Evaluation of miR-21 and miR-150 expression in immune thrombocytopenic purpura pathogenesis: a case-control study

Background

Immune thrombocytopenic purpura (ITP) is a common autoimmune disorder diagnosed with thrombocytopenia and bleeding symptoms due to production of autoantibodies (Abs) against platelets. Nowadays, microRNAs are known as novel biomarkers for diagnosis of diseases. The aim of this study was to investigate the expression levels of miR-21 and miR-150 in ITP patients and determine the role of these miRNAs in ITP pathogenesis.

Materials and Methods

Thirty newly diagnosed patients with acute ITP and 30 healthy subjects( age and sex matched) as controls were enrolled in this study. The expression level of miR-21 and miR-150 was investigated using Real-time-PCR. Comparison of demographic characteristics of the cases was done using independent t-test and chi-square test. Comparison of the expression level of miR-21 and miR-150 with the related parameters was done using independent t-test or Mann–Whitney and Kruskal–Wallis test. Spearman rho correlation coefficient was used to investigate the relationship between the expression of miR-21 and miR-150 with demographic characteristics.

Results

The expression of miR-21, 150 in the patients was not different compared with the control group in general. A significant relationship between the expression of miR-21 with hemoglobin, hematocrit and red blood cell hemoglobin concentration was observed.

Discussion

Expression of miR-21 and miR-150 is not associated with pathogenesis of acute ITP and can involve the synergistic role of other miRNAs. Investigation of miR-21 and miR-150 expression along with other miRNAs and cytokines can be helpful in diagnosis and pathogenesis of ITP.

     

Propofol alleviates hypoxia-induced nerve injury in PC-12 cells by up-regulation of microRNA-153

Background

Although the neuroprotective role of propofol has been identified recently, the regulatory mechanism associated with microRNAs (miRNAs/miRs) in neuronal cells remains to be poorly understood. We aimed to explore the regulatory mechanism of propofol in hypoxia-injured rat pheochromocytoma (PC-12) cells.

Methods

PC-12 cells were exposed to hypoxia, and cell viability and apoptosis were assessed by CCK-8 assay and flow cytometry assay/Western blot analysis, respectively. Effects of propofol on hypoxia-injured cells were measured, and the expression of miR-153 was determined by stem-loop RT-PCR. After that, whether propofol affected PC-12 cells under hypoxia via miR-153 was verified, and the downstream protein of miR-153 as well as the involved signaling cascade was finally explored.

Results

Hypoxia-induced decrease of cell viability and increase of apoptosis were attenuated by propofol. Then, we found hypoxia exposure up-regulated miR-153 expression, and the level of miR-153 was further elevated by propofol in hypoxia-injured PC-12 cells. Following experiments showed miR-153 inhibition reversed the effects of propofol on hypoxia-treated PC-12 cells. Afterwards, we found BTG3 expression was negatively regulated by miR-153 expression, and BTG3 overexpression inhibited the mTOR pathway and AMPK activation. Besides, hypoxia inhibited the mTOR pathway and AMPK, and these inhibitory effects could be attenuated by propofol.

Conclusion

Propofol protected hypoxia-injured PC-12 cells through miR-153-mediataed down-regulation of BTG3. BTG3 could inhibit the mTOR pathway and AMPK activation.

     

Expression and function of microRNA-9 in the mid-hindbrain area of embryonic chick

Background

MiR-9 is a small non-coding RNA that is highly conserved between species and primarily expressed in the central nervous system (CNS). It is known to influence proliferation and neuronal differentiation in the brain and spinal cord of different vertebrates. Different studies have pointed to regional and species-specific differences in the response of neural progenitors to miR-9.

Methods

In ovo and ex ovo electroporation was used to overexpress or reduce miR-9 followed by mRNA in situ hybridisation and immunofluorescent stainings to evaluate miR- expression and the effect of changed miR-9 expression.

