UTR Site Research Articles

Beyond sites 1 and 2, miR-122 target sites in the HCV genome have negligible contributions to HCV RNA accumulation in cell culture.

Hepatitis C virus (HCV) recruits two molecules of the liver-specific microRNA-122 (miR-122) to two adjacent sites (S1 and S2) located at the 5' end of the viral RNA genome. This interaction promotes HCV RNA accumulation by stabilising the viral RNA and resulting in alteration of the secondary structure of the viral genome. In addition to S1 and S2, the HCV genome contains several other putative miR-122 binding sites, one in the IRES region, three in the NS5B coding region, and one in the 3' UTR. We investigated and compared the relative contributions of the S1, S2, IRES, NS5B (NS5B.1, 2 and 3) and 3' UTR sites on protein expression, viral RNA accumulation, and infectious particle production by mutational analysis and supplementation with compensatory mutant miR-122 molecules. We found that mutations predicted to alter miR-122 binding at the IRES and NS5B.2 sites lead to reductions in HCV core protein expression and viral RNA accumulation; with a concomitant decrease in viral particle production for the NS5B.2 mutant. However, supplementation of miR-122 molecules with compensatory mutations did not rescue these site mutants to wild-type levels, suggesting that mutation of these sequences likely disrupts an additional interaction important to the HCV life cycle, beyond direct interactions with miR-122. Thus, S1 and S2 play a predominant role in viral RNA accumulation, while miR-122 interactions with the IRES, NS5B and 3' UTR regions have negligible contributions to viral protein expression, viral RNA accumulation, and infectious particle production.

MiRNA-9 inhibits apoptosis and promotes proliferation in angiotensin II-induced human umbilical vein endothelial cells by targeting MDGA2.

Hypertension is a universal risk factor for a variety of cardiovascular diseases. Investigation of the mechanism for hypertension will benefit around 40% of the world's adult population. MicroRNA is crucial for the initiation and progression of cardiovascular diseases. In this study, angiotensin II-treated human umbilical vein endothelial cells were used as a model to imitate the pathological changes in endothelial cells under hypertensive conditions. We demonstrated that microRNA-9 (miR-9) suppressed angiotensin II-induced apoptosis and enhanced proliferation in human umbilical vein endothelial cells. Direct interaction between miR-9 and mitochondria associated membrance domain containing glycosylphosphatidylinositol anchor 2 (MDGA2) was determined. Moreover, miR-9 suppressed MDGA2 levels by binding to the 3' UTR site of the MDGA2 gene. This negative regulation of MDGA2 by miR-9 significantly increased proliferation and decreased apoptosis. Re-introduction of MDGA2 in the miR-9 overexpressed human umbilical vein endothelial cells and normalized proliferation, apoptosis, and the cell cycle. In summary, the present study demonstrated miR-9 inhibited expression of MDGA2 leading to the inhibition of apoptosis and promotion of proliferation in angiotensin II-treated human umbilical vein endothelial cells.

Mesenchymal stem cells-microvesicle-miR-451a ameliorate early diabetic kidney injury by negative regulation of P15 and P19.

The mechanism of MSCs repairing the injured kidney in diabetic nephropathy is not yet clear. In the research, MVs showed the same surface markers as MSCs but much higher MiR-451a expression. miR-451a was decreased in both injured HK-2 cells and kidneys. MV-miR-451a stimulated the cell proliferation and viability in vitro and promoted structural and functional improvements of injured kidney in vivo. Infusion of MV-miR-451a ameliorated EMT by reducing α-SMA and increasing E-cadherin. These effects relied on the improved cell cycle arrest and the down-regulation of P15 and P19 via miR-451a binding to their 3'-UTR region. This study demonstrated that MSC-MV-miR-451a could specifically inhibit cell cycle inhibitors to restart the blocked cell cycle and reverse EMT in vivo and in vitro. Therefore, miR-451a may be a new target for DN therapy.

Effect of miR-433-3p and miR-883b-5p on murine CYP 3A family enzymes in AML12 cells.

