miRNA Array Research Articles

Identification of atrial fibrillation-associated microRNAs in left and right atria of rheumatic mitral valve disease patients.

MicroRNA (miRNA) is associated with the development and pathology of atrial fibrillation (AF). In this study, we performed miRNA profiling of left and right atrium samples from individuals with AF-associated rheumatic mitral valve disease (RMVD) to identify miRNAs that are differentially expressed between RMVD patients with AF and RMVD with sinus rhythm (SR) as controls, as well as between left and right atrium samples from RMVD with AF patients. We performed hematoxylin and eosin staining as well as scanning and transmission electron microscopy to examine in detail any morphological and physiological changes in cardiomyocytes from RMVD patients with AF or SR. Raman spectroscopy was performed to identify molecular and structural information of left and right atrium samples from RMVD with AF and SR. We also performed miRNA array profiling to separately profile miRNA expression patterns of right and left atrium samples from three independent RMVD patients with AF and in a mixed pool of 10 RMVD patients with SR. Morphological and physiological analysis showed distinct shapes and structures of cardiomyocytes from the left and right atria of RMVD patients with AF or SR. The intensity of Raman spectroscopy of atrial tissues from RMVD patients with AF and with SR was different. miRNA profiling showed differential miRNA expression between RMVD patients with AF or SR, and between the left and right atria of RMVD patients with AF. Importantly, miRNAs showed consistent expression changes among all three patients, suggesting that these miRNAs have potential as markers for AF pathology. Our results revealed potential biomarker miRNAs for atrial fibrillation pathology in patients with RMVD. Meanwhile, our data suggested that miR-10b and miR-138-2, which were both significantly increased in the left atrium, are responsible for morphological and physiological phenotype differences between the left and right atria.

Decreased levels of miR-28-5p and miR-361-3p and increased levels of insulin-like growth factor 1 mRNA in mononuclear cells from patients with hereditary hemorrhagic telangiectasia 1.

Hereditary hemorrhagic telangiectasia (HHT) is a rare vascular disorder inherited in an autosomal dominant manner. Patients with HHT can develop vascular dysplasias called telangiectasias and arteriovenous malformations (AVMs). Our objective was to profile and characterize micro-RNAs (miRNAs), short noncoding RNAs that regulate gene expression posttranscriptionally, in HHT patient-derived peripheral blood mononuclear cells (PBMCs). PBMCs, comprised mostly of lymphocytes and monocytes, have been reported to be dysfunctional in HHT. A total of 40 clinically confirmed HHT patients and 22 controls were enrolled in this study. PBMCs were isolated from 16 mL of peripheral blood and purified for total RNA. MiRNA expression profiling was conducted with a human miRNA array analysis. Select dysregulated miRNAs and miRNA targets were validated with reverse transcription-quantitative polymerase chain reaction. Of the 377 miRNAs screened, 41 dysregulated miRNAs were identified. Both miR-28-5p and miR-361-3p, known to target insulin-like growth factor 1 (IGF1), a potent angiogenic growth factor, were found to be significantly downregulated in HHT patients. Consequently, IGF1 mRNA levels were found to be significantly elevated. Our research successfully identified miRNA dysregulation and elevated IGF1 mRNA levels in PBMCs from HHT patients. This novel discovery represents a potential pathogenic mechanism that could be targeted to alleviate clinical manifestations of HHT.

Persistence of Epigenomic Effects After Recovery From Repeated Treatment With Two Nephrocarcinogens.

The discovery of the epigenetic regulation of transcription has provided a new source of mechanistic understanding to long lasting effects of chemicals. However, this information is still seldom exploited in a toxicological context and studies of chemical effect after washout remain rare. Here we studied the effects of two nephrocarcinogens on the human proximal tubule cell line RPTEC/TERT1 using high-content mRNA microarrays coupled with miRNA, histone acetylation (HA) and DNA methylation (DM) arrays and metabolomics during a 5-day repeat-dose exposure and 3 days after washout. The mycotoxin ochratoxin A (OTA) was chosen as a model compound for its known impact on HA and DM. The foremost effect observed was the modulation of thousands of mRNAs and histones by OTA during and after exposure. In comparison, the oxidant potassium bromate (KBrO3) had a milder impact on gene expression and epigenetics. However, there was no strong correlation between epigenetic modifications and mRNA changes with OTA while with KBrO3 the gene expression data correlated better with HA for both up- and down-regulated genes. Even when focusing on the genes with persistent epigenetic modifications after washout, only half were coupled to matching changes in gene expression induced by OTA, suggesting that while OTA causes a major effect on the two epigenetic mechanisms studied, these alone cannot explain its impact on gene expression. Mechanistic analysis confirmed the known activation of Nrf2 and p53 by KBrO3, while OTA inhibited most of the same genes, and genes involved in the unfolded protein response. A few miRNAs could be linked to these effects of OTA, albeit without clear contribution of epigenetics to the modulation of the pathways at large. Metabolomics revealed disturbances in amino acid balance, energy catabolism, nucleotide metabolism and polyamine metabolism with both chemicals. In conclusion, the large impact of OTA on transcription was confirmed at the mRNA level but also with two high-content epigenomic methodologies. Transcriptomic data confirmed the previously reported activation (by KBrO3) and inhibition (by OTA) of protective pathways. However, the integration of omic datasets suggested that HA and DM were not driving forces in the gene expression changes induced by either chemical.