Results

We have investigated the expression and function of miR-9 during early development of the mid-hindbrain region (MH) in chick. Our analysis reveals a closer relationship of chick miR-9 to mammalian miR-9 than to fish and a dynamic expression pattern in the chick neural tube. Early in development, miR-9 is diffusely expressed in the entire brain, bar the forebrain, and it becomes more restricted to specific areas of the CNS at later stages. MiR-9 overexpression at HH9–10 results in a reduction of FGF8 expression and premature neuronal differentiation in the mid-hindbrain boundary (MHB). Within the midbrain miR-9 does not cause premature neuronal differentiation it rather reduces proliferation in the midbrain.

Conclusion

Our findings indicate that miR-9 has regional specific effects in the developing mid-hindbrain region with a divergence of response of regional progenitors.

     

MiRNA-145 suppresses lung adenocarcinoma cell invasion and migration by targeting N-cadherin

Objective

To investigate the roles of miR-145 in lung adenocarcinoma (LAC) and to clarify the regulation of N-cadherin by miR-145.

Results

In 57 paired clinical LAC tissues, diminished miR-145 was significantly correlated with the lymph node metastasis and was negatively correlated with N-cadherin mRNA level expression. Wound healing and transwell assays revealed a reduced capability of tumor metastasis induced by miR-145 in LAC. miR-145 negatively regulated the invasion of cell lines through targeting N-cadherin by directly binding to its 3′-untranslated region. Silencing of N-cadherin inhibited invasion and migration of LAC cell lines similar to miR-145 overexpression.

Conclusions

MiR-145 could inhibit invasion and migration of lung adenocarcinoma cell lines by directly targeting N-cadherin.

     

Microrna expression signatures predict patient progression and disease outcome in pediatric embryonal central nervous system neoplasms

Background

Although, substantial experimental evidence related to diagnosis and treatment of pediatric central nervous system (CNS) neoplasms have been demonstrated, the understanding of the etiology and pathogenesis of the disease remains scarce. Recent microRNA (miRNA)-based research reveals the involvement of miRNAs in various aspects of CNS development and proposes that they might compose key molecules underlying oncogenesis. The current study evaluated miRNA differential expression detected between pediatric embryonal brain tumors and normal controls to characterize candidate biomarkers related to diagnosis, prognosis and therapy.

Methods

Overall, 19 embryonal brain tumors; 15 Medulloblastomas (MBs) and 4 Atypical Teratoid/Rabdoid Tumors (AT/RTs) were studied. As controls, 13 samples were used; The First-Choice Human Brain Reference RNA and 12 samples from deceased children who underwent autopsy and were not present with any brain malignancy. RNA extraction was carried out using the Trizol method, whilst miRNA extraction was performed with the mirVANA miRNA isolation kit. The experimental approach included miRNA microarrays covering 1211 miRNAs. Quantitative Real-Time Polymerase Chain Reaction was performed to validate the expression profiles of miR-34a and miR-601 in all 32 samples initially screened with miRNA microarrays and in an additional independent cohort of 30 patients (21MBs and 9 AT/RTs). Moreover, meta-analyses was performed in total 27 embryonal tumor samples; 19 MBs, 8 ATRTs and 121 control samples. Twelve germinomas were also used as an independent validation cohort. All deregulated miRNAs were correlated to patients’ clinical characteristics and pathological measures.

Results

In several cases, there was a positive correlation between individual miRNA expression levels and laboratory or clinical characteristics. Based on that, miR-601 could serve as a putative tumor suppressor gene, whilst miR-34a as an oncogene. In general, miR-34a demonstrated oncogenic roles in all pediatric embryonal CNS neoplasms studied.

Conclusions

Deeper understanding of the aberrant miRNA expression in pediatric embryonal brain tumors might aid in the development of tumor-specific miRNA signatures, which could potentially afford promising biomarkers related to diagnosis, prognosis and patient targeted therapy.

     

Integrated analysis of microRNA-target interactions with clinical outcomes for cancers

Background

Clinical statement alone is not enough to predict the progression of disease. Instead, the gene expression profiles have been widely used to forecast clinical outcomes. Many genes related to survival have been identified, and recently miRNA expression signatures predicting patient survival have been also investigated for several cancers. However, miRNAs and their target genes associated with clinical outcomes have remained largely unexplored.

