| Objective: To screen polymorhisms residing in the MicroRNAs binding sites of HCC-related genes and investigate their associations with HCC susceptibility and the underlined molecular mechanism.Methods: (1) HCC-related genes were selected using bioinformatic methods and literature search, then 3′UTRs or 5′UTRs of these genes were identified. We analized putative miRNA-binding sites by means of specialized algorithms. Then we identified polymorphism within the putative binding sites for their ability to affect the binding with miRNA. (2) Based on the polymorphisms obtained from the first step, we performed case-control association studies using PCR-PAGE method. Logistic regression model was used for evaluating the association between polymorphisms and HCC susceptibility. (3) For the polymorphisms discovered in the second step affecting the HCC sucseptibility, putative miRNAs which would bind within polymorphisms were predicted using specialized algorithms. Meanwhile, Renilla luciferase reporter gene containing alternative alleles of specific polymorphisms were constructed using site-directed mutagenesis and molecular cloning methods. Alternative Renilla luciferase reporter genes were cotransfected with specific miRNAs into HepG2 and SMCC7721 cells and Renilla luciferase activities were measured with the Dual Luciferase assay system. (4) Lastly, Taqman? gene expression method was also used to detect target gene mRNA expression levels in different genotypic HCC tissue samples.Results: (1) 105 HCC-related genes were recruited using bioinformatic methods and literature search and 16 candidate polymorphisms (including single nucleotide polymorphism and insertion/deletion polymorphism) were indentified within 3′UTRs or 5′UTRs of these genes. (2) Two insertion/deletion (Indel) polymorphisms (rs3783553 and rs35569394) were chosen from 16 candidate polymorphisms for the following case-control association studies. rs3783553 ("TTCA"Indel) and rs35569394 ("TCCCACTCTTCCCACAGG"Indel) were located within 3′UTR of IL1A and 5′UTR of VEGF, respectively. The genotyping results revealed that the two polymorphisms we studied were highly polymorphic; the minimal allele frequencies for rs3783553 and rs35569394 were 0.385 and 0.255, respectively in our control samples. No polymorphisms genotyped showed significant evidence for deviation from Hardy-Weinberg equilibrium in controls. Significant diffrence was observed for allelic and genotypic frequencies between HCC and control groups after chi-square testing. Using unconditional logistic regression model adjusted for sex, age, smoking status, drinking status and HBV infection, we found that the homozygote 4N Ins/4N Ins of rs3783553 was associated with a significantly reduced risk of HCC compared with its heterozygote 4N Ins/4N Del and homozygote 4N Del/4N Del (odds ratio=0.30, 95% confidence interval: 0.17-0.54, Ptrend<0.001). As HBV infection was one of the major risk factors, a stratified analysis by HBVinfection status was performed using binary logistic regression model. Significant differences were seen between cases and controls after stratification. The overall trend is that the differences between cases and controls were more obvious in HBV positive than the HBV negative population, suggesting a possible role of this polymorphism in the immune regulation during HBV infection. However, we found that rs35569394 was not associated with HCC after analyzing using the above method, at both the allele and genotype levels (Ptrend=0.87). (3) Based on the positive association results, Renilla luciferase reporter gene containing"TTCA"insertion or deletion alleles of rs3783553 were constructed using site-directed mutagenesis. According to the bioinformatic prediction results, two different Renilla luciferase reporter genes were separately cotransfected with Pre-mir-122 or Pre-mir-378 into HepG2 and SMCC7721 cells. Dual Luciferase assay results showed that compared with the constructs containing"TTCA"insertion alleles, the translation of Renilla luciferase from constructs containing the"TTCA"deletion allele was significantly reduced in the presence of Pre-mir-122 in a concentration dependent manner. For miR-378, we observed same change pattern only when >10 pmol pre-miR-378 were added, suggesting miR-378 binding to IL-1αtranscript may be less affected by rs3783553 genotypes. (4) Taqman gene expression analysis showed that"TTCA"insertion homozygous group had the highest IL-1αmRNA level, followed by heterozygous and"TTCA"deletion group. There were 3 of 26 samples in"TTCA" deletion group in which their IL-1αmRNA is under the detection threshold for this method and their Ct value was conservative determined by the maximum cycle number 40. Non-parametric Mann-Whitney U-test ofΔCts of three genotypic groups showed that the P value was 0.042. Compared with TTCA deletion homozygous group, the average IL-1αexpression levels of heterozygous and TTCA insertion homozygous group were 3.76 and 5.57-fold higher, respectively.Conclusion: (1) rs3783553 polymorphism which located within IL1A-3′UTR was significantly associated with HCC susceptibility, stratification analysis by HBV infection status suggested a possible role of this polymorphism in the immune regulation during HBV infection. However, no significant association was observed for rs35569394 which located within VEGF-5′UTR; (2) Our in vitro and in vivo experiments demonstrated the molecular mechanism between rs3783553 and HCC susceptibility: miR-122 binds and negatively regulates the transcription of IL-1αwhich promote anti-tumor immunity and this regulation is negatively influenced by the presence of the"TTCA"insertion allele, presumably affecting miR-122 binding to the IL-1αtranscript. (3) Considering IL-1αaffects not only various phases of the malignant process, such as carcinogenesis, tumor growth and invasiveness, but also patterns of interactions between malignant cells and the host's immune system. Our results indicated that IL-1αmay be a promising target for immunotherapy, early diagnosis and intervention of HCC. |
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