Here we searched for microRNAs that could interact with cytochrome P450 (CYP) enzymes in silico, and then investigated their effects on Cyp gene expressions using the cultured mouse liver cell line AML12. Among the mouse Cyp3a genes, some miRNAs were found to interact with Cyp3a11, 13, 16, and 44 by the in silico analysis using the miRWalk2.0 database. In addition to this software, which included twelve miRNA target prediction algorithms, we also applied our in-house-developed Excel VBA algorithm to obtain predictions more efficiently. Finally, two miRNAs, miR-433-3p and miR-883b-5p, were extracted as candidates that interact with Cyp3a genes. To evaluate the effects of these miRNAs on Cyp3a gene expression, we first examined whether they actually interacted with the Cyp3a 3'-untranslated region (3'-UTR) using a luciferase assay system in AML12 cells. We then evaluated whether the expression of each miRNA affected the expression of Cyp3a mRNAs and their transcribed proteins. We found that the transiently expressed miRNAs significantly reduced the reporter activity of the Cyp3a 3'-UTR site in AML12 cells. In addition, the mRNA and protein expressions of the corresponding Cyp3as were significantly decreased in the miRNA-treated AML12 cells. Using cultured cells, we clearly demonstrated that miR-433-3p and miR-883b-5p, which were identified by in silico prediction, actually bind to Cyp3a mRNAs and regulate Cyp gene expressions.

Abstract 1259: Loss of TFF1 promotes growth and chemotherapeutic resistance in gastric cancer

Abstract Background: Downregulation or loss of TFF1 expression occurs in more than half of human gastric adenocarcinomas through gene deletions, mutation, and a loss of heterozygosity or hypermethylation. Our previous studies have shown that Tff1 knockout (KO) in mice induces gastric lesions from low- grade dysplasia (LGD), high- grade dysplasia (HGD) to adenocarcinoma. BRD2, a family member of BET proteins, promotes aberrant gene expression in a variety of malignant tumors. In this study, with the analysis of Tff1 KO mice and human gastric cancer tissue samples, we discovered that loss of Tff1 promotes gastric cancer proliferation and drug resistance through upregulating BRD2 expression level. Methods and Results: Integration Next Generation Sequencing data analysis in both Tff1 KO mice and human gastric cancer tissue samples demonstrated that miR-143-3p was significantly down-regulated in both mice and human gastric cancer samples (P<0.05). Our findings were further validated by RT-PCR in Tff1 KO mouse gastric legions (LGD and adenocarcinoma) and 3 different cohorts of human gastric cancer tissue samples from USA, Chile, and China. Using different gastric cancer cell models we further confirmed the decrease of miR-143-3p regulated by loss of TFF1. Next, we wanted to find out the downstream effector of miR-143-3p down-regulation. Western blot data showed that BRD2 protein level was regulated by miR-143-3p in 4 gastric cancer cell lines. BRD2 3'UTR luciferase reporter assay further confirmed that miR-143-3p decreased BRD2 protein expression through directly binding to its 3'UTR sites. These data suggested that, for the first time, BRD2 is a direct downstream target of miR-143-3p. At the meantime, the reconstitution of TFF1 in gastric cancer cells up-regulated miR-143-3p expression level, which in turn, decreased the protein expression levels of BRD2, MYC and BCL-2. Using western blot, we showed the synergistic effect of BRD2 inhibitor and CDDP chemotherapy in human gastric cancer cells. Data from 320 human gastric cancer patients demonstrated that high BRD2 expression level in gastric cancer tissues significantly decreased the overall patient survival rate (P<0.0001). Conclusion: This study unveils, for the first time, loss of TFF1 promotes BRD2 activation in gastric cancer through decreasing miR-143-3p. This axis presents novel therapeutic opportunities by using approaches that reconstitute miR-143-3p or utilizing the recently developed clinical trials in human gastric cancer. Citation Format: Zheng Chen, Zheng Li, Mohammed Soutto, Weizhi Wang, Shoumin Zhu, Alejandro Corvalan, Zekuan Xu, Wael El-Rifai. Loss of TFF1 promotes growth and chemotherapeutic resistance in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1259.

MiR-30a attenuates cardiac fibrosis in rats with myocardial infarction by inhibiting CTGF.

