Overexpression of microRNAs miR-9, -98, and -199 Correlates with the Downregulation of <Emphasis Type="Italic">HK2</Emphasis> Expression in Colorectal Cancer

Glycolysis activation is one of the main features of energy metabolism in cancer cells that is associated with the increase in glycolytic enzyme synthesis, primarily, hexokinases (HKs), in many types of tumors. Conversely, in colorectal cancer (CRC) the decrease in the expression of HK2 gene, which encodes one of the key rate-limiting enzyme of glycolysis, was revealed, thus, the study of the mechanisms of its inhibition in CRC is of particular interest. To search for potential microRNAs, inhibiting the expression of HK2 in CRC, we have performed the analysis of data from “The Cancer Genome Atlas” (TCGA) and five microRNA–mRNA target interaction databases (TargetScan, DIANA microT, mirSVR (miRanda), PicTar, and miRTarBase) using original CrossHub software. Seven microRNAs containing binding site on mRNA HK2, which expression is negatively correlated with HK2 expression, were selected for further analysis. The expression levels of these microRNAs and mRNA HK2 were estimated by quantitative PCR on a set of CRC samples. It has been shown, that the expression of three microRNAs (miR-9-5p, -98-5p, and -199-5p) was increased and correlated negatively with mRNA level of HK2 gene. Thus, downregulation of HK2 gene may be caused by its negative regulation through microRNAs miR-9-5p, -98-5p, and -199-5p.     

Involvement of MAP3K8 and miR-17-5p in Poor Virologic Response to Interferon-Based Combination Therapy for Chronic Hepatitis C

Despite advances in chronic hepatitis C treatment, a proportion of patients respond poorly to treatment. This study aimed to explore hepatic mRNA and microRNA signatures involved in hepatitis C treatment resistance. Global hepatic mRNA and microRNA expression profiles were compared using microarray data between treatment responses. Quantitative real-time polymerase chain reaction validated the gene signatures from 130 patients who were infected with hepatitis C virus genotype 1b and treated with pegylated interferon-alpha and ribavirin combination therapy. The correlation between mRNA and microRNA was evaluated using in silico analysis and in vitro siRNA and microRNA inhibition/overexpression experiments. Multivariate regression analysis identified that the independent variables IL28B SNP rs8099917, hsa-miR-122-5p, hsa-miR-17-5p, and MAP3K8 were significantly associated with a poor virologic response. MAP3K8 and miR-17-5p expression were inversely correlated with treatment response. Furthermore, miR-17-5p repressed HCV production by targeting MAP3K8. Collectively, the data suggest that several molecules and the inverse correlation between mRNA and microRNA contributed to a host genetic refractory hepatitis C treatment response.     

Purification and characterization of the DNA cleavage and recognition site of I-ScaI mitochondrial group I intron encoded endonuclease produced in Escherichia coli

The second intron in the mitochondrial cytb gene of Saccharomyces capensis, belonging to group I, encodes a 280 amino acid protein containing two LAGLIDADG motifs. Genetic and molecular studies have previously shown that this protein has a dual function in the wild-type strain. It acts as a specific homing endonuclease I-ScaI promoting intron mobility and as a maturase promoting intron splicing. Here we describe the synthesis of a universal code equivalent to the mitochondrial sequence coding for this protein and the in vitro characterization of I-ScaI endonuclease activity, using a truncated mutant form of the protein p28bi2 produced in Escherichia coli. We have also determined the cleavage pattern as well as the recognition site of p28bi2. It was found that p28bi2 generates a double-strand cleavage downstream from the intron insertion site with 4 nt long 3′-overhangs. Mutational analysis of the DNA target site shows that p28bi2 recognizes a 16–19 bp sequence from positions –11 to +8 with respect to the intron insertion site.     

