Single Nucleotide Polymorphisms In Genes Of Tricarboxylic Acid Cycle Enzymes Are Associated With Outcomes Of Patients With Hepatocellular Carcinoma

Background: Hepatocellular carcinoma (HCC) is one of the most lethal malignanciesand is the third most frequent cause of cancer-related deaths. Hepatic resection is the mostimportant radical treatment for selected HCC patients. Despite of improvements in clinicaloperation and strategies surveillance, the prognosis is still dismal, because of the high ratesof postsurgical recurrence and metastasis. Despite rapid development inoncomolecularbiology and increasing number of new biomarker for postsurgicalrecurrence of HCC, there is no biomarker which is widely accepted. Therefore, it isurgently needed to identify novel markers of HCC progression, which will gain a betterunderstanding on the mechanism of post-surgical recurrence and metastasis, and thencontribute to cancer prevention and extend the life expectancy of HCC patients.The tricarboxylic acid(TCA) cycle, which occurs in mitochondria, is the ultimate wayof energy metabolism and the core pathway for the metabolism of sugars, lipids, and aminoacids. Metabolic reprogramming is a hallmark of cancer cells including the core pathwayof TCA cycle. Recent studies revealed the associations with tumor formation in three coreenzymes involved in the TCA cycle, which are succinate dehydrogenase (SDH), fumaratehydratase (FH), and isocitrate dehydrogenase (IDH). And recent genetic studies showedthat mutations in SDH, FH and IDH genes were associated with various tumors, such asbreast cancer, bladder cancer, renal carcinoma and glioma.Single nucleotide polymorphisms(SNPs) represent the differences of geneticbackgrounds different populations. The detection of SNPs just need peripheral blood whichcan easily obtained, and the detection is easy and low costs high-throughput decection caneasily accomplished, moreover the detection is stable, so SNPs are widely used in researchfor formation, progression and prognosis of various diseases. SNPs are also used intumorigenesis, progression and prognosis of HCC. Increasing number of SNPs in variousgenes were investigated in HCC. However, the significance of SNPs in SDH, FH andIDHgenes has not been well investigated in HCC.Purpose: To assess the association between functional SNPs in SDH, FH and IDHgenes and prognosis of HCC patients under operation, and then apply functionalexperiment to validate the association. Finally novel promising biomarkers may be foundto predict prognosis of HCC patients in Han population.Methods: Firstly based on pubic SNP data bases, we predicted functional SNPs inSDH,FH and IDH genes. A total of492patients with pathological verified HCC were recruited retrospectively from Eastern Hepatobiliary Surgery Hospital affiliated to TheSecondary Military Medical University. Then we collected the Clinical data of all thepatients, containing age, gender, serum HBsAg status, AFP level, tumor size, number oftumor, tumor differentiation, tumor stage (TNM stage and BCLC stage), PVTT status, andprophylactic TACE or not. Standard follow-up was performed through medical chartreview, clinical case history and in-person or telephone interviewing. In this analysis, thelatest follow-up data was obtained in2013January. Signed informed consent was obtainedfrom each participant, and this study was approved by the institutional review boards ofEastern Hepatobiliary Surgery Hospital. Blood sample (5ml) was obtained from eachpatient. As the method showed in part1, we made genome DNA, and Genotyping of492patients was performed on the Sequenom iPLEX platform.The luciferase reporter assay was used to assess functional effects of rs3935401located in the3’UTR of the SDHC gene. First,44-bp double-stranded oligonucleotidescarrying either wildtype or variant genotype of SNP rs3935401were synthesized andcloned into the pMIR-REPORT vector (Ambion, Austin, Tex) using restriction enzymesSpe I and Hind III (Takara, Dalian, China). All the constructs were confirmed by DNAsequencing. Human HCC cell lines SMMC-7721and HepG2and human embryonickidney cell line HEK-293T (American Type Culture Collection) were cotransfected witheither pMIR-rs3935401-A or pMIR-rs3935401-G (200ng/well) and the internal controlplasmid pRLTK (Promega)(20ng/well) using Lipofectamine2000(Invitrogen, Carlsbad,Calif) in a24-well plate with2×105cells per well. After48hours, the cells were harvestedto determine luciferase activity using a dual-luciferase reporter assay system kit (Promega)with a luminometer (Tecan, Mannedorf, Switzerland). All transfections were performed intriplicates, and all experiments were independently repeated3times.The SPSS Statistics19.0software (IBM) was used for all statistical analyses in thisstudy. T test was used to assess the difference of means between different groups.