Expression Of miRNA Targets Research Articles

The Effect of Placenta Growth Factor Knockdown on hsa-miR-22-3p, hsa-let-7b-3p, hsa-miR-451b, and hsa-mir-4290 Expressions in MKN-45- derived Gastric Cancer Stem-like Cells

Background: Placental growth factor is involved in human gastric cancer initiation and progression through stimulating the proliferation, angiogenesis, invasion and metastasis of cancerous cells. Previous studies indicate that the expression profiles of hsa-miR-22-3p, hsa-let-7b-3p, hsa-miR-451b, and hsa-mir-4290 change in MKN-45- derived gastric cancer stem-like cells. Therefore, this study aims to investigate the effect of PlGF knockdown on hsa-miR-22-3p, hsa-let-7b-3p, hsa-miR-451b, and hsa-mir-4290 expressions in MKN-45-derived gastric cancer stem-like cells.Methods: We used a non-adhesive culture system to derive the cancer stem-like cells from MKN-45 cells. PlGF gene silencing was performed by PlGF-specific siRNA. The transcript of PlGF and miRNAs were measured by real-time RT-PCR. We conducted bioinformatics analyses with the online software programs TargetScan, miRanda, miRWalk, PicTar, and the Database for Annotation, Visualization, and Integrated Discovery tools to predict miRNAs’ targets and their signaling pathways.Results: hsa-let-7b-3p had a 2.28-fold up-regulation, whereas we observed downregulation of hsa-mir-451b (25%), hsa-mir-4290 (34%), and hsa-mir-22-3p (9%). Bioinformatics analysis results indicated that the miRNA target genes TGF-β, MAPK, and Wnt, and hedgehog signaling pathways contributed to cancer initiation and progression by influencing different cellular behaviors.Conclusion: We suggest that PlGF signaling may influence miRNA expression profiles in MKN-45-derived cancer stem-like cells, which can influence the expressions of different genes and signaling pathways. However, more empirical studies should determine the exact effect of PlGF knockdown on the expression of miRNA targets in cancer stem-like cells to locate their actual gene targets.

MicroRNA-192-5p is a predictive biomarker of survival for Stage IIIB colon cancer patients.

The poor clinical prognosis of Stage IIIB colon cancer patients is due in part to the current lack of an effective diagnostic method being available and highlights a need for the identification of novel biomarkers like microRNA (miRNA). We used microarray analysis to compare the miRNA expression profiles of eight Stage IIIB colon cancer patients with worse clinical outcome (those who developed liver metastases between 8 and 18 months after surgery) against eight 'cured' Stage IIIB colon cancer patients (those who remained disease free following surgery during the same monitoring period). In addition, quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed examining miRNAs in tumor tissue of 98 patients with Stage IIIB colon cancer. We found, miRNA-192-3p and miRNA-192-5p were down regulated in the patients with worsening disease compared to the control 'cured' Stage IIIB colon cancer patients (P = 0.026 and P = 0.042, respectively). Patients with higher expression of miRNA-192-5p had higher 5-year disease-free survival (DFS) (84.21%) and overall survival (OS) (89.47%) than those with lower targeted miRNA expression DFS (38.8%; hazard ratio (HR): 3.74, 95% confidence interval (CI): 1.52-9.23, P = 0.042) and OS (48.57%; HR: 5.01, 95% CI: 1.75-14.38, P = 0.033). In contrast, patients with higher expression of miRNA-192-3p did not appear to statistically impact the survival of patients in this setting (DFS 73.33% vs 64.7%, HR: 0.68, 95% CI: 0.31-1.52, P = 0.35; OS 76.67% vs 66.17%, HR: 0.62, 95% CI: 0.27-1.45, P = 0.27). The decreased expression of miRNA-192-5p found for patients with relapsing disease might represent a highly predictive marker to use for the prognosis of Stage IIIB colon cancer patients.