The mechanism of miR-30a in myocardial fibrosis in rats with myocardial infarction (MI) was investigated. rAAV9-miR-30a was constructed and transfected to heart via injecting into the left ventricular cavity of MI rats. The sham operation group, control group, miR-30a group and miR-30a-NC group were established. Besides, the 3′-UTR of CTGF was inserted into luciferase expression plasmid (pMir-report), then transfected into COS1 cells. miR-30a and control miRNA were, respectively, cotransfected into COS1 cells. The expression of luciferase was detected before and after knockdown of the binding site of miR-30a and the 3′-UTR of CTGF. Four weeks after MI surgery, cardiac function was measured by color Doppler echocardiography, including short axis shortening (FS) and left ventricular ejection fraction (LVEF); the myocardial collagen volume fraction (CVF) was observed by Masson's staining; deposition of collagen I and collagen III were evaluated by immunohistochemical stain; using real-time PCR to detect expression levels of miR-30a and CTGF; the expression of CTGF was observed by western blotting. In MI group, cardiac function was significantly improved, while the expression levels of CVF, collagen I and III, the ratio of type I/III collagen, CTGF were significantly reduced. After knockdown the binding site of miR-30a and the 3′-UTR of CTGF, luciferase expression in COS1 cells decreased significantly. miR-30a might inhibit the expression of CTGF by directly combining with the 3′-UTR site of CTGF after MI, thereby reduce the production of collagen in myocardia, inhibit myocardial fibrosis, then improve cardiac function.

Post-transcriptional Regulation by 3′ UTRs Can Be Masked by Regulatory Elements in 5′ UTRs

In mRNA sequences, 3' UTRs are thought to contain most elements that specifically regulate localization, turnover, and translation. Although high-throughput experiments indicate that many RNA-binding proteins (RBPs) also bind 5' UTRs, much less is known about specific post-transcriptional control exerted by 5' UTRs. GLD-1 is a conserved RBP and a translational repressor with essential roles in Caenorhabditis elegans germ cell development. Previously, we showed that GLD-1 binds highly conserved sites in both 3' and 5' UTRs. Here, by targeted single-copy insertion of transgenes, we systematically tested invivo functionality of 5' and 3' UTR binding sites individually and in combination. Our data show that sites in 5' UTRs mediate specific and strong translational repression, independent of exact position. Intriguingly, we found that the functionality of 3' UTR sites can be masked by 5' UTR sites and vice versa. We conclude that it is important to study both UTRs simultaneously.

MiR-330-5p/Tim-3 axis regulates macrophage M2 polarization and insulin resistance in diabetes mice

Obesity is associated with a state of low-grade inflammatory response in adipose tissue, and contributes to the development of type 2 diabetes.Immune cells such as macrophages can infiltrate adipose tissue and are responsible for the majority of inflammatory cytokine production. Therefore, adipose tissue promotes macrophage infiltration, resulting in local inflammation and insulin resistance. Tim-3 negatively regulates IFN-γ secretion and influences the ability to induce T cell tolerance in diabetes. MicroRNA contributes to the development of immunological tolerance and involves in macrophage polarization. However, the potential of Tim-3 to regulate macrophage polarization and the related microRNA has not been reported. In this experiment, 8-week-old C57BL/6 mice were fed a high-fat diet for 8 weeks. The adipose tissue macrophages were isolated, miR-330-5p and Tim-3 levels, and M1/M2 polarization were analyzed. In addition, insulin tolerance tests was detected. The results demonstrated that miR-330-5p levels increased but Tim-3 levels decreased, leading to M1 polarization and insulin tolerance in diabetes mice. In addition, inhibition of miR-330-5p enhanced Tim-3 levels, leading to M2 polarization and insulin tolerance attenuation in diabetes mice. Furthermore, we detected the inverse relationship between miR-330-5p and Tim-3. We found that Tim-3 mRNA contained conserved miR-330-5p binding sites in its 3′UTR, and miR-330-5p could directly regulate Tim-3 expression through these 3′UTR sites. Our study demonstrated that miR-330-5p served as a regulator of the M2 polarization and miR-330-5p/Tim-3 axis potentially down-regulated insulin resistance in diabetes, probably through enhancing the M2 polarization of macrophage.

Regulatory role of miR-18a to CCN2 by TGF-β1 signaling pathway in pulmonary injury induced by nano-SiO2.