Methods

Here, we demonstrate a survival analysis based on the regulatory relationships of miRNAs and their target genes. The patient survivals for the two major cancers, ovarian cancer and glioblastoma multiforme (GBM), are investigated through the integrated analysis of miRNA-mRNA interaction pairs.

Results

We found that there is a larger survival difference between two patient groups with an inversely correlated expression profile of miRNA and mRNA. It supports the idea that signatures of miRNAs and their targets related to cancer progression can be detected via this approach.

Conclusions

This integrated analysis can help to discover coordinated expression signatures of miRNAs and their target mRNAs that can be employed for therapeutics in human cancers.

     

Down-regulation of microRNA-126-5p contributes to overexpression of VEGFA in lipopolysaccharide-induced acute lung injury

Objectives

To investigate the role of microRNA-126-5p (miR-126-5p) in acute lung injury induced by bronchial instillation of lipopolysaccharide (LPS), and to explore the potential target(s) of miR-126-5p in acute lung injury.

Results

In the mice with LPS-induced acute lung injury, the level of miR-126-5p in the pulmonary tissues was decreased by 41 % whilst pulmonary vascular endothelial growth factor-A (VEGFA) doubled in its mRNA content and increased threefold in its protein level. Similar results were observed in the alveolar type II (ATII) cells treated with LPS. By using luciferase reporter assay, we found that miR-126-5p inhibited VEGFA expression by targeting its 3′-untranslated region. In addition, overexpression of miR-126-5p attenuated LPS-induced reduction of epithelial sodium channel and aquaporin 1 in ATII cells

Conclusions

MiR-126-5p was down-regulated in LPS-induced acute lung injury in mice. Thus overexpression of miR-126-5p may alleviate acute lung injury by down-regulating VEGFA.

     

miR-124 promotes proliferation and differentiation of neuronal stem cells through inactivating Notch pathway

Background

Neural stem cells (NSCs) are able to differentiate into neurons and astroglia. miRNAs have been demonstrated to be involved in NSC self-renewal, proliferation and differentiation. However, the exact role of miR-124 in the development of NSCs and its underlying mechanism remain to be explored.

Methods

Primary NSCs were isolated from embryos of Wistar rats. Immunocytochemistry was used to stain purified NSCs. miR-124, Delta-like 4 (DLL4), ki-67, Nestin, β-tubulin III, glial fibrillary acidic protein (GFAP), HES1, HEY2, and cyclin D1 (CCND1) expressions were detected by qRT-PCR and western blot. The interaction between miR-124 and DLL4 was confirmed by luciferase reporter assay. Cell proliferation was assessed by MTT assay.

Results

NSCs could self-proliferate and differentiate into neurons and astrocyte. miR-124 was up-regulated and DLL4 was down-regulated during NSC differentiation. DLL4 was identified as a target of miR-124 in NSCs. Ectopic expression of miR-124 or knockdown of DLL4 promoted the proliferation and the formation of NSCs to neurospheres. Moreover, miR-124 overexpression or DLL4 down-regulation improved β-tubulin III expression but decreased GFAP expression in NSCs. Furthermore, enforced expression of DLL4 partially reversed the effects of miR-124 on NSCs proliferation and differentiation. Elevated expression of miR-124 suppressed the expressions of HES1, HEY2, and CCND1 in NSCs, while these effects were attenuated following the enhancement of DLL4 expression.

Conclusion

miR-124 promoted proliferation and differentiation of NSCs through inactivating Notch pathway.

     

Profiling microRNAs in individuals at risk of progression to rheumatoid arthritis

Background

Individuals at risk of rheumatoid arthritis (RA) demonstrate systemic autoimmunity in the form of anti-citrullinated peptide antibodies (ACPA). MicroRNAs (miRNAs) are implicated in established RA. This study aimed to (1) compare miRNA expression between healthy individuals and those at risk of and those that develop RA, (2) evaluate the change in expression of miRNA from “at-risk” to early RA and (3) explore whether these miRNAs could inform a signature predictive of progression from “at-risk” to RA.