CircRNA-Cdr1as Exerts Anti-Oncogenic Functions in Bladder Cancer by Sponging MicroRNA-135a

Background/Aims: CircRNAs regulate gene expression in different malignancies. However, the role of Cdr1as in the tumourigenesis of bladder cancer and its potential mechanisms remain unknown. Methods: qRT-PCR was used to detect Cdr1as and target miRNA expression in bladder cancer tissues and cell lines. Biological functional experiments were performed to detect the effects of Cdr1as on the biological behaviour of bladder cancer cells in vivo and in vitro. Bioinformatic analysis was utilised to predict potential miRNA target sites on Cdr1as. Ago2 RNA binding protein immunoprecipitation assay, RNA antisense purification assay, biotin pull down assay and RNA FISH were performed to detect the interaction between Cdr1as and target miRNAs. Western blot was used to determine the expression level of p21 in bladder cancer cells. Results: Cdr1as was significantly down-regulated in bladder cancer tissues compared with adjacent normal tissues. Overexpression of Cdr1as inhibited the proliferation, invasion and migration of bladder cancer cells in vitro and slowed down tumour growth in vivo. Cdr1as sponged multiple miRNAs in bladder cancer. Moreover, Cdr1as directly bound to miR-135a and inhibited its activity in bladder cancer. Conclusion: Cdr1as is down-regulated and sponges multiple miRNAs in bladder cancer. It exerts anti-oncogenic functions by sponging microRNA-135a.

MiR-31-5p-ACOX1 Axis Enhances Tumorigenic Fitness in Oral Squamous Cell Carcinoma Via the Promigratory Prostaglandin E2.

During neoplastic development, a multitude of changes in genome-encoded information are progressively selected to confer growth and survival advantages to tumor cells. microRNAs-mRNAs regulatory networks, given their role as a critical layer of robust gene expression control, are frequently altered in neoplasm. However, whether and how these gene perturbations impact metabolic homeostasis remains largely unresolved.Methods: Through targeted miRNA expression screening, we uncovered an oral squamous cell carcinoma (OSCC)-associated miRNAome, among which miR-31-5p was identified based on extent of up-regulation, functional impact on OSCC cell migration and invasion, and direct regulation of the rate-limiting enzyme in peroxisomal β-oxidation, ACOX1.Results: We further found that both miR-31-5p and ACOX1 underpin, in an antagonistic manner, the overall cellular lipidome profiles as well as the migratory and invasive abilities of OSCC cells. Interestingly, the extracellular levels of prostaglandin E2 (PGE2), a key substrate of ACOX1, were controlled by the miR-31-5p-ACOX1 axis, and were shown to positively influence the extent of cell motility in correlation with metastatic status. The promigratory effect of this metabolite was mediated by an elevation in EP1-ERK-MMP9 signaling. Of note, functional significance of this regulatory pathway was further corroborated by its clinicopathologically-correlated expression in OSCC patient specimens.Conclusions: Collectively, our findings outlined a model whereby misregulated miR-31-5p-ACOX1 axis in tumor alters lipid metabolomes, consequently eliciting an intracellular signaling change to enhance cell motility. Our clinical analysis also unveiled PGE2 as a viable salivary biomarker for prognosticating oral cancer progression, further underscoring the importance of lipid metabolism in tumorigenesis.

MicroRNA-146b-5p Identified in Porcine Liver Donation Model is Associated with Early Allograft Dysfunction in Human Liver Transplantation.

BackgroundPoor transplant outcome was observed in donation after brain death followed by circulatory death (DBCD), since the donor organs suffered both cytokine storm of brain death and warm ischemia injury. MicroRNAs (miRNAs) have emerged as promising disease biomarkers, so we sought to establish a miRNA signature of porcine DBCD and verify the findings in human liver transplantation.Material/MethodsMiRNA expression was determined with miRNA sequencing in 3 types of the porcine model of organ donation, including donation after brain death (DBD) group, donation after circulatory death (DCD) group, and DBCD group. Bioinformatics analysis was performed to reveal the potential regulatory behavior of target miRNA. Human liver graft biopsy samples after reperfusion detected by fluorescence in situ hybridization were used to verify the expression of target miRNA.ResultsWe compared miRNA expression profiles of the 3 donation types. The porcine liver graft miR-146b was significantly increased and selected in the DBCD group versus in the DBD and DCD groups. The donor liver expression of human miR-146b-5p, which is homologous to porcine miR-146b, was further examined in 42 cases of human liver transplantations. High expression of miR-146b-5p successfully predicted the post-transplant early allograft dysfunction (EAD) with the area under the ROC curve (AUC) 0.759 (P=0.004).ConclusionsOur results revealed the miRNA signature of DBCD liver grafts for the first time. The miR-146b-5p may have important clinical implications for monitoring liver graft function and predicating transplant outcomes.