This research is designed to investigate the regulatory effect of miR-18a to the target gene connective tissue growth factor (CTGF, or CCN2), by participating in TGF-β1 signaling pathway and explore the pathogenic mechanism of miR-18a in pulmonary injury induced by nano-SiO2 based on our early study. miR-18a and expression of TGF-β1 in Chinese hamster lung (CHL) fibroblasts cells stimulated by supernatants of NR8383 cells exposed to 40μg/ml nano-SiO2 for 24h demonstrated 1.58±0.22-fold and 1096.00±2.60pg/ml increase compared with blank control group analyzed by real-time quantitative PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Expression increase of miR-18a and reduction of CCN2 mRNA expression levels and protein gray value ratio detected by Western blotting in CHL cells transfect miR-18a mimics for 48h. The reverse of CHL cell transfection miR-18a inhibit is also true. The result of miR-18a and CCN2 binding sites tested by luciferase reporter gene assay shows that the report relative fluorescence value of miR-18a mimics wild type on CCN2 is 0.50±0.02 with the control of mimics NC and mutant relative fluorescence report value 0.86±0.04 (P<0.05). Expression levels of miR-18a, CCN2 mRNA, and protein gray value ratio decreased in CHL cells treated by TGF-β1, respectively, and vice versa treated by TGF-β1corepressor. The results suggest that CCN2 is the target gene regulated by miR-18a and miR-18a participates in TGF-β1 signaling pathway by regulating the expression of CCN2 negatively through CCN2 3'UTR site, and thus may be involved in the development process of pulmonary injury.

Knockdown of long non-coding RNA MALAT1 inhibits growth and motility of human hepatoma cells via modulation of miR-195.

The metastasis-associated lung adenocarcinoma transcription 1 (Malat1) is a long non-coding RNA (lncRNA), exerts oncogenic role in multiple cancers, including hepatocellular carcinoma (HCC). This study was aimed to investigate its posttranscriptional regulation in HCC cells. RT-PCR was performed to monitor the expression levels of Malat1 in normal liver and HCC cell lines. The expression of Malat1, microRNA (miR)-195, and epidermal growth factor receptor (EGFR) in HepG2 and MHCC97 cells was respectively or synchronously altered by transfection. Then the changes in cell viability, apoptotic cell rate, cell cycle distribution, migration, and invasion were respectively assessed. As a result, we found that Malat1 was highly expressed in HCC cell lines when compared to normal liver cells. Malat1 silence suppressed HCC cells viability, migration and invasion, induced apoptosis, and arrested more cells in G0/G1 phase. Malat1 acted as a circular endogenous RNA (ceRNA) for miR-195. Malat1 silence could not suppress HCC cell growth and motility when miR-195 was knocked down. EGFR was a direct target of miR-195. miR-195 overexpression could not suppress HCC cell growth and motility when the 3'UTR site of EGFR was overexpressed. Furthermore, Malat1 silence blocked the activation of PI3K/AKT and JAK/STAT pathways, while EGFR overexpression activated them. Our study demonstrates Malat1-miR-195-EGFR axis plays a critical role in HCC cells which provided a better understanding of Malat1 in HCC.

Abstract 12: M6A Modification in RNA Regulates Cardiomyocyte and Cardiac Function in Heart Failure

Background: Adenosine in RNA is a substrate for addition or removal of methyl group. Reported five decades ago, methylated adenosine (m6A), the most abundant and functionally relevant chemical modification in RNA, whose transcriptome-wide mapping became possible only recently due to next generation sequencing (NGS). Coupled with NGS, m6A-methylated RNA capture (MeRIP-seq) identified widespread m6A distribution in ~8000 mRNA and ~1000 lncRNA transcripts in human and mouse transcriptome. Methods and Results: In a first of its kind approach, we examined m6A RNA methylation in both failing and non-failing hearts. We discovered that global m6A RNA methylation is significantly higher in left ventricles (LV) of failing human, swine and mouse hearts as compared to non-failing controls. Increase in m6A was associated with significantly lower expression of one of the key m6A demethylases, FTO, in the ischemic heart. siRNA-mediated silencing of FTO resulted in significant arrhythmias, loss of Ca 2+ dynamics such as Ca 2+ transient decay (Tau) and cardiomyocyte relaxation time in isolated adult rat cardiomyocytes. Conversely, FTO gene transfer reduced m6A and improved Ca 2+ transients and contractile function in primary cardiomyocytes under hypoxia. In a mouse model of MI, AAV-mediated gene transfer of FTO significantly improved cardiac function post-MI. We identified transcriptome-wide m6A distribution signatures and conserved methylated sites of several mRNAs and lncRNAs using MeRIP-seq in both human and mouse failing and non-failing LV. Detailed MeRIP map of individual transcripts identified differentially methylated 3’-UTR, 5’-UTR and exon sites within several cardiac mRNAs that are important for cardiac function. Conclusion: Our data provide first evidence that m6A modification in RNA is a regulator of cardiomyocyte Ca 2+ dynamics and cardiac function. Our findings on the dynamic nature of the cardiac m6A-epitranscriptome will add another portfolio to mRNA and lncRNA regulation of cardiac remodeling.