Pro-tumorigenic Effects of miR-31 Loss in Mesothelioma

The human genome encodes several hundred microRNA (miRNA) genes that produce small (21–23n) single strand regulatory RNA molecules. Although abnormal expression of miRNAs has been linked to cancer progression, the mechanisms of this dysregulation are poorly understood. Malignant mesothelioma (MM) of pleura is an aggressive and highly lethal cancer resistant to conventional therapies. We and others previously linked loss of the 9p21.3 chromosome in MM with short time to tumor recurrence. In this study, we report that MM cell lines derived from patients with more aggressive disease fail to express miR-31, a microRNA recently linked with suppression of breast cancer metastases. We further demonstrate that this loss is due to homozygous deletion of the miR-31-encoding gene that resides in 9p21.3. Functional assessment of miR-31 activity revealed its ability to inhibit proliferation, migration, invasion, and clonogenicity of MM cells. Re-introduction of miR-31 suppressed the cell cycle and inhibited expression of multiple factors involved in cooperative maintenance of DNA replication and cell cycle progression, including pro-survival phosphatase PPP6C, which was previously associated with chemotherapy and radiation therapy resistance, and maintenance of chromosomal stability. PPP6C, whose mRNA is distinguished with three miR-31-binding sites in its 3′-untranslated region, was consistently down-regulated by miR-31 introduction and up-regulated in clinical MM specimens as compared with matched normal tissues. Taken together, our data suggest that tumor-suppressive propensity of miR-31 can be used for development of new therapies against mesothelioma and other cancers that show loss of the 9p21.3 chromosome.     

Microarray analysis of defined Mycobacterium tuberculosis populations using RNA amplification strategies

Background

Halibuts are commercially important flatfish species confined to the North Pacific and North Atlantic Oceans. We have determined the complete mitochondrial genome sequences of four specimens each of Atlantic halibut (Hippoglossus hippoglossus), Pacific halibut (Hippoglossus stenolepis) and Greenland halibut (Reinhardtius hippoglossoides), and assessed the nucleotide variability within and between species.

Results

About 100 variable positions were identified within the four specimens in each halibut species, with the control regions as the most variable parts of the genomes (10 times that of the mitochondrial ribosomal DNA). Due to tandem repeat arrays, the control regions have unusually large sizes compared to most vertebrate mtDNAs. The arrays are highly heteroplasmic in size and consist mainly of different variants of a 61-bp motif. Halibut mitochondrial genomes lacking arrays were also detected.

Conclusion

The complexity, distribution, and biological role of the heteroplasmic tandem repeat arrays in halibut mitochondrial control regions are discussed. We conclude that the most plausible explanation for array maintenance includes both the slipped-strand mispairing and DNA recombination mechanisms.     

TNFα induces the expression of genes associated with endothelial dysfunction through p38MAPK-mediated down-regulation of miR-149

MicroRNAs have been proposed as novel regulators of vascular inflammation and dysfunction. This study aimed to evaluate the role of miR-149 in regulating the expression of key molecules associated with TNFα-induced endothelial activation. miR-149 was selected by in silico analysis and microRNA target prediction. Endothelial dysfunction was induced by TNFα treatment in Eahy926 endothelial cells and HUVEC. miR-149 level was evaluated by quantitative real time-polymerase chain reaction (RT-qPCR). Metalloproteinase-9 (MMP-9) was measured by zymography, Inducible Nitric Oxide Synthase (iNOS) by immunoblotting, Interleukin-6 (IL-6) and Interleukin-8 (IL-8) by ELISA. miR-149 regulatory effect was evaluated by gain-of-function technique upon miR-149 mimics transfection.     

Apparent digestibility coefficients and accumulation of astaxanthin E/Z isomers in Atlantic salmon (Salmo salar L.) and Atlantic halibut (Hippoglossus hippoglossus L.)

Apparent astaxanthin (3,3'-dihydroxy-beta,beta-carotene-4,4'-dione) digestibility coefficients (ADC) and carotenoid compositions of the muscle, liver, whole kidney and plasma were compared in Atlantic salmon (Salmo salar) and Atlantic halibut (Hippoglossus hippoglossus) fed a diet supplemented with 66 mg astaxanthin kg(-1) dry matter for 112 days. The astaxanthin source consisted of 75% all-E-, 3% 9Z- and 22% 13Z-astaxanthin, of (3R,3'R)-, (3R,3'S; meso)-, and (3S,3'S)-astaxanthin in a 1:2:1 ratio. The ADC of astaxanthin was significantly higher in Atlantic halibut than in Atlantic salmon after 56 and 112 days of feeding (P < 0.05). The ADC of all-E-astaxanthin was significantly higher than ADC of 9Z-astaxanthin (P < 0.05). Considerably more carotenoids were present in all plasma and tissue samples of salmon than in halibut. Retention of astaxanthin in salmon muscle was 3.9% in salmon and 0 in halibut. All-E-astaxanthin accumulated selectively in the muscle of salmon, and in plasma of salmon and halibut compared with diet. 13Z-astaxanthin accumulated selectively in liver and whole kidney of salmon and halibut, when compared with plasma. A reductive pathway for astaxanthin metabolism in halibut similar to that of salmon was shown by the presence of 3',4'-cis and trans glycolic isomers of idoxanthin (3,3',4'-trihydroxy-beta,beta-carotene-4'-one) in plasma, liver and whole kidney. In conclusion, the higher ADC of astaxanthin in halibut than Atlantic salmon may be explained by lower feed intake in halibut, and the lower retention of astaxanthin by a higher capacity to transform astaxanthin metabolically.     

