Evolution of coding and non-coding genes in HOX clusters of a marsupial

ABSTRACT: BACKGROUND: The HOX gene clusters are thought to be highly conserved amongst mammals and othervertebrates, but the long non-coding RNAs have only been studied in detail in human andmouse. The sequencing of the kangaroo genome provides an opportunity to use comparativeanalyses to compare the HOX clusters of a mammal with a distinct body plan to those ofother mammals. RESULTS: Here we report a comparative analysis of HOX gene clusters between an Australian marsupialof the kangaroo family and the eutherians. There was a strikingly high level of conservationof HOX gene sequence and structure and non-protein coding genes including the microRNAsmiRNA-196a, miRNA-196b, miRNA-10a and miRNA-10b and the long non-coding RNAsHOTAIR, HOTAIRM1 and HOXA11AS that play critical roles in regulating gene expressionand controlling development. By microRNA deep sequencing and comparative genomicanalyses, two conserved microRNAs (miR-10a and miR-10b) were identified and one newcandidate microRNA with typical hairpin precursor structure that is expressed in bothfibroblasts and testes was found. The prediction of microRNA target analysis showed thatseveral known microRNA targets, such as mir-10, mir-414 and mir-464, were found in thetammar HOX clusters. In addition, several novel and putative miRNAs were identified thatoriginated from elsewhere in the tammar genome and that target the tammar HOXB andHOXD clusters. CONCLUSIONS: This study confirms that the emergence of known long non-coding RNAs in the HOXclusters clearly predate the marsupial-eutherian divergence 160 Ma ago. It also identified anew potentially functional microRNA as well as conserved miRNAs. These non-codingRNAs may participate in the regulation of HOX genes to influence the body plan of thismarsupial.     

Genome-wide aberrant DNA methylation of microRNA host genes in hepatocellular carcinoma

Mature microRNAs (miRNAs) are a class of small non-coding RNAs involved in posttranslational gene silencing. Previous studies found that downregulation of miRNAs is a common feature observed in solid tumors, including hepatocellular carcinoma (HCC). We employed a genome-wide approach to test the hypothesis that DNA methylation alterations in miRNA host genes may cause deregulated miRNA expression in HCC. We analyzed tumor and adjacent non-tumor tissues from 62 Taiwanese HCC cases using Infinium HumanMethylation27 DNA Analysis BeadChips that include 254 CpG sites covering 110 miRNAs from 64 host genes. Expression levels of three identified miRNAs (miR-10a, miR-10b and miR-196b) were measured in a subset of 37 HCC tumor and non-tumor tissues. After Bonferroni adjustment, a total of 54 CpG sites from 27 host genes significantly differed in DNA methylation levels between tumor and adjacent non-tumor tissues with 53 sites significantly hypermethylated in tumor tissues. Among the 54 significant CpG sites, 15 sites had more than 2-fold tumor/non-tumor changes, 17 sites had differences > 10%, and 10 sites had both features [including 8 significantly hypermethylated CpG sites in the host genes of miR-10a, miR-10b and miR-196b (HOXB4, HOXD4 and HOXA9, respectively)]. Significant downregulation of miR-10a was observed in tumor compared with non-tumor tissues (0.50 vs. 1.73, p = 0.031). The concordance for HOXB4 methylation alteration and dysregulation of miR-10a was 73.5%. No significant change was observed for miR-10b expression. Unexpectedly, miR-196b was significantly upregulated in tumor compared with non-tumor tissues (p = 0.0001). These data suggest that aberrant DNA methylation may lead to dysregulation of miR-10a in HCC tumor tissues.     

Genome-wide aberrant DNA methylation of microRNA host genes in hepatocellular carcinoma

Mature microRNAs (miRNAs) are a class of small non-coding RNAs involved in posttranslational gene silencing. Previous studies found that downregulation of miRNAs is a common feature observed in solid tumors, including hepatocellular carcinoma (HCC). We employed a genome-wide approach to test the hypothesis that DNA methylation alterations in miRNA host genes may cause deregulated miRNA expression in HCC. We analyzed tumor and adjacent non-tumor tissues from 62 Taiwanese HCC cases using Infinium HumanMethylation27 DNA Analysis BeadChips that include 254 CpG sites covering 110 miRNAs from 64 host genes. Expression levels of three identified miRNAs (miR-10a, miR-10b and miR-196b) were measured in a subset of 37 HCC tumor and non-tumor tissues. After Bonferroni adjustment, a total of 54 CpG sites from 27 host genes significantly differed in DNA methylation levels between tumor and adjacent non-tumor tissues with 53 sites significantly hypermethylated in tumor tissues. Among the 54 significant CpG sites, 15 sites had more than 2-fold tumor/non-tumor changes, 17 sites had differences > 10%, and 10 sites had both features [including 8 significantly hypermethylated CpG sites in the host genes of miR-10a, miR-10b and miR-196b (HOXB4, HOXD4 and HOXA9, respectively)]. Significant downregulation of miR-10a was observed in tumor compared with non-tumor tissues (0.50 vs. 1.73, p = 0.031). The concordance for HOXB4 methylation alteration and dysregulation of miR-10a was 73.5%. No significant change was observed for miR-10b expression. Unexpectedly, miR-196b was significantly upregulated in tumor compared with non-tumor tissues (p = 0.0001). These data suggest that aberrant DNA methylation may lead to dysregulation of miR-10a in HCC tumor tissues.     