Upregulation of microRNA 344a-3p is involved in curcumin induced apoptosis in RT4 schwannoma cells

BackgroundSchwannoma arising from peripheral nervous sheaths is a benign tumor.MethodsTo evaluate cell cytotoxicity, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction and terminal deoxynucleotidyltransferase UTP nick-end labeling (TUNEL) assays were used. A microRNA (miRNA) array was used to identify the miRNAs involved in curcumin-induced apoptosis. To examine miRNA expression, quantitative RT-PCR was used.ResultsIn this study, curcumin exerted cellular cytotoxicity against RT4 schwannoma cells, with an increase in TUNEL-positive cells. Curcumin also activated the expression of apoptotic proteins, such as polyADP ribose polymerase, caspase-3, and caspase-9. The miRNA array revealed that seven miRNAs (miRNA 350, miRNA 17-2-3p, let 7e-3p, miRNA1224, miRNA 466b-1-3p, miRNA 18a-5p, and miRNA 322-5p) were downregulated following treatment with both 10 and 20 μM curcumin in RT4 cells, while four miRNAs (miRNA122-5p, miRNA 3473, miRNA182, and miRNA344a-3p) were upregulated. Interestingly, transfection with a miRNA 344a-3p mimic downregulated the mRNA expression of Bcl2 and upregulated that of Bax, Curcumin treatment in RT 4 cells also reduced the mRNA expression of Bcl2 and enhanced expression of Bax, Overexpression of miRNA344a-3p mimic combined with curcumin treatment activated the expression of apoptotic proteins, including procaspase-9 and cleaved caspase-3 while inhibition of miRNA 344a-3p using miR344a-3p inhibitor repressed cleaved caspase-3 and -9 in curcumin treated RT-4 cells compared to control.ConclusionsOur findings demonstrate that curcumin induces apoptosis in schwannoma cells via miRNA 344a-3p. Thus, curcumin may serve as a potent therapeutic agent for the treatment of schwannoma.

S-Adenosylmethionine regulates apoptosis and autophagy in MCF-7 breast cancer cells through the modulation of specific microRNAs

BackgroundTo get insight into the molecular mechanisms underlying the anti-tumor activity of S-adenosyl-l-methionine (AdoMet), we analyzed AdoMet-induced modulation of microRNAs (miRNAs) expression profile in MCF-7 breast cell line and its correlation with cancer-related biological pathways.MethodsMiRNA expression profiling was performed using a TaqMan MiRNA Array, following 500 µM AdoMet-treatment. The results were confirmed by Quantitative real-time PCR analysis. MCF-7 were transfected with miR-34a, miR-34c and miR-486-5p, mimics and inhibitors in presence or not of 500 µM AdoMet for 72 h. Apoptosis and autophagy were analyzed by flow cytometry and the modulation of the main antiproliferative signaling pathways were evaluated by Western blotting. The potential mRNA targets for each miRNA were identified by the TargetScan miRNA target prediction software.ResultsTwenty-eight microRNAs resulted differentially expressed in AdoMet-treated MCF-7 cells compared to control cells. Among them, miRNA-34a and miRNA-34c were up-regulated while miRNA-486-5p was down-regulated. Moreover, we confirmed the ability of AdoMet to regulate these miRNAs in MDA-MB 231 breast cancer cell line. We demonstrate that, in MCF7 cells, the combination of either miR-34a or miR-34c mimic with AdoMet greatly potentiated the pro-apoptotic effect of AdoMet, by a caspase-dependent mechanism and activates p53 acetylation by inhibiting SIRT1 and HDAC1 expression. We also showed that miR-486-5p inhibitor induces autophagy and enhances AdoMet-induced autophagic process by increasing PTEN expression and by inhibiting AKT signaling.ConclusionsOur findings provide the first evidence that AdoMet can regulate miRNA expression in MCF-7 increasing our knowledge on the molecular basis of the antitumor effect of the sulfonium compound and suggest the use of AdoMet as an attractive miRNA-mediated chemopreventive and therapeutic strategy in breast cancer.