Abstract 4244: Detection and characterization of extrachromosomal circular DNA in human plasma

Abstract Background: eccDNA (extrachromosomal circular DNA) has been reported in most eukaryotes, including in human cells. However, little is known about the eccDNA profiles in human circulating system such as blood. Method: We extracted plasma cell free DNA (cfDNA) from three patients with advanced stage cancers. For each patient, we tested 10ng, 1ng and 0.1ng cfDNA, each with a technical replicate. The cfDNA samples were subjected to ATP-dependent DNase digestion and whole genome amplification (WGA). An additional 2ng cfDNA from each patient skipped the DNase digestion and went directly to WGA step. We sequenced the amplified DNAs using the Illumina 100bp PE read protocol. We analyzed sequencing data using a custom split reads-based strategy to retrieve circular DNA molecules. We further analyzed eccDNAs for their biofeatures using functional annotation tools. Results: We received sequence reads at ~14.8 million (11.4~22.4) for each sample. When read depth≥ 3 as a cutoff, we detected a total number of 2542, 1578, and 57 unique eccDNAs in three patients while negative controls showed &amp;lt;=5 eccDNAs. For non-DNase digested cfDNAs, we detected only 3 eccDNAs. By comparing read numbers mapped to mitochondria and human genome, we found that mitochondrial sequence reads increased significantly from 0.34-1.1% before digestion to 24.1-50.1% after digestion. Numbers of detected eccDNAs were similar between technical replicates and proportional to initial input cfDNA. However, eccDNAs detected also showed few overlaps between technical replicates. Size distribution ranged from 31bp to 19740bp (median 1801-1962bp). To test if eccDNAs were generated in specific genomic regions and if eccDNA-involved genes were enriched in specific pathways, we performed enrichment analysis and observed higher GC content in smaller eccDNAs (&amp;lt;500 bp, 48%) than larger ones (&amp;gt;500bp, 41%) (p&amp;lt;0.001). When compared to random distribution, eccDNAs were more likely located at CpG islands (1.36-1.53 fold enrichment). The eccDNAs were also enriched in exons, 5’UTR, 3’UTR and DNase hypersensitive sites (enrichment folds from 1.39 to 2.3). Using DAVID annotation tool, we observed significant enrichment of the eccDNA-related genes in several categories including the Coiled coil, phosphoprotein, fibronectin, suggesting a potential involvement in gene expression regulation and cell adhesion events. Using GREAT (Genomic Regions Enrichment of Annotations Tool), we found that the eccDNAs were involved in genes associated with invasive cancer cell gene expression regulation (p&amp;lt;0.001). Conclusion: Human plasma contains an abundant number of eccDNAs. Distribution of these eccDNAs is nonrandom event and is likely enriched in genomic regions with known functional consequences and genes that play a role in gene regulations. Further characterization of circulating eccDNAs in peripheral blood will facilitate understanding of their molecular mechanisms and potential clinical utilities. Citation Format: Jing Zhu, Meijun Du, Peng Zhang, Liang Wang. Detection and characterization of extrachromosomal circular DNA in human plasma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4244. doi:10.1158/1538-7445.AM2017-4244

Estimating the parameters of background selection and selective sweeps in Drosophila in the presence of gene conversion

We used whole-genome resequencing data from a population of Drosophila melanogaster to investigate the causes of the negative correlation between the within-population synonymous nucleotide site diversity (πS ) of a gene and its degree of divergence from related species at nonsynonymous nucleotide sites (KA ). By using the estimated distributions of mutational effects on fitness at nonsynonymous and UTR sites, we predicted the effects of background selection at sites within a gene on πS and found that these could account for only part of the observed correlation between πS and KA We developed a model of the effects of selective sweeps that included gene conversion as well as crossing over. We used this model to estimate the average strength of selection on positively selected mutations in coding sequences and in UTRs, as well as the proportions of new mutations that are selectively advantageous. Genes with high levels of selective constraint on nonsynonymous sites were found to have lower strengths of positive selection and lower proportions of advantageous mutations than genes with low levels of constraint. Overall, background selection and selective sweeps within a typical gene reduce its synonymous diversity to ∼75% of its value in the absence of selection, with larger reductions for genes with high KA Gene conversion has a major effect on the estimates of the parameters of positive selection, such that the estimated strength of selection on favorable mutations is greatly reduced if it is ignored.