Epigenetic inactivation of the hsa-miR-203 in haematological malignancies

miR-203 is a tumour suppressor microRNA (miRNA). We studied the methylation of hsa-miR-203 in 150 samples including acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myeloid leukaemia (CML), chronic lymphocytic leukaemia (CLL) and non-Hodgkin's lymphoma (NHL) by methylation-specific PCR, and miRNA expression by stem-loop RT-qPCR. hsa-miR-203 promoter was unmethylated in normal controls but homozygously methylated in two AML and four lymphoma cell lines, in which 5-Aza-2'-deoxycytidine treatment led to promoter demethylation and miR-203 re-expression. Restoration of miR-203 expression in lymphoma cells inhibited cellular proliferation and increased cell death, suggesting an inherent tumour suppressor activity. In primary samples, hsa-miR-203 methylation was absent in CML but detected in 5.0% ALL, 10.0% AML, 42.0% CLL and 38.8% of NHL (including six [60.0%] natural killer-cell, nine [40.9%] B-cell and four [23.5%] T cell NHL). Moreover, hsa-miR-203 methylation was associated with hypermethylation of hsa-miR-34a, -124a and -196b in NHL but not CLL. In CLL, hsa-miR-203 methylation was associated with a higher presenting Hb level (P = 0.033). The projected 10 year overall survival of the CLL patients was 58.2%, which was impacted by Rai stage and high-risk karyotypes but not hsa-miR-203 methylation. hsa-miR-203 was more frequently methylated in lymphoid than myeloid malignancies (P = 0.002). In conclusion, miR-203, a tumour suppressor gene, was hypermethylated in a tumour-specific manner with gene silencing. hsa-miR-203 was more frequently hypermethylated in lymphoid than myeloid malignancies. In NHL, hsa-miR-203 methylation was associated with concomitant methylation of other tumour suppressor miRNAs. The frequent hsa-miR-203 methylation in lymphoid malignancies suggested a pathogenetic role of hsa-miR-203 methylation.     

MicroRNAs differentially expressed in postnatal aortic development downregulate elastin via 3' UTR and coding-sequence binding sites

Elastin production is characteristically turned off during the maturation of elastin-rich organs such as the aorta. MicroRNAs (miRNAs) are small regulatory RNAs that down-regulate target mRNAs by binding to miRNA regulatory elements (MREs) typically located in the 3' UTR. Here we show a striking up-regulation of miR-29 and miR-15 family miRNAs during murine aortic development with commensurate down-regulation of targets including elastin and other extracellular matrix (ECM) genes. There were a total of 14 MREs for miR-29 in the coding sequences (CDS) and 3' UTR of elastin, which was highly significant, and up to 22 miR-29 MREs were found in the CDS of multiple ECM genes including several collagens. This overrepresentation was conserved throughout mammalian evolution. Luciferase reporter assays showed synergistic effects of miR-29 and miR-15 family miRNAs on 3' UTR and coding-sequence elastin constructs. Our results demonstrate that multiple miR-29 and miR-15 family MREs are characteristic for some ECM genes and suggest that miR-29 and miR-15 family miRNAs are involved in the down-regulation of elastin in the adult aorta.     

miR-148 targets human DNMT3b protein coding region

MicroRNAs (miRNAs) are small noncoding RNA molecules of 20-24 nucleotides that regulate gene expression. In animals, miRNAs form imperfect interactions with sequences in the 3' Untranslated region (3'UTR) of mRNAs, causing translational inhibition and mRNA decay. In contrast, plant miRNAs mostly associate with protein coding regions. Here we show that human miR-148 represses DNA methyltransferase 3b (Dnmt3b) gene expression through a region in its coding sequence. This region is evolutionary conserved and present in the Dnmt3b splice variants Dnmt3b1, Dnmt3b2, and Dnmt3b4, but not in the abundantly expressed Dnmt3b3. Whereas overexpression of miR-148 results in decreased DNMT3b1 expression, short-hairpin RNA-mediated miR-148 repression leads to an increase in DNMT3b1 expression. Interestingly, mutating the putative miR-148 target site in Dnmt3b1 abolishes regulation by miR-148. Moreover, endogenous Dnmt3b3 mRNA, which lacks the putative miR-148 target site, is resistant to miR-148-mediated regulation. Thus, our results demonstrate that the coding sequence of Dnmt3b mediates regulation by the miR-148 family. More generally, we provide evidence that coding regions of human genes can be targeted by miRNAs, and that such a mechanism might play a role in determining the relative abundance of different splice variants.     