Circulating Plasma Extracellular Vesicles from Septic Mice Induce Inflammation via MicroRNA- and TLR7-Dependent Mechanisms.

We have previously reported that a group of host cellular microRNAs (miRNAs; miR-34a-5p, miR-122-5p, miR-145-5p, miR-146a-5p, miR-210-3p) are released into the blood during sepsis, some of which are capable of inducing complement activation, cytokine production, and leukocyte migration. Extracellular vesicles (EVs) have been proposed as vehicles for extracellular miRNA-mediated intercellular communication. However, the biological function of plasma EVs and the associated miRNAs in sepsis are largely unknown. In this study, we tested the hypothesis that plasma EVs in sepsis are proinflammatory and EV-associated miRNAs are responsible for EV-induced cytokine production. Compared with those of sham mice, the plasma EVs from septic mice were slightly smaller (157 ± 2 versus 191 ± 6 nm, p < 0.0001), but more abundant [(1.6 ± 0.14) × 1010 versus (0.93 ± 0.14) × 1010/ml plasma, p < 0.003]. miRNA array revealed that among 65 miRNAs, 8 miRNAs exhibited >1.5-fold increase in septic EVs compared with sham EVs, including miR-126-3p, miR-122-5p, miR-146a-5p, miR-145-5p, miR-26a-5p, miR-150-5p, miR-222-3p, and miR-181a-5p. Septic but not sham EVs were proinflammatory, promoting IL-6, TNF-α, IL-1β, and MIP-2 production. The effects of EVs were resistant to polymyxin B (an endotoxin inhibitor) but significantly inhibited by anti-miR inhibitors against miR-34a, miR-122, and miR-146a. Moreover, the septic EV-induced cytokine production was attenuated in TLR7-/- or MyD88-/- cells but remained the same in TLR3-/- or Trif-/- cells. In vivo, mice i.p. injected with septic EVs had marked peritoneal neutrophil migration, which was significantly attenuated in MyD88-/- mice. Taken together, these data demonstrate that plasma EVs of septic animals play an important role in inflammation, and EV-associated miRNAs likely mediate the cytokine production via TLR7-MyD88 signaling.

MicroRNA cluster miR199a/214 are differentially expressed in female and male rats following nicotine self-administration

Previous research has established sex differences associated with nicotine intake, however a significant gap in knowledge remains regarding the molecular mechanisms that govern these differences at the transcriptional level. One critical regulator of transcription are microRNAs (miRNAs). miRNAs are a family of non-coding RNAs that regulate an array of important biological functions altered in several disease states, including neuroadaptive changes within the brain associated with drug dependence. We examined the prefrontal cortex (PFC) from male and female Sprague-Dawley rats following self-administration (22 days) of nicotine or yoked saline controls using next generation RNA-Sequencing (RNA-Seq) technology and found an array of miRNAs to be significantly and differentially regulated by nicotine self-administration. Of these, we found the expression of miR-199a and 214, which are expressed on the same cluster of chromosome 1, to be upregulated in the female rats exposed to nicotine; upregulation in this group was further validated by real time polymerase chain reaction (RT-PCR). Bioinformatics analysis to assess common targets of miR-199/214 identified Sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide (NAD)- dependent deacetylase that plays a role in apoptosis, neuron survival, and stress resistance. Using western-blot, we confirmed downregulation of SIRT1 and increased cleaved caspase 3 expression in the brains of nicotine-exposed female rats and no change in expression levels in the other groups. Collectively, our findings highlight a miR-199/214 regulatory network that, through SIRT1, may be associated with nicotine seeking in females which may serve as a potential therapeutic target for sex-specific treatment approaches.