Newer Insights in Personalized and Evidence Based Medicine- the Role of MicroRNAs

Background: MicroRNAs are non-coding RNAs which regulate gene expression by binding to 3’ UTR site on mRNA. These small RNAs repress gene expression by interfering with the process of translation or by degrading the target mRNA. Abnormal expression of miRNAs has been implicated in a wide spectrum of human diseases like atherosclerosis, cancers and diabetes. Traditionally, pharmacogenomics focuses on single nucleotide polymorphisms and copy number variants of genes implicated in pharmacokinetics and pharmacodynamics. Recently, drug response has been proposed to be regulated by microRNAs. Mutations involving miRNAs can contribute to disease pathogenesis and modulation of drug response. Keywords: MicroRNA, pharmacogenomics, evidence based medicine, personalized medicine, microRNA therapeutics, circulating microRNA, Pharmaco-miR.

Identification of Novel Rosavirus Species That Infects Diverse Rodent Species and Causes Multisystemic Dissemination in Mouse Model.

While novel picornaviruses are being discovered in rodents, their host range and pathogenicity are largely unknown. We identified two novel picornaviruses, rosavirus B from the street rat, Norway rat, and rosavirus C from five different wild rat species (chestnut spiny rat, greater bandicoot rat, Indochinese forest rat, roof rat and Coxing's white-bellied rat) in China. Analysis of 13 complete genome sequences showed that “Rosavirus B” and “Rosavirus C” represent two potentially novel picornavirus species infecting different rodents. Though being most closely related to rosavirus A, rosavirus B and C possessed distinct protease cleavage sites and variations in Yn-Xm-AUG sequence in 5’UTR and myristylation site in VP4. Anti-rosavirus B VP1 antibodies were detected in Norway rats, whereas anti-rosavirus C VP1 and neutralizing antibodies were detected in Indochinese forest rats and Coxing's white-bellied rats. While the highest prevalence was observed in Coxing's white-bellied rats by RT-PCR, the detection of rosavirus C from different rat species suggests potential interspecies transmission. Rosavirus C isolated from 3T3 cells causes multisystemic diseases in a mouse model, with high viral loads and positive viral antigen expression in organs of infected mice after oral or intracerebral inoculation. Histological examination revealed alveolar fluid exudation, interstitial infiltration, alveolar fluid exudate and wall thickening in lungs, and hepatocyte degeneration and lymphocytic/monocytic inflammatory infiltrates with giant cell formation in liver sections of sacrificed mice. Since rosavirus A2 has been detected in fecal samples of children, further studies should elucidate the pathogenicity and emergence potential of different rosaviruses.

MicroRNA-122 target sites in the hepatitis C virus RNA NS5B coding region and 3' untranslated region: function in replication and influence of RNA secondary structure.

We have analyzed the binding of the liver-specific microRNA-122 (miR-122) to three conserved target sites of hepatitis C virus (HCV) RNA, two in the non-structural protein 5B (NS5B) coding region and one in the 3' untranslated region (3'UTR). miR-122 binding efficiency strongly depends on target site accessibility under conditions when the range of flanking sequences available for the formation of local RNA secondary structures changes. Our results indicate that the particular sequence feature that contributes most to the correlation between target site accessibility and binding strength varies between different target sites. This suggests that the dynamics of miRNA/Ago2 binding not only depends on the target site itself but also on flanking sequence context to a considerable extent, in particular in a small viral genome in which strong selection constraints act on coding sequence and overlapping cis-signals and model the accessibility of cis-signals. In full-length genomes, single and combination mutations in the miR-122 target sites reveal that site 5B.2 is positively involved in regulating overall genome replication efficiency, whereas mutation of site 5B.3 showed a weaker effect. Mutation of the 3'UTR site and double or triple mutants showed no significant overall effect on genome replication, whereas in a translation reporter RNA, the 3'UTR target site inhibits translation directed by the HCV 5'UTR. Thus, the miR-122 target sites in the 3'-region of the HCV genome are involved in a complex interplay in regulating different steps of the HCV replication cycle.

