Increasing evidence indicates that deregulation of microRNAs (miRNAs) is involved in tumorigenesis. Downregulation of microRNA-503 has been observed in various types of diseases, including cancer. However, the biological function of miR-503 in hepatocellular carcinoma (HCC) is still largely unknown. In this study we aimed to elucidate the prognostic implications of miR-503 in HCC and its pathophysiologic role. Quantitative reverse transcriptase polymerase chain reaction was used to evaluate miR-503 expression in HCC tissues and cell lines. Western blotting was performed to evaluate the expression of the miR-503 target genes. In vivo and in vitro assays were performed to evaluate the function of miR-503 in HCC. Luciferase reporter assay was employed to validate the miR-503 target genes. miR-503 was frequently downregulated in HCC cell lines and tissues. Low expression levels of miR-503 were associated with enhanced malignant potential such as portal vein tumor thrombi, histologic grade, TNM stage, AFP level and poor prognosis. Multivariate analysis indicated that miR-503 downregulation was significantly associated with worse overall survival of HCC patients. Functional studies showed miR-503 suppressed the proliferation of HCC cells by induction of G1 phase arrest through Rb-E2F signaling pathways, and thus may function as a tumor suppressor. Further investigation characterized two cell cycle-related molecules, cyclin D3 and E2F3, as the direct miR-503 targets. Our data highlight an important role for miR-503 in cell cycle regulation and in the molecular etiology of HCC, and implicate the potential application of miR-503 in prognosis prediction and miRNA-based HCC therapy.

/pdf/microrna-503-inhibits-the-g1-s-transition-by-downregulating-11ko0b3j84.pdf

MicroRNA-503 inhibits the G1/S transition by downregulating cyclin D3 and E2F3 in hepatocellular carcinoma

https://opus.lib.uts.edu.au/bitstream/10453/36885/4/Final%20submission%20YSCDB-D-14-00168R1.pdf

Microparticles in cancer: A review of recent developments and the potential for clinical application

Since the discovery and classification of non-coding RNAs, their roles have gained great attention. In this respect, microRNAs and long non-coding RNAs have been firmly demonstrated to be linked to regulation of gene expression and onset of human diseases, including rare genetic diseases; therefore they are suitable targets for therapeutic intervention. This issue, in the context of rare genetic diseases, is being considered by an increasing number of research groups and is of key interest to the health community. In the case of rare genetic diseases, the possibility of developing personalized therapy in precision medicine has attracted the attention of researchers and clinicians involved in developing “orphan medicinal products” and proposing these to the European Medicines Agency (EMA) and to the Food and Drug Administration (FDA) Office of Orphan Products Development (OOPD) in the United States. The major focuses of these activities are the evaluation and development of products (drugs, biologics, devices, or medical foods) considered to be promising for diagnosis and/or treatment of rare diseases or conditions, including rare genetic diseases. In an increasing number of rare genetic diseases, analysis of microRNAs and long non-coding RNAs has been proven a promising strategy. These diseases include, but are not limited to, Duchenne muscular dystrophy, cystic fibrosis, Rett syndrome, and β-thalassemia. In conclusion, a large number of approaches based on targeting microRNAs and long non-coding RNAs are expected in the field of molecular diagnosis and therapy, with a facilitated technological transfer in the case of rare genetic diseases, in virtue of the existing regulation concerning these diseases.

/pdf/micrornas-and-long-non-coding-rnas-in-genetic-diseases-1pxdnd5alz.pdf

MicroRNAs and Long Non-coding RNAs in Genetic Diseases.