MiR-155, a potential serum marker of extramammary Paget\u2019s disease

BackgroundExtramammary Paget’s disease (EMPD), a rare skin malignancy with non-specific manifestations, is often misdiagnosed as eczema of scrotum or tinea cruris. Although the diagnosis of EMPD could be confirmed by biopsy, it can be delayed as patients are reluctant to receive invasive operations. Herein, we investigated the serum miRNA expressions of EMPD patients and compared to that of the eczema of scrotum or tinea cruris patients as well as health volunteers for potential diagnostic markers for EMPD.MethodsAltogether 45 subjects including 16 patients diagnosed with EMPD, 12 patients diagnosed with eczema of scrotum or tinea cruris and 17 healthy volunteers were enrolled in this study. Serum from all of subjects were collected to identify miRNAs (by miRNA array global normalization, RT-PCR validation, and receiver operating characteristic curve analysis) that could be potential diagnostic markers for EMPD.ResultsThe miRNA array analyses revealed that the expressions of 37 miRNAs from the EMPD patients were different (change ≥4-fold) from health volunteers. Among these miRNAs, the expression of miR-155 was significantly increased (p < 0.01) in the EMPD patients as compared with that of the health volunteers and the eczema of scrotum or the tinea cruris patients (no difference between these two control groups). In addition, receiver operating characteristic (ROC) curve analysis showed that diagnostic capacities (defined as the area under curve of ROC) of miR-155 are 0.85 (as compared with health volunteers group) and 0.81 (as compared with the eczema of scrotum or the tinea cruris patients group), respectively.ConclusionThe serum miRNA expression of gene miR-155 in the EMPD patients was differentiated from that of other subjects warranting further validation of miR-155 as a diagnostic marker of EMPD.

MiR-148a suppressed cell invasion and migration via targeting WNT10b and modulating β-catenin signaling in cisplatin-resistant colorectal cancer cells

Cancer stem cells (CSCs) are suggested to be responsible for high recurrence rate and metastasis of colorectal cancer (CRC). Identifying novel targets that can suppress CSCs proliferation and metastasis may provide novel approach to combat against CRC. In the present study, we examined the role of miR-148a in cisplatin-resistant CRC cells with enhanced stem cell marker expression and explored the underlying mechanisms. In this study, we used cisplatin to selectively enrich cisplatin-resistant CRC cells from SW480 cell line, and these selected cisplatin-resistant SW480 cells were with significantly enhanced expression of stem cell markers and increased chemoresistance. MicroRNA (miRNA) array and qRT-PCR assay identified the down-regulation of miR-148a in cisplatin-resistant SW480 cells. Overexpression of miR-148a suppressed expression of stem cell markers, inhibited sphere formation, invasion and migration, induced apoptosis, and reduced chemo-resistance in cisplatin-resistant SW480 cells. Bioinformatics prediction and luciferase reporter assay revealed that WNT10b was a downstream target of miR-148a, and overexpression of miR-148a suppressed WNT10b expression and β-catenin signaling activities. Enforced expression WNT10b attenuated the effects of miR-148a on cisplatin-resistant SW480 cells sphere formation, invasion and migration. Further study showed that overexpression of miR-148a also suppressed in vivo tumor growth, and WNT10b expression and β-catenin signaling activities in tumor tissues were suppressed by miR-148a overexpression. In the clinical samples, miR-184a was found to be down-regulated in CRC tissues, down-regulation of miR-148a predicted poor prognosis in CRC patients. In conclusion, our study for the first time enriched the cisplatin-resistant CRC cells with enhanced stem cell marker expression from sphere-forming and chemo-resistant SW480-derived tumor xenografts in immune-deficient mice, and miR-148a suppressed the expression of stem cell markers, increased chemo-sensitivity, cell invasion and migration at least partly via regulating WNT10b and β-catenin signaling pathway.

MiRNA array analysis of plasma miRNA alterations in rats exposed to a high altitude hypoxic environment.

In the present study, the microRNA (miRNA) expression profiles of rats exposed to high altitude hypoxia and normal conditions were obtained from miRNA array analysis. Bioinformatics analyses, including the use of the Gene Oncology and Kyoto Encyclopedia of Genes and Genomes databases, were used to identify the genes and pathways, which were specifically associated with high altitude hypoxic environment-associated miRNAs. A total of 26 miRNAs were differentially expressed in the two groups, comprising six upregulated and 20 downregulated miRNAs. In the present study, a novel pattern of upregulated miRNAs and their associated pathways were constructed, including proteoglycans in cancer, spliceosome, gluamatergic synapse, glycolysis/gluconeogenesis, Foxo, cGMP-PKG and p53 signaling pathways, which may provide novel targets for diagnosing and understanding the mechanism of high altitude hypoxia-induced disease.

MiR-122-5p as a novel biomarker for alpha-fetoprotein-producing gastric cancer.