Methods

We performed global profiling of 754 miRNAs per patient on a matched serum sample cohort of 12 anti-cyclic citrullinated peptide (CCP)?+?“at-risk” individuals that progressed to RA. Each individual had a serum sample from baseline and at time of detection of synovitis, forming the matched element. Healthy controls were also studied. miRNAs with a fold difference/fold change of four in expression level met our primary criterion for selection as candidate miRNAs. Validation of the miRNAs of interest was conducted using custom miRNA array cards on matched samples (baseline and follow up) in 24 CCP+ individuals; 12 RA progressors and 12 RA non-progressors.

Results

We report on the first study to use matched serum samples and a comprehensive miRNA array approach to identify in particular, three miRNAs (miR-22, miR-486-3p, and miR-382) associated with progression from systemic autoimmunity to RA inflammation. MiR-22 demonstrated significant fold difference between progressors and non-progressors indicating a potential biomarker role for at-risk individuals.

Conclusions

This first study using a cohort with matched serum samples provides important mechanistic insights in the transition from systemic autoimmunity to inflammatory disease for future investigation, and with further evaluation, might also serve as a predictive biomarker.

     

Targeting of HER3 with Functional Cooperative miRNAs Enhances Therapeutic Activity in HER2-Overexpressing Breast Cancer Cells

Background

The HER3 receptor functions as a major cause of drug resistance in cancer treatment. It is believed that therapeutic targeting of HER3 is required to improve patient outcomes. It is not clear whether a novel strategy with two functional cooperative miRNAs would effectively inhibit erbB3 expression and potentiate the anti-proliferative/anti-survival effects of a HER2-targeted therapy (trastuzumab) and chemotherapy (paclitaxel) on HER2-overexpressing breast cancer cells.

Results

Combination of miR-125a and miR-205, as compared to either miRNA alone, potently inhibited expression of HER3 in HER2-overexpressing breast cancer BT474 cells. Co-expression of the two miRNAs not only reduced the levels of phosphorylated erbB3 (P-erbB3), Akt (P-Akt), and Src (P-Src), it also inhibited cell proliferation and increased cells at G1 phase. A multi-miRNA lentiviral vector - the cluster of miR-125a and miR-205 - was constructed to simultaneously express the two miRNAs in HER2-overexpressing breast cancer cells. Concurrent expression of miR-125a and miR-205 via the miRNA cluster transfection significantly enhanced trastuzumab-mediated growth inhibition and cell cycle G1 arrest in BT474 cells and markedly increased paclitaxel-induced apoptosis in another HER2-overexpressing breast cancer cell line HCC1954.

Conclusions

Here, we showed that functional cooperative miRNAs effectively suppressed erbB3 expression. This novel approach targeting of HER3 was able to enhance the therapeutic efficacy of trastuzumab and paclitaxel against HER2-overexpressing breast cancer.

     

Deregulation of neuronal miRNAs induced by amyloid-β or TAU pathology

Background

Despite diverging levels of amyloid-β (Aβ) and TAU pathology, different mouse models, as well as sporadic AD patients show predictable patterns of episodic memory loss. MicroRNA (miRNA) deregulation is well established in AD brain but it is unclear whether Aβ or TAU pathology drives those alterations and whether miRNA changes contribute to cognitive decline.

Methods

miRNAseq was performed on cognitively intact (4 months) and impaired (10 months) male APPtg (APP^swe/PS1^L166P) and TAUtg (THY-Tau22) mice and their wild-type littermates (APPwt and TAUwt). We analyzed the hippocampi of 12 mice per experimental group (n =?96 in total), and employed a 2-way linear model to extract differentially expressed miRNAs. Results were confirmed by qPCR in a separate cohort of 4 M and 10 M APPtg and APPwt mice (n =?7–9 per group) and in human sporadic AD and non-demented control brain. Fluorescent in situ hybridization identified their cellular expression. Functional annotation of predicted targets was performed using GO enrichment. Behavior of wild-type mice was assessed after intracerebroventricular infusion of miRNA mimics.