Target regulation of micro-RNA-203 to the expression of collagen type Ⅳ alpha 4 and its role in oral submucous fibrosis

To explore the effect of microRNA-203(miR-203) on the expression of collagen type IV alpha 4(COL4A4) and its role in oral submucous fibrosis(OSF). It has been revealed that COL4A4 is the putative target of miR-203 by retrieving a variety of bioinformatics software tools. The expression of COL4A4 protein in the indicated tissues was examined by Western blotting analysis and the potential binding sites of miR-203 and COL4A4-3'UTR was analyzed by using bioinformatics. This study integrated a fragment of COL4A4 3'UTR containing the target sequence into the pMIR-REPORT luciferase vector. Then, the COL4A4 3'UTR-luciferase and miR-203 were contransfected into 293T cells. Beta-gal was used as a control to monitor transfection efficiency. Cells were harvested and luciferase activity was measured after 24 h of transfection. The relative expression mean valves of COL4A4 were 2.46 ± 2.26 in OSF and 0.70 ± 0.49 in normal control. The expression of COL4A4 in OSF was higher than normal control(P<0.05) and the difference between groups was statistically significant. The relative expression mean valves of miR-203 were 2.57 ± 2.09 in OSF and 154.07 ± 191.11 in normal control. The expression of miR-203 in OSF was lower than in normal control(P<0.01) and the difference between groups was statistically significant. Bioinformatics analysis revealed that the COL4A4 3'UTR had a conserved site (site 1) and 3 non-conserved sites(site 2, site 3, site4) complementary to the miR-203. MiR-203 could significantly inhibit site 1-luciferase reporter activity, partially inhibited site 3- and site 4-luciferase reporter activities, while no significant effect on site 3-luciferase reporter activity. MiR-203 was negatively correlated with the expression of COL4A4 protein in OSF. Moreover, miR-203 repressed the expression of COL4A4 by targeting 3'UTR site of COL4A4 mRNA.

MicroRNA-197 Mediates the Overgrowth and Anti-Apoptotic Effects by Downregulating Insulin-Like Growth Factor-Binding Protein-3 During Nephroblastoma Tumorigenesis

ABSTRACTMiR-197 is frequently upregulated to induce a series of oncogenic effects, which is closely associated with poor survival and prognosis of multiple malignancies. However, the roles of miR-197 in tumorigenesis and the detailed molecular mechanism in Wilms tumor (WT) have rarely been reported. This study aimed to evaluate the expression of miR-197 in WT in vivo and the potential effects of miR-197 on the proliferation and apoptosis in SK-NEP-1 cells. A total of 15 patients with a pathologically confirmed diagnosis of WT and 15 paraneoplastic controls were enrolled. Real-time quantitative PCR (RT-qPCR) identified the upregulation of miR-197 and downregulation of insulin-like growth factors binding protein 3 (IGFBP3) in WT tissues in comparison with adjacent normal tissue (p < 0.001). CCK-8 and flow cytometry assay found that inhibition of miR-197 caused a significantly reduced proliferation along with a dramatically enhanced apoptosis of SK-NEP-1 cells (p < 0.01). IGFBP3 was overexpressed in SK-NEP-1 cells by pEGFP-C1-IGFBP3 plasmid transfection. Overexpression of IGFBP3 suppressed the proliferation and induced the apoptosis of SK-NEP-1 cells (p < 0.01). Further study detected the decreased IGFBP3 expression with miR-197 mimics SK-NEP-1 cells and increased IGFBP3 expression with miR-197 inhibitor SK-NEP-1 cells compared with mock (p < 0.01). Dual luciferase reporter assay revealed a direct interaction between miR-197 and 3′-UTR site of IGFBP3. Overall, the above results indicated that miR-197 targeted IGFBP3 to induce the overgrowth and anti-apoptotic effects of WT cells, which could promote nephroblastoma tumorigenesis. Therefore, miR-197 may be further assessed as a potential target for the treatment of WT.

Characterization of the Ryanodine Receptor Gene With a Unique 3'-UTR and Alternative Splice Site From the Oriental Fruit Moth.