AIMTo investigate the clinical utility of alpha-fetoprotein (AFP)-producing gastric cancer (AFPGC)-specific microRNA (miRNA) for monitoring and prognostic prediction of patients.METHODSWe performed a comprehensive miRNA array-based approach to compare miRNA expression levels between AFP-positive and AFP-negative cells in three patients with primary AFPGC. We next examined the expression levels of the selected miRNAs in five AFPGC and ten non-AFPGC tissue samples by quantitative reverse transcription-polymerase chain reaction to validate their utility. We also investigated the expression levels of the selected miRNA not only in tissue but also in plasma samples. Moreover, we investigated the relationship between plasma AFP levels and plasma selected miRNA expression levels, and also investigated the correlation of the selected miRNA expression levels and malignant potential.RESULTSAmong the five miRNAs selected from the miRNA array results, the expression levels of miR-122-5p were significantly higher in the AFPGC patients than in the non-AFPGC patients (P < 0.05). In tissue samples, miR-122-5p expression level tended to be lower in the non-AFPGC tissue than the normal gastric mucosa. Conversely, in the AFPGC tissue, miR-122-5p expression level was significantly higher in the AFPGC tissue than both the normal gastric mucosa and the non-AFPGC tissue samples (P < 0.05). Plasma miR-122-5p expression levels were also significantly higher in the AFPGC patients than the health volunteers and the non-AFPGC patients (P < 0.05) and were strongly correlated with plasma AFP levels (r = 0.7975, P < 0.0001). Moreover, the correlation of miR-122-5p expression in tissue samples with malignant potential was stronger than that of plasma AFP level in the AFPGC patients. In contrast, no correlation was found between miR-122-5p expression levels and liver metastasis in the non-AFPGC patients.CONCLUSIONmiR-122-5p might be a useful biomarker for early detection and disease monitoring in AFPGC.

MiRNA expression profiles in healthy OSAHS and OSAHS with arterial hypertension: potential diagnostic and early warning markers

BackgroundObstructive sleep apnea-hypopnea syndrome (OSAHS) is prone to being complicated with various cardiovascular, cerebrovascular and metabolic conditions. OSAHS, due to its multifactorial nature, entails individualized and comprehensive treatment. So far, no well-established diagnostic criteria for the disease are available. In recent years, miRNA has been shown to be a sensitive biomarker suggestive of the progression and prognosis of many diseases. In this study, we examined some serum miRNAs in healthy OSAHS (OSAHS patients without complication) and OSAHS with arterial hypertension, with an attempt to understand the potential effects on the disease, improve the diagnosis of OSAHS and find OSAHS-related diagnostic markers.MethodsAgainst various diagnostic criteria, participants were divided into three groups: healthy OSAHS, OSAHS with arterial hypertension and healthy controls. Their serum miRNA profiles were assessed by microarray technology, and then differentially expressed miRNAs were verified by quantitative real-time PCR (qRT-PCR). The receiver operating characteristic (ROC) curves of miRNAs were constructed and the areas under the curve (AUC) were calculated. Meanwhile, the miRNAs were subjected to logistic regression analysis. The target genes were bioinformatically assessed, their functions and signaling pathways further determined and eventually an miRNA-gene network was established.ResultsAnalysis with the miRNA array exhibited that, compared with the control group, 12 differentially expressed miRNAs were found in healthy OSAHS, and 33 were found in OSAHS with arterial hypertension. The expression of miR-126-3p, let-7d-5p, miR-7641 and miR-1233-5p, miR-320b, miR-145-5p, miR-107, miR-26a-5p were validated by using qRT-PCR. Bioinformatics analysis predicted that the potential target genes of these miRNAs might be involved in metabolism, and the regulation of endothelial cells and nervous system. Moreover, the ROC analysis showed that the using miR-145-5p and let-7d-5p in combination can identify the healthy OSAHS, presence of miR-126-3p, miR-26a-5p and miR-107 was well indicative of OSAHS with arterial hypertension.ConclusionsA cluster of dysregulation miRNAs have been found to be involved in the development of OSAHS patients. Moreover, these miRNAs might be used to be potential diagnostic and early warning markers.

Regulation of cell proliferation and metastasis by microRNA-593-5p in human gastric cancer.

