterion for significance.Results: 1) We measured RYR3 expression by immunohistochemistry in all HCC tissues collected. 88 cases were analyzed for RYR3 staining, and the HSCORE was calculated. The distributions of RYR3 expression for each haplotype in patients with HCC are AA low level vs AA high level(29 vs 4),AG low level vs AG high level(8 vs 33), GG low level vs GG high level(3 vs11). We found that the patients with the RYR3 AA genotype had lower levels of RYR3 expression than AG and GG genotype by χ2 test(P=0.001). 2) Thesurvival rate of the patients with HCC with low and high RYR3 levels was compared to Kaplan-Meier method subsequently. The patients with low levels of RYR3 displayed longer survival length than high levels(3-year survival rate, 54.91% vs 27.45%, P=0.032). 3) To analyze the functional effect of rs1044129 on RYR3 expression, we constructed a vector containing the AA or GG genotype of rs1044129 in the 3′UTR region of the Renilla luciferase gene and transfected them into He La cells. A dramatic reduction in Renilla luciferase activity was observed in the AA genotype. The Renilla luciferase activities were defined as the ratio of Renilla luciferase activities versus firefly luciferase activities. The Renilla luciferase activities were AA vs GG(1.61±0.09 vs 2.01±0.14, P =0.019).Part three Effect of polymorphism at the miRNA binding site in the3′untranslated region of RYR3 on the function of human hepatocarcinoma cell line SMMC-7721Objective: In this part, we researched human hepatocarcinoma cell line SMMC-7721 using small interfering RNA silencing RYR3, found changes of cell proliferation and apoptosis after gene transfection, understood the mechanism of rs1044129 at the mi RNA binding site in the 3′UTR of RYR3 on hepatocarcinoma cell, and proved the effect on outcomes of patients with HCC.Methods: 1) We transfected into human hepatocarcinoma cell line SMMC-7721 cells with the mixture used plasmid and Lipofectamine2000. We used to constructed knockdown plasmid of RYR3 used vector of psi-U6,synthesized 4 candidates RYR3-sh RNA and 1 negative control NC-sh RNA.We prepared the stated Escherichia coli, and converted and extracted plasmids,and transfected them into human hepatocarcinoma cell line SMMC-7721 cells with the mixture using plasmid and lipofectamine 2000. It was successed when we observed green fluorescence by an inverted microscope after transfected. We set up 3 groups, blank-control, control-si RNA/lipofactamine2000 and RYR3-si RNA/lipofactamine 2000. 2) Western Blotting techniques were used to screen the effecive si RNA sequences on the expressions of RYR3protein. 3) The proliferation of human hepatocarcinoma cell line SMMC-7721 transfected RYR3 has been detected by MTS method. 4) The apoptosis of human hepatocarcinoma cell line SMMC-7721 transfected RYR3 has been detected by flow cytometry method. 5) The student t-test was used to compare the different expression levels between three groups. All statistical analyses were performed using the SPSS 18.0 software package. A probability level less than 0.05 were used as the criterion for significance.Results: 1) We transfected the plasmid into human hepatocarcinoma cell line SMMC-7721 cells, and we found the green fluorescence cells cultured by inverted fluorescence microscope at the excitation wavelength of 480 nm after3 days. The transfection efficiency could reach 91%. 2) We detected SMMC-7721 cells by Western blotting, and found that all of the si RNAs,including si RNA-1, si RNA-2, si RNA-3 and si RNA-4, were effectively decrease the protein expression level of RYR3 compared with control group after transfected 48 h(48.6%, 54.7%, 36.9% and 38.2%). Therefore, we selected si RNA-2 for subsequent experiments. 3) We detected absorption value(OD) of the human hepatocarcinoma cell line SMMC-7721 cells transfected with RYR3 gene 12 h, 24 h, 36 h, 48 h, 60 h, 72 h. The OD values of RYR3-si RNA group were 0.867±0.010, 1.184±0.059, 1.441±0.082,1.659±0.102, 1.744±0.065, 1.862±0.033, 1.998±0.093. The Control-si RNA group were 0.868±0.080, 1.327±0.052, 1.837±0.076, 2.220±0.138,2.449±0.072, 2.535±0.035, 2.613±0.176; Blank-control group of 0.863±0.107,1.598±0.131, 1.919±0.059, 2.554±0.082, 2.715±0.078, 2.804±0.112,2.986±0.086. The proliferation of SMMC-7721 cell lines was inhibited during different observation period, and increased the inhibition rate with prolonged incubation time after transfection. The RYR3-si RNA group was significant difference compared with Control-si RNA group and Blank-control(P<0.05). 4)The apoptosis rates after transfection of 3 groups, RYR3-si RNA,Control-si RNA and Blank-control, were 39.010±1.677, 12.379±1.075 and5.373±0.456. The apoptosis rate of RYR3-si RNA group decreased significantly compared with the other groups(P<0.05).Conclusions: 1) In this study, we selected the clinical features and 4polymorphisms at the micro RNA binding site in the 3′untranslated region associated with 3-year survival rate in HCC. These factors, RYR3(rs1044129),Child-Pugh classification, TNM classification and Portal vein thrombosis were associated with outcomes in HCC. 2) AA type of RYR3 decreased obviously in liver tissues of patients with HCC. AA type reduced the He La cell activity after RYR3 plasmid transfected than GG type. 3) RYR3 silencing could significantly inhibit the proliferation and induce apoptosis of hepatocarcinoma cells. A polymorphism rs1044129 at the micro RNA binding site in the 3′UTR of RYR3 changed its binding affinity with mi RNA. Thus affected its expression, then RYR3 affected the proliferation and apoptosis of hepatocarcinoma cell, and finally regulated outcome in HCC. In conclusion, a SNP in the RYR3 mi RNA binding site was found to be a biomarker for HCC outcomes.