The 1000 Genome project paved the way for sequencing diverse human populations. New genome projects are being established to sequence underrepresented populations helping in understanding human genetic diversity. The Kuwait Genome Project an initiative to sequence individual genomes from the three subgroups of Kuwaiti population namely, Saudi Arabian tribe; “tent-dwelling” Bedouin; and Persian, attributing their ancestry to different regions in Arabian Peninsula and to modern-day Iran (West Asia). These subgroups were in line with settlement history and are confirmed by genetic studies. In this work, we report whole genome sequence of a Kuwaiti native from Persian subgroup at >37X coverage. We document 3,573,824 SNPs, 404,090 insertions/deletions, and 11,138 structural variations. Out of the reported SNPs and indels, 85,939 are novel. We identify 295 ‘loss-of-function’ and 2,314 ’deleterious’ coding variants, some of which carry homozygous genotypes in the sequenced genome; the associated phenotypes include pharmacogenomic traits such as greater triglyceride lowering ability with fenofibrate treatment, and requirement of high warfarin dosage to elicit anticoagulation response. 6,328 non-coding SNPs associate with 811 phenotype traits: in congruence with medical history of the participant for Type 2 diabetes and β-Thalassemia, and of participant’s family for migraine, 72 (of 159 known) Type 2 diabetes, 3 (of 4) β-Thalassemia, and 76 (of 169) migraine variants are seen in the genome. Intergenome comparisons based on shared disease-causing variants, positions the sequenced genome between Asian and European genomes in congruence with geographical location of the region. On comparison, bead arrays perform better than sequencing platforms in correctly calling genotypes in low-coverage sequenced genome regions however in the event of novel SNP or indel near genotype calling position can lead to false calls using bead arrays. We report, for the first time, reference genome resource for the population of Persian ancestry. The resource provides a starting point for designing large-scale genetic studies in Peninsula including Kuwait, and Persian population. Such efforts on populations under-represented in global genome variation surveys help augment current knowledge on human genome diversity.

/pdf/sequence-and-analysis-of-a-whole-genome-from-kuwaiti-511vb2xehi.pdf

Sequence and analysis of a whole genome from Kuwaiti population subgroup of Persian ancestry

MicroRNA Expression in β-Thalassemia and Sickle Cell Disease: A Role in The Induction of Fetal Hemoglobin.

In search of genetic alterations responsible for high fetal hemoglobin (Hb F) phenotypes in the population of eastern India, 91 probands were screened for four polymorphisms by sequencing and/or restriction fragment length polymorphism (RFLP) analysis. These are the A>G allele on the rs4895441 locus in the intergenic region between HBS1L and MYB on chromosome 6, the G>A allele on the rs4671393 locus on chromosome 2 (BCL11A gene), the A>C allele on the rs2071348 (HBBP1 gene) and the XmnI polymorphism (rs7482144, -158 position of HBG2) on chromosome 11. We found a significant association (p = 0.002 and 0.0013) of Hb F levels with rs2071348 and rs4895441, respectively. However, the polymorphism rs4671393 gene did not show significant association with Hb F levels (p = 0.0655). As is well known, the XmnI polymorphism (p <0.0001) showed the strongest association.

Influence of BCL11A, HBS1L-MYB, HBBP1 single nucleotide polymorphisms and the HBG2 XmnI polymorphism on Hb F levels

5(G 1 C). All probands were aged between 20 and 30 years. Written informed consent was obtained from them, and the institutional human research committee approved the study. The ethical principles followed by the institute are guided by rules as formulated by the Indian Council of Medical Research and these are in agreement with the Helsinki rules. The CD34+ hematopoietic stem cells were first isolated from peripheral blood samples of these probands via the MACS cell separation technique and they were then grown following a two-step culture system to achieve the unilineage differentiation into mature erythrocytes [6] . Ninety percent of the cells became benzidine positive by day 13 in normal samples. The cells were harvested on the 13th day of culture by selecting for CD235a expression using an immunomagnetic isolation procedure and processed for miRNA and mRNA isolation ( mir VanaTM miRNA Isolation Kit; Ambion). Solution hybridization was performed with an mir Vana miRNA Probe Construction Kit (Ambion) and an mir Vana miRNA Detection Kit (Ambion) following the manufacturer’s instructions. For probe labeling, 32 P CTP (BRIT, India) was used. The intensity of the bands was analyzed using Imagequant software. U6 MicroRNAs (miRNAs) are small, 22to 25-nt-long, endogenous, non-proteincoding RNA molecules which have the potential to downregulate gene expression. The involvement of miRNAs in several biological processes has been demonstrated, and diseased states are characterized by altered expression of miRNAs [1] . We intended to study how the miRNA expression profile is modulated in patients with thalassemia major. Thalassemia is characterized by an enhanced rate of growth of erythropoietic progenitors leading to marrow expansion and resultant iron absorption [2] . Of the various miRNAs expressed during erythropoiesis, hsa-miR-503 has been implicated in regulation of genes involved in cell cycle arrest and apoptosis [3–5] . To investigate whether there is a variance in the expression level of hsa-miR-503 in red blood cells of thalassemic and hematologically normal individuals, four patients with  thalassemia major, four with Ethalassemia major, and two with Ethalassemia intermedia, as well as four normal healthy adult volunteers, were recruited. All  major patients studied here are homozygous for the mutation IVS-I nt 5(G 1 C). The intermedia and severe patients of HbE/ thalassemia are compound heterozygous for HbE and IVS-I nt Received: March 5, 2012 Accepted after revision: May 8, 2012 Published online: August 11, 2012

hsa-miR-503 is downregulated in β thalassemia major.