BackgroundMicroRNA (miRNA) array analysis has reported that the expression of miR-593-5p is associated with lymph node metastasis in gastric cancer (GC); however, the function and mechanism of miR-593-5p in GC have not been described yet. miR-593-5p has also not been elucidated widely in other cancers.MethodsmiR-593-5p expression was detected by quantitative RT-PCR (qRT-PCR) in human GC tissues and cell lines. Cell proliferation was investigated using CCK-8 assays, cell cycle was detected by flow cytometric method, and cell migration and invasion abilities were evaluated by wound-healing and transwell assays. miR-593-5p-influenced gene expression profiles were detected by total gene expression chip method in MGC-803 cells, and miR-593-5p candidate target genes were predicted using bioinformatics methods. The candidate target gene and downstream of miR-593-5p were determined by qRT-PCR, Western blot, and dual-luciferase reporter assays. The effects of miR-593-5p on the growth and metastasis of GC were evaluated by tumor xenograft experiment in vivo.ResultsmiR-593-5p was frequently downregulated in GC patients and GC cell lines. miR-593-5p was significantly correlated with tumor size and distant metastasis in GC patients. miR-593-5p inhibited cell proliferation, migration, and invasion and also arrested cell cycle at the G0/G1 phase in SGC-7901 and MGC-803 cells in vitro. miR-593-5p also suppressed tumor growth and metastasis in vivo. miR-593-5p influenced gene expression profile in MGC-803 cells. MST4 was indirectly targeted by miR-593-5p. miR-593-5p also downregulated FAK, MMP12, and JUN protein expression.ConclusionOur study suggests that miR-593-5p may function as a tumor suppressor in GC through a mechanism that regulates JUN pathway via indirectly targeting the MST4 gene.

Cervical squamous cell carcinoma-secreted exosomal miR-221-3p promotes lymphangiogenesis and lymphatic metastasis by targeting VASH1

Cancer-secreted exosomal miRNAs are emerging mediators of cancer-stromal cross-talk in the tumor environment. Our previous miRNAs array of cervical squamous cell carcinoma (CSCC) clinical specimens identified upregulation of miR-221-3p. Here, we show that miR-221-3p is closely correlated with peritumoral lymphangiogenesis and lymph node (LN) metastasis in CSCC. More importantly, miR-221-3p is characteristically enriched in and transferred by CSCC-secreted exosomes into human lymphatic endothelial cells (HLECs) to promote HLECs migration and tube formation in vitro, and facilitate lymphangiogenesis and LN metastasis in vivo according to both gain-of-function and loss-of-function experiments. Furthermore, we identify vasohibin-1 (VASH1) as a novel direct target of miR-221-3p through bioinformatic target prediction and luciferase reporter assay. Re-expression and knockdown of VASH1 could respectively rescue and simulate the effects induced by exosomal miR-221-3p. Importantly, the miR-221-3p-VASH1 axis activates the ERK/AKT pathway in HLECs independent of VEGF-C. Finally, circulating exosomal miR-221-3p levels also have biological function in promoting HLECs sprouting in vitro and are closely associated with tumor miR-221-3p expression, lymphatic VASH1 expression, lymphangiogenesis, and LN metastasis in CSCC patients. In conclusion, CSCC-secreted exosomal miR-221-3p transfers into HLECs to promote lymphangiogenesis and lymphatic metastasis via downregulation of VASH1 and may represent a novel diagnostic biomarker and therapeutic target for metastatic CSCC patients in early stages.

The regulation of miRNAs by reconstituted high-density lipoproteins in diabetes-impaired angiogenesis

Diabetic vascular complications are associated with impaired ischaemia-driven angiogenesis. We recently found that reconstituted high-density lipoproteins (rHDL) rescue diabetes-impaired angiogenesis. microRNAs (miRNAs) regulate angiogenesis and are transported within HDL to sites of injury/repair. The role of miRNAs in the rescue of diabetes-impaired angiogenesis by rHDL is unknown. Using a miRNA array, we found that rHDL inhibits hsa-miR-181c-5p expression in vitro and using a hsa-miR-181c-5p mimic and antimiR identify a novel anti-angiogenic role for miR-181c-5p. miRNA expression was tracked over time post-hindlimb ischaemic induction in diabetic mice. Early post-ischaemia when angiogenesis is important, rHDL suppressed hindlimb mmu-miR-181c-5p. mmu-miR-181c-5p was not detected in the plasma or within HDL, suggesting rHDL specifically targets mmu-miR-181c-5p at the ischaemic site. Three known angiogenic miRNAs (mmu-miR-223-3p, mmu-miR-27b-3p, mmu-miR-92a-3p) were elevated in the HDL fraction of diabetic rHDL-infused mice early post-ischaemia. This was accompanied by a decrease in plasma levels. Only mmu-miR-223-3p levels were elevated in the hindlimb 3 days post-ischaemia, indicating that rHDL regulates mmu-miR-223-3p in a time-dependent and site-specific manner. The early regulation of miRNAs, particularly miR-181c-5p, may underpin the rescue of diabetes-impaired angiogenesis by rHDL and has implications for the treatment of diabetes-related vascular complications.