The ryanodine receptor (RyR), the largest calcium channel protein, has been studied because of its key roles in calcium signaling in cells. Insect RyRs are molecular targets for novel diamide insecticides. The target has been focused widely because of the diamides with high activity against lepidopterous pests and safety for nontarget organisms. To study our understanding of effects of diamides on RyR, we cloned the RyR gene from the oriental fruit moth, Grapholita molesta, which is the most serious pest of stone and pome tree fruits throughout the world, to investigate the modulation of diamide insecticides on RyR mRNA expression in G. molesta (GmRyR). The full-length cDNAs of GmRyR contain a unique 3′-UTR with 625 bp and an open reading frame of 15,402 bp with a predicted protein consisting of 5,133 amino acids. GmRyR possessed a high level of overall amino acid homology with insect and vertebrate isoforms, with 77–92% and 45–47% identity, respectively. Furthermore, five alternative splice sites were identified in GmRyR. Diagnostic PCR showed that the inclusion frequency of one optional exon (f) differed between developmental stages, a finding only found in GmRyR. The lowest expression level of GmRyR mRNA was in larvae, the highest was in male pupae, and the relative expression level in male pupae was 25.67 times higher than that of in larvae. The expression level of GmRyR in the male pupae was 8.70 times higher than in female pupae, and that in male adults was 5.70 times higher than female adults.

Genome-Wide Crispr-Cas9 Screen Identifies Functionally Relevant Micro-RNAs in FLT3-ITD+ AML

Mammalian microRNA expression is dysregulated in many human cancers, including hematologic malignancies. MicroRNAs are small non-coding RNAs that repress their target genes by binding to the 3' UTR sites in their respective mRNA targets, leading to transcript degradation and/or preventing translation. MicroRNAs play a variety of physiologic roles within the hematopoietic compartment, and a few microRNAs have even been shown to drive leukemogenesis when deleted or expressed at abnormally high levels. Among hematologic malignancies, acute myeloid leukemia (AML) carries a particularly poor prognosis, leading to over 10,000 deaths each year. The most common genetic aberration in AML is a gain-of-function mutation in the FMS-like tyrosine kinase 3 (FLT3) receptor. FLT3 internal tandem duplication (ITD) occurs in ~25% of all AML diagnoses, and confers a poor prognosis due to increased cellular proliferation and survival of the hematopoietic stem and progenitor cells (HSPCs). MicroRNA expression has been shown to be highly dysregulated in FLT3-ITD+ AML, however the functional relevance of many of these microRNAs on leukemic phenotypes remains unclear. In this study, we took an unbiased approach to determine which microRNAs, and which of their putative targets, are involved in FLT3-ITD+ AML cellular growth.Using CRISPR-Cas9 technology, we performed a global loss-of-function screen to simultaneously test the functions of individual microRNAs and protein-coding genes during the growth of FLT3-ITD+ leukemia cells (MV4-11 cells) over a 23-day time course. By comparing library representation from genomic DNA isolated from the final time point versus the initial time point, we were able to identify a number of protein-coding gene and microRNA candidates that either promoted or suppressed FLT3-ITD+ AML cellular growth.Our screen identified both evolutionarily conserved and non-conserved human microRNAs that function to suppress or promote FLT3-ITD+ AML cellular growth, revealing that microRNAs are extensively integrated into the molecular networks that control tumor cell physiology. We also performed anti-correlation functional profiling to predict relevant microRNA-tumor suppressor gene or microRNA-oncogene interactions in FLT3-ITD+ cells. We validated one of our targets, miR-155, as a critical regulator of FLT3-ITD+ AML cell growth in vitro. miR-155 knockout cells displayed a competitive growth disadvantage compared to miR-155 wild type cells. Further analysis revealed that deletion of miR-155 in MV4-11 cells led to decreased STAT5 activation, a key signaling intermediate known to promote cell survival and proliferation in FLT3-ITD+ AML. Finally, we found that miR-155 promotes FLT3-ITD-mediated myeloproliferation in vivo. FLT3-ITD miR-155-/- mice exhibited decreased myeloid expansion in the bone marrow, reduced splenomegaly, and decreased peripheral blood monocytosis compared to their FLT3-ITD miR-155+/+ counterparts. This phenotype was attributed to miR-155's role in promoting proliferation of the HSPC and myeloid progenitor cell compartments in the bone marrow.Our CRISPR-Cas9 screen identified a subset of microRNAs that regulate FLT3-ITD+ cell growth, and extensively validated miR-155 as a promoter of FLT3-ITD+ cell proliferation. These findings were validated both in vitro and in vivo, and suggest that miR-155 inhibitors may warrant clinical consideration as therapeutics in FLT3-ITD+ AML. Taken together, our study describes a powerful genetic approach by which the function of individual microRNAs can be assessed on a global level, and its use could rapidly advance our understanding of how microRNAs contribute to human hematological diseases. DisclosuresNo relevant conflicts of interest to declare.