Characterisation of Saccharomyces cerevisiae ARO8 and ARO9 genes encoding aromatic aminotransferases I and II reveals a new aminotransferase subfamily

The ARO8 and ARO9 genes of Saccharomyces cerevisiae were isolated by complementation of the phenylalanine/tyrosine auxotrophy of an aro8 aro9 double-mutant strain that is defective in aromatic aminotransferases I (aro8) and II (aro9). The genes were sequenced, and deletion mutants were constructed and analysed. The expression of ARO8 and ARO9 was studied. The deduced amino acid sequences of Aro8p and Aro9p suggest that the former is a 500-residue, 56168-Da polypeptide and the latter a 513-residue, 58516-Da polypeptide. They correspond, respectively, to Ygl202p and Yhr137p, two putative proteins of unknown function revealed by systematic sequencing of the yeast genome. We show that aromatic aminotransferases I and II are homologous proteins, members of aminotransferase subgroup I, and, together with three other proteins, they constitute within the subgroup a new subfamily of enzymes specialised for aromatic amino acid and α-aminoadipate transamination. ARO8 expression is subject to the general control of amino acid biosynthesis. ARO9 expression is induced when aromatic amino acids are present in the growth medium and also in aro8 mutants grown on minimal ammonia medium. An autonomously replicating sequence (ARS) element is located between the ARO8 gene and YGL201c which encodes a protein of the minichromosome maintenance family.     

miR-485-5p Binding Site SNP rs8752 in HPGD Gene Is Associated with Breast Cancer Risk

Background

Single nucleotide polymorphisms (SNPs) that reside in microRNA target sites may play an important role in breast cancer development and progression. To reveal the association between microRNA target site SNPs and breast cancer risk, we performed a large case-control study in China.

Methods

We performed a two-stage case-control study including 2744 breast cancer cases and 3125 controls. In Stage I, we genotyped 192 SNPs within microRNA binding sites identified from the “Patrocles” database using custom Illumina GoldenGate VeraCode assays on the Illumina BeadXpress platform. In Stage II, genotyping was performed on SNPs potentially associated with breast cancer risk using the TaqMan platform in an independent replication set.

Results

In stage I, 15 SNPs were identified to be significantly associated with breast cancer risk (P<0.05). In stage II, one SNP rs8752 was replicated at P<0.05. This SNP is located in the 3’ untranslated region (UTR) of the 15-hydroxyprostaglandin dehydrogenase (HPGD) gene at 4q34-35, a miR-485-5p binding site. Compared with the GG genotype, the combined GA+AA genotypes has a significantly higher risk of breast cancer (OR = 1.18; 95% CI: 1.06-1.31, P = 0.002). Specifically, this SNP was associated with estrogen receptor (ER) positive breast cancer (P = 0.0007), but not with ER negative breast cancer (P = 0.23), though p for heterogeneity not significant.

Conclusion

Through a systematic case-control study of microRNA binding site SNPs, we identified a new breast cancer risk variant rs8752 in HPGD in Chinese women. Further studies are warranted to investigate the underling mechanism for this association.     

