Genetic Associations Of MiRNA-SNPs With The Common Diseases And Integrative Analysis Of " OMICS" Data Of Hepatocellular Carcinoma

ChapterΙ: Genetic associations of single nucleotide polymorphisms in microRNAs and genes of microRNA biogenesis pathway with the common diseasesSingle nucleotide polymorphisms (SNPs) within protein-coding genes have been shown to be associated with susceptibility to and/or clinical outcome of human common diseases. However, few study has been involved in clarifying the genetic associations between the common diseases and SNPs within non-coding RNA genes (e.g. microRNA) and genes in microRNA biogenesis pathway. Recently, examinations of microRNAs (miRNAs) expression pro?les have highlighted the importance of miRNA expression alterations in etiology, classi?cation, progression, and prognosis of multiple common diseases, including hepatocellular carcinoma (HCC), nasopharyngeal carcinoma (NPC) and sepsis. SNPs or mutations in miRNA sequence may change the property of miRNAs through altering its expression and/or maturation, which in turn influences numerous functionally important target protein-encoding genes, broadly affecting miRNA function. Therefore, it is appealing to propose that SNPs occurring in miRNAs and genes in microRNA biogenesis pathway are novel sources of genetic variation that may contribute to disease susceptibility and prognosis. In the present study, we therefore performed the genetic association studies to examine whether the SNPs within miRNAs and genes in microRNA biogenesis pathway have any bearing on the risk or progression of HCC, NPC and sepsis. The main results are as follows:1. The selection of SNPs in microRNA and microRNA biogenesis pathway genes. We first found 186 SNPs in 142 pre-miRNAs by searching 705 pre-miRNAs in miRBase (Release 13.0: April 2009). Then by resequencing, 52 SNPs in pre-miRNAs were confirmed to having the minor allele frequency (MAF) higher than 1% in Han Chinese. In addition, 97 SNPs in 14 microRNA biogenesis pathway genes were selected if they are functional or haplotype-tagging SNPs (htSNP). Totally, 149 SNPs were selected for the subsequent association studies. 2. Several SNPs were significantly associated with the hepatitis B virus-related HCC (HBV-related HCC). 149 SNPs were genotyped in a case-control population of 340 patients with HBV-related HCC and 360 controls, and in a case-control population of 107 patients with non-HBV-related HCC and 108 controls recruited from Guangxi by SNPstream and TaqMan genotyping methods. By unconditional logistic regression analyses controlling for confounding factors, we identified 2 SNPs in pre-miRNAs (i.e. hsa-mir-1283-1 rs57111412 and hsa-mir-1908 rs174561), and 3 SNPs in microRNA biogenesis pathway genes (i.e. EIF2C2 rs4961226, RAN rs7132224 and PACT rs3752689) were significantly associated with the risk of HBV-related HCC. These ?ndings indicate that the hsa-mir-1283-1, hsa-mir-1908, EIF2C2, RAN and PACT may contribute to the risk of HCC onset in Chinese population. In addition, 5 SNPs in pre-miRNA and 4 SNPs in miRNA biogenesis pathway genes were significantly associated with the risk of non-HBV-related HCC, However, these 5 SNPs were not replicated in another independent case-control population, including 174 patients with non-HBV-related HCC and 179 controls recruited from Guangdong. This is the first systematic genetic association study between SNPs in microRNA and microRNA biogenesis pathway genes and the risk of HCC, indicating the pivotal roles of genetic variation of miRNA in the development of HCC.3. Several SNPs were significantly associated with the risk or severity of NPC. 149 SNPs were genotyped in a case-control population of 855 patients with NPC and 1036 controls recruited from Guangxi by SNPstream, Sequenom and TaqMan genotyping methods. Among the 149 SNPs, 51 were significantly associated with the risk or severity of NPC. Then, the 51 SNPs were further genotyped in another independent case-control population recruited from Guangdong, including 997 patients with NPC and 972 controls. For the 52 SNPs in pre-miRNAs, 2 SNPs (i.e. hsa-mir-548j rs4822739 and hsa-mir-149 rs2292832) were significantly associated with the risk of NPC in both populations, 4 SNPs (i.e. hsa-mir-1307 rs7911488, hsa-mir-149 rs2292832, hsa-mir-196a-2 rs11614913 and hsa-mir-453 rs56103835) were significantly associated with advanced lymph node metastasis in both populations. For the 97 SNPs in miRNA biogenesis pathway genes, 2 SNPs (RNASEN rs524138 and rs7731057) were significantly associated with the risk of NPC, 2 SNPs (EIF2C2 rs2977469 and rs3928672) were significantly associated with advanced lymph node metastasis, and 3 SNPs (DGCR8 rs11089328, EIF2C2 rs2977464 and RAN rs7132224) were significantly associated with advanced tumor invasion. It is expected that these SNPs may change the property of miRNAs through altering its expression and/or maturation, which in turn influences numerous functionally important target protein-encoding genes, broadly affecting miRNA function, and ultimately contributed to the development of NPC. Further studies are needed to systematically verify the function and mechanisms of these "susceptibility genes" and "susceptibility polymorphisms" in the development of NPC. 4. Functional polymorphism in hsa-mir-146a was significantly associated with the risk of sepsis. 