Differential miRNA expression in the three-spined stickleback, response to environmental changes

miRNAs play important role in the various physiological and evolutionary processes, however, there is no data allowing comparison of evolutionary differences between various ecotypes adapted to different environmental conditions and specimen demonstrating immediate physiological response to the environmental changes. We compared miRNA expression profiles between marine and freshwater stickleback populations of the three-spined stickleback to identify the evolutionary differences. To study the immediate physiological response to foreign environment, we explored the changes induced by transfer of marine sticklebacks into freshwater environment and vice versa. Comparative analysis of changes in miRNA expression suggested that they are driven by three independent factors: (1) non-specific changes in miRNA expression under different environmental conditions; (2) specific response to freshwater conditions in the marine stickleback ecotype; (3) specific response to extreme osmotic conditions for both marine and freshwater ecotypes during the contact with non-native environment. Gene Ontology enrichment analysis of differential expressed miRNA targets supports our current hypothesis.

Differentially expressed miRNAs in hepatocellular carcinoma cells under hypoxic conditions are associated with transcription and phosphorylation.

Hypoxia is a critical aspect of tumor biology and has been associated with poor prognosis and resistance to traditional therapy. In the present study, differentially expressed genes and microRNAs (miRNAs/miRs) were screened for in the hepatocellular carcinoma (HCC) cell line Huh7 under hypoxic conditions. On the basis of microarray data, 11,508 mRNAs and 58 miRNAs exhibiting ≥1.5-fold change in expression under hypoxic conditions were identified. Gene Ontology (GO) and Kyoto Encyclopedia or Genes and Genomes pathway analysis revealed that the differentially expressed genes were primarily involved in cell cycle regulation, cell division, transcription and G-protein-coupled receptor signaling pathways. Using the TargetScan and miRanda software packages with the miRNA-mRNA negative expression network, differentially expressed miRNA targets were predicted. GO analysis revealed that the primary function of these miRNAs was to regulate transcription and phosphorylation. The miRNA-mRNA networks for transcription and phosphorylation were analyzed. Network analysis revealed that the key miRNAs in these networks were miR-19a, miR-34a, miR-29a, mir-196a, miR-25 and miR-1207, whose potential gene targets include DNA-binding proteins, zinc-finger proteins and transcription factors. Certain protein kinases, includingmitogen-activated protein kinase (MAPK) 1, MAPK kinase kinase4 and cyclin-dependent kinase 18, were also revealed to be present in the network. In hypoxic HCC tissue, levels of several key miRNAs implicated in the network analyses (miR-19a, miR-34a, miR-25 and miR-1207) were revealed to exhibit increased expression levels compared with the surrounding tissue. The results of the present study provide evidence that miRNAs serve an important function in transcription and phosphorylation in the hypoxic response of HCC cells.

Identification of competing endogenous RNAs of the tumor suppressor gene PTEN: A probabilistic approach