χ2testwas used to assess the differences of categorical variables. The association between SNPsand overall survival and recurrence free survival was estimated by multivariate Coxproportional hazard regression model，Kaplan-Meier curve and log-rank test were used toassess overall survival and recurrence free survival between different groups. Three geneticmodels (additive, dominant, and recessive) were used to assess the associations，which onecan get the smallest P value is defined as best fitting model. All P values were two-sided,and P <0.05was considered as the threshold of statistical significance. Results: We selected18SNPs in three genes, because the call rate of rs9708193inIDH gene was lower than90%，rs9708193was not included in further analysis. In clinicalvariables, patients who had up-level of AFP, tumor larger than5cm, multiple lesions, poortumor differentiation, later tumor stage and PVTT also had early tumor recurrence andhigher death rate.In overall recurrence analysis, we found that variant genotype of rs12064957andrs3935401in SDHC gene significantly associated with high recurrence rate of HCCpatients in recessive and additive model, respectively (HR,2.01;95%CI,1.02-3.96, P=0.044; HR,1.24;95%CI,1.00-1.53, P=0.049respectively). Variant genotype ofrs544184、 rs7121782and rs10789859in SDHD gene all showed significantly associationwith high recurrence rate in dominant model. Linkage disequilibrium analysis showedthat the three SNPs have strong linkage disequilibrium, so we selected rs10789859forfurther analysis. Variant genotype of rs1414493in FH gene indicated a good prognosis ofHCC patients (HR,0.77;95%CI,0.61-0.97, P=0.030). rs11540478in IDH2gene wassignificantly associated with recurrence of HCC (HR,0.76;95%CI,0.61-0.95, P=0.015).And cumulative effect analysis showed that the cumulative recurrence risk ofunfavorable genotypes exhibited a significant dose-dependent effect （P=1.69x10-6).Survival tree analysis indicated that rs10789859in SDHD gene was the initial recurrencefactor in all the unfavorable SNPs. Further stratified analysis revealed that recurrence freesurvival (RFS) of patients with low recurrence risk of unfavorable SNPs were longer thanpatients with high risk in single surgery patients group（log-rank P=2.2x10-5）andpatients in TNM Ⅰand Ⅱ stages（log-rank P=7.4x10-5）; Patients treated withoperation combined with TACE had a longer RFS than operation only in high recurrencerisk of unfavorable SNPs subgroup （log-rank P=6.6*10-4）.In overall survival analysis, we found that variant genotype of rs3935401in SDHCgene significantly associated with poor prognosis of HCC patients in additive model (HR,1.64;95%CI,1.27-2.12, P=1.5x10-4)； variant genotype of rs4131826in SDHC geneshowed a better prognosis in dominant model(HR,0.72;95%CI,0.53-0.97, P=0.032);FHrs1414493in FH gene was significantly associated with prognosis of HCC patients indominant model(HR,0.73;95%CI,0.54-0.98, P=0.035)， variant genotype ofIDrs12478653in IDH1gene (HR,0.73;95%CI,0.53-1.00, P=0.047) and rs11540478inIDH2gene(HR,0.68;95%CI,0.50-0.92, P=0.011) indicated a good prognosis of HCCpatients in dominant and additive model respectively. Then cumulative effect was evaluated by the combination of unfavorable genotypes identified from the main effectanalysis of individual SNP, the results showed that the cumulative death risk ofunfavorable genotypes exhibited a significant dose-dependent effect (P=5.8x10-8).Survival tree analysis indicated that rs3935401in SDHC gene was the initial prognosticfactor in all the unfavorable SNPs. rs3935401located in3’UTR，which can change thecombination of SDHC gene with mi-RNA. Double fluorescence report revealed thattransfected with variant (GG) genotype of rs3935401exhibited a significant reduction ofnormalized luciferase activity when compared with cells transfected with plasmidscarrying wild-type genotype in SMMC-7721(P <0.001)、 HepG2(P=0.046) andHEK-293T(P=0.022) cell lines.Conclusions: Our data suggest that genetic polymorphisms in TCA cycle enzymegenes (SDH, FH and IDH gene) were significantly associated with the OS and RFS ofHCC patients treated with TACE. Further functional double fluorescence report exhibited asignificant reduction of normalized luciferase activity when compared with cellstransfected with plasmids carrying wild-type genotype, which also demonstrated the effortsof SNPs on gene expression. Further functional and prospective studies are needed tovalidate our findings and promote the usage in clinical works.