Generation of PTEN‑knockout (‑/‑) murine prostate cancer cells using the CRISPR/Cas9 system and comprehensive gene expression profiling

Phosphatase and tensin homolog (PTEN) deficiency is associated with development, progression, and metastasis of various cancers. However, changes in gene expression associated with PTEN deficiency have not been fully characterized. To explore genes with altered expression in PTEN‑deficient cells, the present study generated a PTEN‑knockout cell line (ΔPTEN) from a mouse prostate cancer‑derived cell line using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‑associated protein9 (CRISPR/Cas9) gene editing system. Following transfection of the CRISPR/Cas9 construct, DNA sequencing was performed to identify deletion of the Pten locus and PTEN inactivation was verified by western blotting. The ΔPTEN cell line exhibited enhanced RAC‑alpha serine/threonine‑protein kinase phosphorylation and cyclinD1 expression. In addition, an increase in cell proliferation and colony formation was observed in the ΔPTEN cell line. Gene expression profiling experiments were analyzed with microarray and microRNA (miRNA) arrays. In the microarray analysis, 111genes exhibited ≥10‑fold increased expression compared with the parent strain and mock cell line and 23genes were downregulated. The only miRNA with increased expression of 10‑fold or more was mmu‑miR‑210‑3p. Genes with enhanced expression included genes involved in the development, progression, and metastasis of cancer such as Tet methylcytosine dioxygenase1, twist family BHLH transcription factor2, C‑fos‑induced growth factor and Wingless‑Type MMTV Integration Site Family, Member3, and genes involved in immunosuppression such as Arginase1. The results of the present study suggest that PTEN deficiency mobilizes a variety of genes critical for cancer cell survival and host immune evasion.

Osteogenic differentiation of fibroblast-like synovial cells in rheumatoid arthritis is induced by microRNA-218 through a ROBO/Slit pathway

BackgroundFibroblast-like synovial cells (FLS) have multilineage differentiation potential including osteoblasts. We aimed to investigate the role of microRNAs during the osteogenic differentiation of rheumatoid arthritis (RA)-FLS.MethodsRA-FLS were differentiated in osteogenic medium for 21 days. Osteogenic differentiation was evaluated by alkaline phosphatase (ALP) staining and Alizarin Red staining. MicroRNA (miRNA) array analysis was performed to investigate the differentially expressed miRNAs during osteogenic differentiation. Expression of miR-218-5p (miR-218) during the osteogenic differentiation was determined by quantitative real-time PCR. Transfections with an miR-218 precursor and inhibitor were used to confirm the targets of miR-218 and to analyze the ability of miR-218 to induce osteogenic differentiation. Secreted Dickkopf-1 (DKK1) from FLS transfected with miR-218 precursor/inhibitor or roundabout 1 (ROBO1) knockdown FLS established using ROBO1-small interfering RNA (siRNA) were measured by ELISA.ResultsThe miRNA array revealed that 12 miRNAs were upregulated and 24 miRNAs were downregulated after osteogenic differentiation. We observed that the level of miR-218 rose in the early phase of osteogenic differentiation and then decreased. Pro-inflammatory cytokines modified the expression of miR-218. The induction of miR-218 in RA-FLS decreased ROBO1 expression, and promoted osteogenic differentiation. Both the overexpression of miR-218 and the knockdown of ROBO1 in RA-FLS decreased DKK1 secretion.ConclusionWe identified miR-218 as a crucial inducer of the osteogenic differentiation of RA-FLS. MiR-218 modulates the osteogenic differentiation of RA-FLS through the ROBO1/DKK-1 axis. The induction of the osteogenic differentiation of proliferating RA-FLS through the provision of miR-218 into RA-FLS or by boosting the cellular reservoir of miR-218 might thus become a therapeutic strategy for RA.