Regulation by microRNAs (miRNAs) and modulation of miRNA activity are critical components of diverse cellular processes. Recent research has shown that miRNA-based regulation of the tumor suppressor gene PTEN can be modulated by the expression of other miRNA targets acting as competing endogenous RNAs (ceRNAs). However, the key sequence-based features enabling a transcript to act as an effective ceRNA are not well understood and a quantitative model associating statistical significance to such features is currently lacking. To identify and assess features characterizing target recognition by PTEN-regulating miRNAs, we analyze multiple datasets from PAR-CLIP experiments in conjunction with RNA-Seq data. We consider a set of miRNAs known to regulate PTEN and identify high-confidence binding sites for these miRNAs on the 3′ UTR of protein coding genes. Based on the number and spatial distribution of these binding sites, we calculate a set of probabilistic features that are used to make predictions for novel ceRNAs of PTEN. Using a series of experiments in human prostate cancer cell lines, we validate the highest ranking prediction (TNRC6B) as a ceRNA of PTEN. The approach developed can be applied to map ceRNA networks of critical cellular regulators and to develop novel insights into crosstalk between different pathways involved in cancer.

Exosomal miR-9-3p suppresses HBGF-5 expression and is a functional biomarker in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer-related death worldwide. Exosomes are secreted membrane vesicles that play important roles in various diseases by transporting proteins and RNAs, including microRNAs, between cells. However, the function of exosomal miRNA in HCC has not been fully investigated. Exosomes were obtained from the sera by ultracentrifugation and were processed for transmission electron microscopy (TEM). Real time PCR were revealed changes of miRNA between patients and normal donors. Predicted targets of miRNA were described by bioinformatics analysis, luciferase reporter assay was used to confirmed whether miR-9-3p regulates target expression. And then miRNA were over-expressed in HCC cell line to study its function, western blotting were used to test expression of miRNA targets, Cell viability and proliferation were analyzed after over-expressed miR-9-3p using MTT and BrdU assay. Serum exosomes from patients with HCC contained significantly lower levels of the miR-9-3p than did serum exosomes from normal donors, suggesting a potential role for this microRNA in HCC. Bioinformatics analysis identified fibroblast growth factor 5 (HBGF-5), which plays an important role in cell proliferation, as a potential miR-9-3p target mRNA. Luciferase reporter assay confirming that miR-9-3p can directly regulate HBGF-5 expression. Consistent with this finding, overexpression of miR-9-3p in three HCC cell lines significantly downregulated HBGF-5 expression at both the mRNA and protein levels. Finally, overexpression of miR-9-3p reduced HCC cell viability and proliferation, and additionally reduced ERK1/2 expression, suggesting a potential mechanism by which miR-9-3p acts. These results provide new insight into the functions of miR-9-3p and HBGF-5 in HCC and identify miR-9-3p as a potential therapeutic target for HCC.

Hypoxia in CNS Pathologies: Emerging Role of miRNA-Based Neurotherapeutics and Yoga Based Alternative Therapies.

Cellular respiration is a vital process for the existence of life. Any condition that results in deprivation of oxygen (also termed as hypoxia) may eventually lead to deleterious effects on the functioning of tissues. Brain being the highest consumer of oxygen is prone to increased risk of hypoxia-induced neurological insults. This in turn has been associated with many diseases of central nervous system (CNS) such as stroke, Alzheimer's, encephalopathy etc. Although several studies have investigated the pathophysiological mechanisms underlying ischemic/hypoxic CNS diseases, the knowledge about protective therapeutic strategies to ameliorate the affected neuronal cells is meager. This has augmented the need to improve our understanding of the hypoxic and ischemic events occurring in the brain and identify novel and alternate treatment modalities for such insults. MicroRNA (miRNAs), small non-coding RNA molecules, have recently emerged as potential neuroprotective agents as well as targets, under hypoxic conditions. These 18–22 nucleotide long RNA molecules are profusely present in brain and other organs and function as gene regulators by cleaving and silencing the gene expression. In brain, these are known to be involved in neuronal differentiation and plasticity. Therefore, targeting miRNA expression represents a novel therapeutic approach to intercede against hypoxic and ischemic brain injury. In the first part of this review, we will discuss the neurophysiological changes caused as a result of hypoxia, followed by the contribution of hypoxia in the neurodegenerative diseases. Secondly, we will provide recent updates and insights into the roles of miRNA in the regulation of genes in oxygen and glucose deprived brain in association with circadian rhythms and how these can be targeted as neuroprotective agents for CNS injuries. Finally, we will emphasize on alternate breathing or yogic interventions to overcome the hypoxia associated anomalies that could ultimately lead to improvement in cerebral perfusion.