HRP2 determines the efficiency and specificity of HIV-1 integration in LEDGF/p75 knockout cells but does not contribute to the antiviral activity of a potent LEDGF/p75-binding site integrase inhibitor

The binding of integrase (IN) to lens epithelium-derived growth factor (LEDGF)/p75 in large part determines the efficiency and specificity of HIV-1 integration. However, a significant residual preference for integration into active genes persists in Psip1 (the gene that encodes for LEDGF/p75) knockout (KO) cells. One other cellular protein, HRP2, harbors both the PWWP and IN-binding domains that are important for LEDGF/p75 co-factor function. To assess the role of HRP2 in HIV-1 integration, cells generated from Hdgfrp2 (the gene that encodes for HRP2) and Psip1/Hdgfrp2 KO mice were infected alongside matched control cells. HRP2 depleted cells supported normal infection, while disruption of Hdgfrp2 in Psip1 KO cells yielded additional defects in the efficiency and specificity of integration. These deficits were largely restored by ectopic expression of either LEDGF/p75 or HRP2. The double-KO cells nevertheless supported residual integration into genes, indicating that IN and/or other host factors contribute to integration specificity in the absence of LEDGF/p75 and HRP2. Psip1 KO significantly increased the potency of an allosteric inhibitor that binds the LEDGF/p75 binding site on IN, a result that was not significantly altered by Hdgfrp2 disruption. These findings help to rule out the host factor-IN interactions as the primary antiviral targets of LEDGF/p75-binding site IN inhibitors.     

Genetic sex identification and the potential evolution of sex determination in Pacific halibut (Hippoglossus stenolepis)

The discovery of genetic markers linked to physiological traits in wild populations is increasingly desired for ecological and evolutionary studies, as well as to inform management decisions. However, identifying such markers often requires a large investment of both time and money. Serendipitously, in a recent microsatellite survey, we discovered three out of 16 microsatellite loci that were correlated to the female sex in Pacific halibut (Hippoglossus stenolepis). These three loci were screened in 550 Pacific halibut to determine their accuracy at identifying sex. Genetic assignment successfully identified sex in 92% of individuals from sample collections spanning 3,000 km and 9 years. All but two of 287 females had one copy of a characteristic allele for at least one of the three microsatellite loci, resulting in consistent heterozygote excess in females. This pattern is consistent with the hypothesis that females are the heterogametic sex in Pacific halibut, which thus may have a different sex-determination pattern than the closely related Atlantic halibut (Hippoglossus hippoglossus). A rapid divergence of sex-determining mechanisms could be either a cause or consequence of speciation between Pacific and Atlantic halibut. In either case, the ability to genetically identify sex in individual Pacific halibut provides a new tool for ecological studies, fisheries management, and insight into the evolution of sex determination in flatfish.     

Hepatitis C Virus Core Protein Down-Regulates p21Waf1/Cip1 and Inhibits Curcumin-Induced Apoptosis through MicroRNA-345 Targeting in Human Hepatoma Cells

Background

Hepatitis C virus (HCV) has been reported to regulate cellular microRNAs. The HCV core protein is considered to be a potential oncoprotein in HCV-related hepatocellular carcinoma, but HCV core-modulated cellular microRNAs are unknown. The HCV core protein regulates p21^Waf1/Cip1 expression. However, the mechanism of HCV core-associated p21^Waf1/Cip1 regulation remains to be further clarified. Therefore, we attempted to determine whether HCV core-modulated cellular microRNAs play an important role in regulating p21^Waf1/Cip1 expression in human hepatoma cells.

Methods

Cellular microRNA profiling was investigated in core-overexpressing hepatoma cells using TaqMan low density array. Array data were further confirmed by TaqMan real-time qPCR for single microRNA in core-overexpressing and full-length HCV replicon-expressing cells. The target gene of microRNA was examined by reporter assay. The gene expression was determined by real-time qPCR and Western blotting. Apoptosis was examined by annexin V-FITC apoptosis assay. Cell cycle analysis was performed by propidium iodide staining. Cell proliferation was analyzed by MTT assay.

Results

HCV core protein up- or down-regulated some cellular microRNAs in Huh7 cells. HCV core-induced microRNA-345 suppressed p21^Waf1/Cip1 gene expression through targeting its 3′ untranslated region in human hepatoma cells. Moreover, the core protein inhibited curcumin-induced apoptosis through p21^Waf1/Cip1-targeting microRNA-345 in Huh7 cells.

Conclusion and Significance

HCV core protein enhances the expression of microRNA-345 which then down-regulates p21^Waf1/Cip1 expression. It is the first time that HCV core protein has ever been shown to suppress p21^Waf1/Cip1 gene expression through miR-345 targeting.