Defective RNA-mediated c-myc gene silencing pathway in Burkitt's lymphoma

Keeping in view the fact that molecular basis of Burkitt's lymphoma (BL) is poorly understood, we attempted to explore the small interfering RNA (siRNA) mediated c-myc gene regulation using BL-derived EB-3 cell line as archetype cellular model. Such a study revealed that EB-3 cells possess 4-fold higher expression of Dicer gene coupled with 2-fold higher activity of RNA polymerase III than that observed in normal human lymphocytes. siRNAs derived from EB-3 cells had the inherent capacity to suppress c-myc gene expression in normal cells but not in native cells. Based on these findings we have proposed a novel RNA-mediated c-myc gene regulation pathway that may be responsible for BL.     

靶向调控血管生成素miRNA的筛选和验证

血管生成素是一个重要的促血管生成因子,在细胞增殖、迁移和凋亡等过程中均发挥重要作用,但其具体的分子机制尚待阐明.miRNA是一类长约22 nt的小RNA,在转录后水平调控基因的表达,广泛参与各种生物学过程.本文探索了可直接调控血管生成素表达的miRNA,希望为阐明血管生成素的作用机制提供线索.首先,我们利用数据库预测得到8个可能靶向结合血管生成素mRNA 3′端非编码区的miRNA;然后,用实验方法验证它们与血管生成素的靶向关系,发现miR-1208、miR-196b、miR-296、miR-409-3p、miR-570和miR-641这6个miRNA可以不同程度地抑制血管生成素的mRNA和蛋白质表达水平,但只有miR-196b、miR-296、miR-409-3p和miR-641可以直接结合血管生成素mRNA的3′端非编码区;进而,在血管内皮细胞中分别过表达这4个miRNA,发现miR-196b、miR-409-3p和miR-641可以抑制血管内皮细胞的细胞增殖,而miR-196b、miR-296和miR-409-3p可以抑制血管内皮细胞的管腔形成.以上结果表明,细胞内有多个miRNA调控血管生成素的表达,它们可能协调调节血管生成,抑或在血管生成的不同阶段发挥作用.我们的工作还为“一种mRNA可被多种microRNA调节,而一种microRNA可调节多种mRNA”假说提供了部分证据.     

Low Expression of miR-196b Enhances the Expression of BCR-ABL1 and HOXA9 Oncogenes in Chronic Myeloid Leukemogenesis

MicroRNAs (miRNAs) can function as tumor suppressors or oncogene promoters during tumor development. In this study, low levels of expression of miR-196b were detected in patients with chronic myeloid leukemia. Bisulfite genomic sequencing PCR and methylation-specific PCR were used to examine the methylation status of the CpG islands in the miR-196b promoter in K562 cells, patients with leukemia and healthy individuals. The CpG islands showed more methylation in patients with chronic myeloid leukemia compared with healthy individuals (P<0.05), which indicated that low expression of miR-196b may be associated with an increase in the methylation of CpG islands. The dual-luciferase reporter assay system demonstrated that BCR-ABL1 and HOXA9 are the target genes of miR-196b, which was consistent with predictions from bioinformatics software analyses. Further examination of cell function indicated that miR-196b acts to reduce BCR-ABL1 and HOXA9 protein levels, decrease cell proliferation rate and retard the cell cycle. A low level of expression of miR-196b can cause up-regulation of BCR-ABL1 and HOXA9 expression, which leads to the development of chronic myeloid leukemia. MiR-196b may represent an effective target for chronic myeloid leukemia therapy.     

MicroRNAs associated with mitogen-activated protein kinase in human pancreatic cancer

Aberrant expression of microRNAs (miRNA) is associated with phenotypes of various cancers, including pancreatic cancer. However, the mechanism of the aberrant expression is largely unknown. Activation of the mitogen-activated protein kinase (MAPK) signaling pathway plays a crucial role in gene expression related to the malignant phenotype of pancreatic cancer. Hence, we studied the role of MAPK in the aberrant expression of miRNAs in pancreatic cancer cells. The alterations in expression of 183 miRNAs induced by activation or inactivation of MAPK were assayed in cultured pancreatic cancer cells and HEK293 cells by means of the quantitative real-time PCR method. We found that four miRNAs, namely, miR-7-3, miR-34a, miR-181d, and miR-193b, were preferentially associated with MAPK activity. Among these miRNAs, miR-7-3 was upregulated by active MAPK, whereas the others were downregulated. Promoter assays indicated that the promoter activities of the host genes of miR-7-3 and miR-34a were both downregulated by alteration in MAPK activity. Exogenous overexpression of the MAPK-associated miRNAs had the effect of inhibition of the proliferation of cultured pancreatic cancer cells; miR-193b was found to exhibit the most remarkable inhibition. A search for target genes of miR-193b led to identification of CCND1, NT5E, PLAU, STARD7, STMN1, and YWHAZ as the targets. Translational suppression of these genes by miR-193b was confirmed by reporter assay. These results indicate that activation of MAPK may play a significant role in aberrant expression of miRNAs and their associated phenotypes in pancreatic cancer.