Enhanced antiproliferative activity of antibody-functionalized polymeric nanoparticles for targeted delivery of anti-miR-21 to HER2 positive gastric cancer.

MiR-21 is an oncogenic miR frequently elevated in gastric cancer. Overexpression of miR-21 decreases the sensitivity of gastric cancer cells to trastuzumab, which is a humanized monoclonal antibody targeting human epidermal growth factor receptor 2. However, optimization of miRNA or its anti-miRNA oligonucleotides (AMOs) for delivery is a challenge. Receptor-mediated endocytosis plays a crucial role in the delivery of biotherapeutics including AMOs. This study is a continuation of our earlier findings involving poly(ε-caprolactone) (PCL)-poly (ethylene glycol) (PEG) nanoparticles (PEG-PCL NPs), which were coated with trastuzumab to target gastric cancer cells with HER2 receptor over-expression using anti-miRNA-21 antisense oligonucleotides (AMO-21). The antibody conjugates (HER-PEG-PCL NPs) act against target cells via antibody-dependent mechanisms and also based on encapsutalated AMO-21. X-ray photoelectron spectroscopy validated the presence of trastuzumab on NP surface. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a stable antibody expression. The cell line specificity, cellular uptake, AMO-21 delivery, and cytotoxicity of the HER-PEG-PCL NPs were investigated. We found that the antibody conjugates significantly enhanced the cellular uptake of NPs. The HER-PEG-PCL NPs effectively suppressed the target miRNA expression in gastric cancer cells, which further up-regulated phosphatase and tensin homolog (PTEN). As a result, the sensitivity of HER2-expressing gastric cancer cells to trastuzumab was enhanced. The approach enhances the targeting by trastuzumab as well as antibody-dependent cellular cytotoxicity of immune effector cells. The antitumor effects of AMO-21-HER-PEG-PCL NPs were compared with trastuzumab in xenograft gastric cancer mice. The results provide insight into the biological and clinical potential of targeted AMO-21 delivery using modified trastuzumab for gastric cancer treatment.

Prognostic microRNAs in upper tract urothelial carcinoma: multicenter and international validation study.

ObjectiveTo validate previously discovered miRNAs (miR-31-5p and miR-149-5p) as prognostic factors for UTUC in an independent cohort of UTUC patients.Patients and MethodsMulticenter, international and retrospective study of formalin-fixed paraffin-embedded tissue samples from 103 UTUC patients (45 progressing and 58 non-progressing) who underwent radical nephroureterectomy. Total RNA was isolated and reverse transcribed. The expression of target miRNAs (miR-31-5p and miR-149-5p) and the endogenous control miR-218-5p was evaluated in all samples by reverse transcription quantitative PCR. Normalized miRNA expression values were evaluated by multivariate forward stepwise Cox regression analysis. Kaplan Meier curves were used to discriminate between two groups of patients with a different probability of tumour progression.ResultsThe mean age (range) of the series was 67 (33-94) years. Overall, 45 patients (43.7%) developed tumour progression and 32 patients (31.2%) died, 20 of these (62.5%) due to their UTUC, after a median follow-up of 36 months. The mean time for tumour progression and cancer-specific survival were 15 and 20 months, respectively. Five year tumour progression free survival and cancer-specific survival were 58% for ≤ pT2, 36% for pT3 and 0% for pT4 and 67.8% for ≤ pT2, 50.6% for pT3 and 0% for pT4, respectively. In the multivariate analysis, expression of miR-31-5p was found to be an independent prognostic factor of tumour progression (HR 1.1; 95% CI 1.039-1.273; p=0.02). Kaplan Meier curve shows that miR-31-5p expression values are able to discriminate between two groups of UTUC patients with a different probability of tumour progression (p=0.007).ConclusionsWe have been able to validate our previous results in an independent multicentre international cohort of UTUC patients, suggesting that miRNA-31-5p could be a useful prognostic marker of UTUC progression. The application of miRNA expression values to clinical practice could refine the currently used clinicopathological-based approach for predicting UTUC patients’ outcome.