Different signatures of miR-16, miR-30b and miR-93 in exosomes from breast cancer and DCIS patients

Loading of microRNAs (miRNAs) into exosomes that are involved in cellular communication is a selective process. The current study investigates whether the enrichment of miRNAs in exosomes reflects the pathogenesis of breast cancer (BC) and ductal carcinoma in situ (DCIS). The levels of miRNAs were quantified in exosomes from plasma of 32 BC patients, 8 DCIS patients and 8 healthy women by TaqMan real-time PCR-based miRNA array cards containing 47 different miRNAs. Then, exosomal miR-16, miR-30b and miR-93 that displayed deregulation in the arrays were selected and analyzed in 111 BC patients, 42 DCIS patients and 39 healthy women by TaqMan real-time PCR. Identification of exosomes was performed by Western blot. The levels of exosomal miR-16 were higher in plasma of BC (p = 0.034) and DCIS (p = 0.047) patients than healthy women, and were associated with estrogen (p = 0.004) and progesterone (p = 0.008) receptor status. Particularly, in estrogen-positive patients miR-16 was significantly enriched in exosomes (p = 0.0001). Lower levels of exosomal miR-30b were associated with recurrence (p = 0.034). Exosomal miR-93 was upregulated in DCIS patients (p = 0.001). Our findings suggest that different signatures of miR-16, miR-30b and miR-93 in exosomes from BC and DCIS patients are associated with a particular biology of breast tumors.

Dynamics of circulating microRNAs as a novel indicator of clinical response to neoadjuvant chemotherapy in breast cancer.

BackgroundCirculating microRNAs (miRNAs) have been indicated as predictive biomarkers in breast cancer. We aimed to explore the association of plasma miRNA dynamics with response to neoadjuvant chemotherapy (NCT) and disclose early markers for predicting sensitivity.MethodsOne hundred and nine patients with operable or locally advanced breast cancer, who participated in a prospective clinical trial and received NCT, were analyzed. Blood samples were collected before random assignment, after two cycles of chemotherapy (C2) and before surgery. Based on their clinical response, the patients were defined as chemo‐sensitive or insensitive. First, baseline and preoperative samples of selected cases from both groups were screened via TaqMan miRNA array for candidate miRNAs. Afterward all the biospecimens were tested for the candidate miRNAs (miR‐222, miR‐20a, miR‐451, miR‐9, miR‐34a, miR‐155, and miR‐145) by quantitative real‐time PCR. Finally, logistic regression model was utilized to determine the predictive value of baseline/C2 expression of these miRNAs.ResultsBased on the results of microRNA profiling, seven miRNAs were selected for further validation. In the HR+/HER2‐ cohort (n = 51) dynamics of three miRNAs, including miR‐222, miR‐20a, and miR‐451, were associated with chemo‐sensitivity. Importantly, across all the three subtypes we consistently identified chemo‐induced decrease in plasma miR‐34a in the insensitive patients. Finally, baseline miR‐222 overexpression (OR = 6.422, P = 0.049), C2 miR‐20a up‐regulation (OR = 0.144, P = 0.021) and C2 miR‐451 down‐regulation (OR = 8.213, P = 0.012) were predictive markers of response to NCT in HR+/HER2‐ breast cancer.ConclusionsWe described that dynamics of circulating miRNAs might help predict clinical response to NCT in breast cancer.

Circulating microRNA-636 is associated with the elimination of hepatitis C virus by ombitasvir/paritaprevir/ritonavir.

Hepatitis C virus (HCV) infection causes sustained inflammation and fibrosis. Several oral direct-acting antivirals (DAAs) including ombitasvir/paritaprevir/ritonavir (OBV/PTV/r) were recently developed for HCV elimination. The combination of DAAs brought a higher sustained viral response (SVR) rate to anti-HCV therapy compared to interferon (IFN)-based regimens. However, 5% of hepatitis C patients who undergo DAA therapy still suffer from a sustained HCV infection. MicroRNA (miRNA) is essentially interfering, endogenous noncoding RNA that has been investigated as a new biomarker for the response to DAA in hepatitis C patients. Here we used a miRNA array and real-time polymerase chain reaction (PCR) to determine the targetable miRNA before and 12 weeks after OBV/PTV/r treatment for refractory hepatitis C. We used replicon cells, in which genotype 1b type HCV is stably transfected in Huh7 cells, to determine whether miRNA can inhibit HCV replication. Among 2,555 miRNAs, three were significantly up-regulated and eight miRNAs were down-regulated in serum 12 weeks after OBV/PTV/r treatment. An unsupervised hierarchical clustering analysis, using Pearson's correlation, showed that the miRNA profiles between before and 12 weeks after OBV/PTV/r treatment were clustered separately. At 12 weeks after OBV/PTV, miR-636 was targeted among the eight down-regulated miRNAs, and the expression level of circulating miR-636 was significantly diminished. The amount of HCV-RNA was significantly diminished 48 hours after miR-636 inhibitor transfection in HCV replicon cells. In conclusion, miR-636 might be one of the essential targetable molecules in HCV patients who undergo DAA therapy and still suffer from a sustained HCV infection.