Point mutations of α-globin genes in homozygous or in compound heterozygous states cause severe α-thalassemia (α-thal). Here we describe a polymerase chain reaction-restriction fragment length polymorphism-based method for easy detection of the point mutation Hb Sallanches [α104(G11)Cys→Tyr, TGC>TAC], earlier detected by a sequencing technique. In a cohort of 104 unrelated putative α-thal patients, nine carried the mutation and two were homozygotes. The mutation occurred on both the α2- or α1-globin genes. The phenotypes, in conjunction with other point mutations or deletions, are presented. Earlier detected in Pakistan and Punjab of India, it is probably present all over the Indian subcontinent.

Hb Sallanches [α104(G11)Cys→Tyr, TGC>TAC] Occurs Frequently on the Indian Subcontinent

The microRNAs (miRNAs) are small, 20–22 nt long, endogenous, non protein-coding RNA molecules which have potential to down regulate gene expression [1]. We studied the involvement of miRNA in disease severity of β thalassemia major patients. Initially, miRNA expression in one thalassemiamajor patient requiring regular transfusion was compared to another patient of intermedia phenotype. Both had the same mutations of the β globin gene, IVS-I nt 5(G>C) mutation and β globin gene mutation encoding for HbE [cd 26 (GAG>AAG) (Glu>Lys)], which produces a β plus thalassemia phenotype. The intermedia patient was never transfused and had Hb value of 8.5 g/dl with hemoglobins A 24.2%, A2+E 59.7% and F 10.2%. The average HbF level of the thalassemia major patient prior to regular transfusions was 1.8%, and Hb 8.2 g/dl, A 74.1%, A2+E 12.2%. Both were females of age 30 and 25 years old. Written informed consent was obtained from both of them. Glycophorin A (CD 235a+) positive cells from the blood of these patients were isolated by density gradient centrifugation followed by immunomagnetic labeling using Glycophorin A MicroBeads, and the miRNA expression profile of these CD235a+ cells was analyzed. When the normalized values of signal intensity were considered, expression levels of only 4 miRNAs differed more than 10 fold, hsamiR-503 showing the largest change. It was expressed 449 times more in the intermedia patient. The miRNA microarray data was submitted in Gene Expression Omnibus (GEO) database and the Accession no. is GSE24428. The marker used in this experiment, CD235a or glycophorin A is present on all erythroid cells including its precursors and progenitors, but is not known to be expressed in any other cell types. The samples used in this experiment included the whole spectrum of early to late erythroid progenitors; hence, the miRNA expression profile presented here is an average representation of the miRNA expression profile in both cases. The array data is also limited in scope as it deals with only one sample of each thalassemic phenotype. Thus, we chose an alternative strategy to verify the observed modulation of hsa-miR-503 expression in thalassemic individuals, by disease severity. The expression of hsa-miR-503 in CD235a+ cells of β thalassemia major, Eβ thalassemia major and Eβ thalassemia intermedia patients was comparedwith that of hematologically normal adults by solution hybridization, and was shown to be reduced significantly as the clinical severity of the patients increased [2], severity being assessed by age of first transfusion and frequency of transfusion. The median value of age at first transfusion is 1 year and 6 months and 2 years and 7 months for the β thalassemia major and Eβ thalassemia major groups respectively. Average frequency of transfusion was 2 and 1.5 per month respectively. The E-β intermedia patients were never transfused. The hsa-miR-503 expression pattern is in inverse relationship with the expression level of CDC25A, one of the targets of hsa-

Papai Roy

Papers

Influence of BCL11A, HBS1L-MYB, HBBP1 single nucleotide polymorphisms and the HBG2 XmnI polymorphism on Hb F levels

hsa-miR-503 is downregulated in β thalassemia major.

Hb Sallanches [α104(G11)Cys→Tyr, TGC>TAC] Occurs Frequently on the Indian Subcontinent

The microRNA hsa-miR-503 inhibits growth of K562 cell line.