Pharmacoepigenomic responses of antipsychotic drugs on pharmacogenes are likely to be modulated by miRNAs.

It is imperative to differentiate the role of host epigenetics from pharmacoepigenetics in resolving therapeutic response. Therefore, the objective was to identify how antipsychotic drugs influence epigenetic response on pharmacogenes. The study design was based on in vitro evaluation of pharmacoepigenetic response of haloperidol, clozapine and olanzapine. Post antipsychotic treatment, the alterations in expression of ABCB1, CYP1A2 and CYP3A4 were monitored, and followed up by promoter methylation and their target miRNA expression studies. Critical observations were followed up in a restrictive clinical setting. Under in vitro conditions increased expression of ABCB1, CYP1A2 and CYP3A4 was observed which seems to be regulated by miR-27a and miR-128a and not by methylation. A similar pattern was observed in clinical setting with ABCB1, which was reflective of good therapeutic response. The study demonstrates that antipsychotic drugs can influence miRNA-mediated epigenetic response in pharmacogenes resulting in modulating therapeutic response.

Comparison of microRNA expression profiles of Kashin-Beck disease, osteoarthritis and rheumatoid arthritis

Kashin-Beck disease (KBD) is a chronic osteochondropathy with unclear pathogeny. In this study, we compared the microRNA expression profiles of 16 KBD patients, 16 osteoarthritis (OA) patients and 16 rheumatoid arthritis (RA) patients and 16 healthy controls in their blood specimens. miRNAs expression profiling was performed using Exiqon miRCURY LNATM miRNAs Array. miRNAs target genes were predicted using miRror suite. Another independent mRNA expression profile dataset of 20 KBD patients and 15 healthy controls were integrated with the miRNA expression profiles of KBD. We identified 140 differently expressed miRNAs in KBD vs. Controls. GO enrichment analysis found that hypoxia, Wnt receptor signaling pathway and vitamin B6 biosynthesis related GO terms were significantly overrepresented in the target genes of differently expressed miRNAs in KBD vs. Control. 18 differently expressed common miRNAs were identified in KBD vs. Control, KBD vs. OA and KBD vs. RA. Integrating the lists of differently expressed miRNA target genes and mRNA differently expressed genes detected 6 common genes for KBD. Our results demonstrated the altered miRNAs expression profiles of KBD comparing to healthy controls, OA and RA, which provide novel clues for clarifying the mechanism of KBD.

MiRNAs in the vitreous humor of patients affected by idiopathic epiretinal membrane and macular hole

PurposeThe aim of the present study was to assess the expression of miRNAs in the Vitreous Humor (VH) of patients with Macular Hole (MH) and Epiretinal Membrane (ERM) compared to a control group.MethodsIn this prospective, comparative study, 2-ml of VH was extracted from the core of the vitreous chamber in consecutive patients who underwent standard vitrectomy for ERM and MH. RNA was extracted and TaqMan® Low Density Arrays (TLDAs) were used to profile the transcriptome of 754 miRNAs. Results were validated by single TaqMan® assays. Finally, we created a biological network of differentially expressed miRNA targets and their nearest neighbors.ResultsOverall 10 eyes with MH, 16 eyes with idiopathic ERM and 6 controls were enrolled in the study. Profiling data identified 5 miRNAs differentially expressed in patients affected by MH and ERM with respect to controls. Four were downregulated (miR-19b, miR-24, miR-155, miR-451) and 1 was downregulated (miR-29a); TaqMan® assays of the VH of patients affected by MH and ERM, with respect to controls, showed that the most differentially expressed were miR-19b (FC -9.13, p:<0.00004), mir-24 (FC -7.52, p:<0.004) and miR-142-3p (FC -5.32, p:<0.011). Our network data showed that deregulation of differentially expressed miRNAs induces an alteration of several pathways associated with genes involved in both MH and ERM.ConclusionThe present study suggests that disregulation of miR-19b, miR-24 and miR-142-3p, might be related to the alterations that characterize patients affected by MH and ERM.