52 SNPs in pre-miRNAs were genotyped in a case-control population of 255 patients with sepsis and 260 controls recruited from Zhengzhou by SNPstream and TaqMan genotyping methods, and among which 13 SNPs were significantly associated with the risk of sepsis. Then the 13 SNPs were genotyped in an independent case-control study (including 299 cases and 299 controls), and only one SNP (i.e. mir-146a rs2910164) was significantly associated with the risk of sepsis. Compared with ones bearing C/C + G/G genotype, the subjects bearing rs2910164 C/G genotype were significantly associated with an increased susceptibility to sepsis. Previous studies have shown that the miR-146a can negatively regulate the NF-κB mediated inflammatory response through the regulation of IRAK1 and TRAF6. Further, other studies showed that the NF-κB pathway was significantly upregulated, and the inflammatory response was promoted in the subjects bearing C/G genotype. We therefore conclude that the hsa-mir-146a rs2910164 can affect susceptibility to sepsis in the Chinese population.ChapterⅡ: Integrative analysis of"OMICS"data of hepatocellular carcinomaHCC is one of the most common malignant tumors, and 50% of HCC patients worldwide are in China. The highly malignant, easy to recurrence and metastasis and short survival make HCC a big human threat. The identification of genes contributing to the development of HCC may define important therapeutic targets for prevention and treatment of this malignancy.According to the strict inclusion and exclusion criteria, primary HCC tissues, adjacent non-tumor tissues, peripheral blood samples and related clinical and demographic data were collected from 40 HCC patients. In both tumor tissues and adjacent non-tumor tissues, the genomic copy number alteration (CNA), mRNA expression profile and miRNA expression profile were identified by Affymetrix SNP 6.0 array. Affymetrix Human Exon 1.0 ST array and Solexa deep sequencing technology, respectively. After the strict quality control (QC), the "omics" data of the genome and transcriptome were analyzed and integrated.37 significant somatic genomic CNAs (15 amplifications and 27 deletions) were identified in tumors by GISTIC and JISTIC analysis, which consist of 662 genes, including several known cancer genes (e.g. MYC, CCND1, RB1, CDKN2A, AXIN1, PTEN and AXIN1). 543 significant differentially expressed genes (279 up-regulated and 264 down-regulated) were identified between tumors and adjacent non-tumor tissues through analyzing the mRNA expression profiles by SVA method and Bonferroni correction. Additionally, 79 significant differentially expressed miRNAs (41 up-regulated and 38 down-regulated) were identified between tumors and adjacent non-tumor tissues by analyzing the miRNA expression profiles using SAM method, in which 44 miRNAs (e.g. miR-192, -21, -30d, -199a-3p, -199b-3p, and let-7c) were reported previously to be significant differentially expressed in HCC.When the genomic CNVs and mRNA expression profile were integrated, 40 candidate driver genes and their target gene modules were identified by CONEXIC analysis, among which includes one known HCC gene (MTAP) and three known tumor genes (FZR1, RRM2B and CLPTM1L). The other 36 candidate driver genes in HCC were newly identified in this study. For an example, EXOC2 and ILF3 regulate a module consisting of 168 genes that are involved in the Src pathway, MAPK pathway, Cyclin pathway, p53 pathway and Ras pathway etc, indicating their pivotal role on the development of HCC. In addition, we integrated the miRNA expression profiles and mRNA expression profile, and found 414 differentially expressed miRNA/mRNA pairs, among which includes 26 differentially expressed miRNAs and 310 differentially expressed genes. The most significant differentially expressed miRNA was miR-128, which regulates 65 target genes (including BMI1, previously reported target gene of miR-128), and involves in the VEGF pathway, AKT pathway, MAPK pathway, and EGF pathway. This result strongly indicates that the miR-128 may contribute to the development of HCC.In conclusion, several HCC candidate driver genes and their target gene modules were identified. These findings may contribute to the early diagnosis, molecular typing and the discovery of new candidate drug targets of HCC. Further studies were needed to clarifying the mechanisms of these CNAs, miRNA and mRNA underlying the development of HCC.