MicroRNAs in the Lymphatic Endothelium: Master Regulators of Lineage Plasticity and Inflammation.

microRNAs (miRNAs) are highly conserved, small non-coding RNAs that regulate gene expression at the posttranscriptional level. They have crucial roles in organismal development, homeostasis, and cellular responses to pathological stress. The lymphatic system is a large vascular network that actively regulates the immune response through antigen trafficking, cytokine secretion, and inducing peripheral tolerance. Here, we review the role of miRNAs in the lymphatic endothelium with a particular focus on their role in lymphatic endothelial cell (LEC) plasticity, inflammation, and regulatory function. We highlight the lineage plasticity of LECs during inflammation and the importance of understanding the regulatory role of miRNAs in these processes. We propose that targeting miRNA expression in lymphatic endothelium can be a novel strategy in treating human pathologies associated with lymphatic dysfunction.

Antitumor activity of miR-34a in peritoneal mesothelioma relies on c-MET and AXL inhibition: persistent activation of ERK and AKT signaling as a possible cytoprotective mechanism

BackgroundThe value of microRNAs (miRNAs) as novel targets for cancer therapy is now widely recognized. However, no information is currently available on the expression/functional role of miRNAs in diffuse malignant peritoneal mesothelioma (DMPM), a rapidly lethal disease, poorly responsive to conventional treatments, for which the development of new therapeutic strategies is urgently needed. Here, we evaluated the expression and biological effects of miR-34a—one of the most widely deregulated miRNAs in cancer and for which a lipid-formulated mimic is already clinically available—in a large cohort of DMPM clinical samples and a unique collection of in house-developed preclinical models, with the aim to assess the potential of a miR-34a-based approach for disease treatment.MethodsmiR-34a expression was determined by qRT-PCR in 45 DMPM and 7 normal peritoneum specimens as well as in 5 DMPM cell lines. Following transfection with miR-34a mimic, the effects on DMPM cell phenotype, in terms of proliferative potential, apoptotic rate, invasion ability, and cell cycle distribution, were assessed. In addition, three subcutaneous and orthotopic DMPM xenograft models were used to examine the effect of miR-34a on tumorigenicity. The expression of miRNA targets and the activation status of relevant pathways were investigated by western blot.ResultsmiR-34a was found to be down-regulated in DMPM clinical specimens and cell lines compared to normal peritoneal samples. miR-34a reconstitution in DMPM cells significantly inhibited proliferation and tumorigenicity, induced an apoptotic response, and declined invasion ability, mainly through the down-regulation of c-MET and AXL and the interference with the activation of downstream signaling. Interestingly, a persistent activation of ERK1/2 and AKT in miR-34a-reconstituted cells was found to counteract the antiproliferative and proapoptotic effects of miRNA, yet not affecting its anti-invasive activity.ConclusionsOur preclinical data showing impressive inhibitory effects induced by miR-34a on DMPM cell proliferation, invasion, and growth in immunodeficient mice strongly suggest the potential clinical utility of a miR-34a-replacement therapy for the treatment of such a still incurable disease. On the other hand, we provide the first evidence of a potential cytoprotective/resistance mechanism that may arise towards miRNA-based therapies through the persistent activation of RTK downstream signaling.

Integration of MicroRNA, mRNA, and Protein Expression Data for the Identification of Cancer-Related MicroRNAs.

MicroRNAs (miRNAs) are responsible for the regulation of target genes involved in various biological processes, and may play oncogenic or tumor suppressive roles. Many studies have investigated the relationships between miRNAs and their target genes, using mRNA and miRNA expression data. However, mRNA expression levels do not necessarily represent the exact gene expression profiles, since protein translation may be regulated in several different ways. Despite this, large-scale protein expression data have been integrated rarely when predicting gene-miRNA relationships. This study explores two approaches for the investigation of gene-miRNA relationships by integrating mRNA expression and protein expression data. First, miRNAs were ranked according to their effects on cancer development. We calculated influence scores for each miRNA, based on the number of significant mRNA-miRNA and protein-miRNA correlations. Furthermore, we constructed modules containing mRNAs, proteins, and miRNAs, in which these three molecular types are highly correlated. The regulatory interactions between miRNA and genes in these modules have been validated based on the direct regulations, indirect regulations, and co-regulations through transcription factors. We applied our approaches to glioblastomas (GBMs), ranked miRNAs depending on their effects on GBM, and obtained 52 GBM-related modules. Compared with the miRNA rankings and modules constructed using only mRNA expression data, the rankings and modules constructed using mRNA and protein expression data were shown to have better performance. Additionally, we experimentally verified that miR-504, highly ranked and included in the identified modules, plays a suppressive role in GBM development. We demonstrated that the integration of both expression profiles allows a more precise analysis of gene-miRNA interactions and the identification of a higher number of cancer-related miRNAs and regulatory mechanisms.

MicroRNA-499-5p regulates porcine myofiber specification by controlling Sox6 expression

MicroRNAs (miRNAs) are a class of small non-coding RNAs that negatively regulate gene expression of target messenger RNAs (mRNAs) and miRNAs have been proven to play vital roles in skeletal muscle development. The miRNA-499-5p has been reported to be negatively related with the expression of Sox6, a critical transcription factor for the maintenance of fast-twitch skeletal muscle. In this study, we amplified a length of 2012-bp mRNA that contains a 1512-bp porcine Sox6 (pSox6) 3'UTR from skeletal muscle of a Duroc×Landrace×Yorkshire pig. By luciferase reporter assay we verified that pSox6 is a target of miR-499-5p. In extensor digitorum longus and Soleus muscles of pigs, the expression levels of miR-499-5p and pSox6 mRNA were also inversely correlated. Besides, overexpression of miR-499-5p in porcine satellite cells promoted the expression of MyHC I and MyHC IIa mRNA, along with a reduction of pSox6 mRNA. Taken together, these results indicate that miR-499-5p may facilitate the oxidative myofibers formation by downregulating pSox6 expression.

Transcriptome analysis reveals dynamic changes in coxsackievirus A16 infected HEK 293T cells

BackgroundCoxsackievirus A16 (CVA16) and enterovirus 71 (EV71) are two of the major causes of hand, foot and mouth disease (HFMD) world-wide. Although many studies have focused on infection and pathogenic mechanisms, the transcriptome profile of the host cell upon CVA16 infection is still largely unknown.ResultsIn this study, we compared the mRNA and miRNA expression profiles of human embryonic kidney 293T cells infected and non-infected with CVA16. We highlighted that the transcription of SCARB2, a cellular receptor for both CVA16 and EV71, was up-regulated by nearly 10-fold in infected cells compared to non-infected cells. The up-regulation of SCARB2 transcription induced by CVA16 may increase the possibility of subsequent infection of CVA16/EV71, resulting in the co-infection with two viruses in a single cell. This explanation would partly account for the co-circulation and genetic recombination of a great number of EV71 and CVA16 viruses. Based on correlation analysis of miRNAs and genes, we speculated that the high expression of SCARB2 is modulated by down-regulation of miRNA has-miR-3605-5p. At the same time, we found that differentially expressed miRNA target genes were mainly reflected in the extracellular membrane (ECM)-receptor interaction and circadian rhythm pathways, which may be related to clinical symptoms of patients infected with CVA16, such as aphthous ulcers, cough, myocarditis, somnolence and potentially meningoencephalitis. The miRNAs hsa-miR-149-3p and hsa-miR-5001-5p may result in up-regulation of genes in these morbigenous pathways related to CVA16 and further cause clinical symptoms.ConclusionsThe present study elucidated the changes in 293T cells upon CVA16 infection at transcriptome level, containing highly up-regulated SCARB2 and genes in ECM-receptor interaction and circadian rhythm pathways, and key miRNAs in gene expression regulation. These results provided novel insight into the pathogenesis of HFMD induced by CVA16 infection.