Results

Six miRNAs (miR-10a-5p, miR-142a-5p, miR-146a-5p, miR-155-5p, miR-211-5p, miR-455-5p) are commonly upregulated between APPtg and TAUtg mice, and four of these (miR-142a-5p, miR-146a-5p, miR-155-5p and miR-455-5p) are altered in AD patients. All 6 miRNAs are strongly enriched in neurons. Upregulating these miRNAs in wild-type mice is however not causing AD-related cognitive disturbances.

Conclusion

Diverging AD-related neuropathologies induce common disturbances in the expression of neuronal miRNAs. 4 of these miRNAs are also upregulated in AD patients. Therefore these 4 miRNAs (miR-142a-5p, miR-146a-5p, miR-155-5p and miR-455-5p) appear part of a core pathological process in AD patients and APPtg and TAUtg mice. They are however not causing cognitive disturbances in wild-type mice. As some of these miRNA target AD relevant proteins, they may be, in contrast, part of a protective response in AD.

     

miR-149 promotes human osteocarcinoma progression via targeting bone morphogenetic protein 9 (BMP9)

Objectives

To investigate the roles of miR-149 in the progression of human osteosarcoma (OS).

Results

miR-149 level was upregulated in tissues from OS patients more than in normal subjects. Cell proliferation and apoptosis assays revealed that miR-149 increased cell proliferation and inhibited cell apoptosis in OS cell line (MG63). An increase of Bcl-2 gene expression and a decrease of cleaved-caspase-3, and cleaved-PARP expression were observed in MG63 cells with transfection of miR-149. Additionally, bone morphogenetic protein 9 (BMP9) was identified as a target of miR-149 in MG63 cells, and BMP9 expression was negatively correlated with miR149 level in OS clinical samples. Co-overexpression of BMP9 with miR-149 in MG63 cells prohibited miR-149-mediated promotive effects on OS progression. Importantly, overexpression of miR-149 conferred chemoresistance in MG63 cells.

Conclusions

miR-149 promotes OS progression via targeting BMP9.

     

The microRNA miR-29a is associated with human immunodeficiency virus latency

Background

Latent reservoirs of HIV-1 provide a major challenge to its cure. There are increasing reports of interplay between HIV-1 replication and host miRNAs. Several host miRNAs, which potentially target the nef-3′LTR region of HIV-1 RNA, including miR-29a, are proposed to promote latency.

Findings

We used two established cellular models of HIV-1 latency – the U1 monocytic and J1.1 CD4+ T cell lines to show an inverse relationship between HIV-1 replication and miR-29a levels, which was mediated by the HIV-1 Nef protein. Using a miR-29a responsive luciferase reporter plasmid, an expression plasmid and an anti-miR29a LNA, we further demonstrate increased miR-29a levels during latency and reduced levels following active HIV replication. Finally, we show that miR-29a levels in the PBMCs and plasma of HIV infected persons also correlate inversely with latency and active viral replication.

Conclusions

The levels of miR-29a correlate inversely with active HIV-1 replication in cell culture models and in HIV infected persons. This links miR-29a to viral latency and suggests another approach to activate and destroy latent HIV-1 reservoirs.

     

MicroRNA-190b confers radio-sensitivity through negative regulation of Bcl-2 in gastric cancer cells

Objectives

To determine the role of miR-190b in radio-sensitivity of gastric cancer (GC).

Results

In radio-resistant GC cells, down-regulation of miR-190b and up-regulation of Bcl-2 were observed. The protein expression of Bcl-2 was negatively regulated by miR-190b. Overexpression of miR-190b significantly decreased cell viability and enhanced radio-sensitivity of GC cells. Of note, these effects of miR-190b on GC cells radio-sensitivity were abolished by Bcl-2.

Conclusion

miR-190b confers radio-sensitivity of GC cells, possibly via negative regulation of Bcl-2.

     

Non-small cell lung cancer: miR-30d suppresses tumor invasion and migration by directly targeting NFIB

Objective

To evaluate the role and the molecular mechanism of miR-30d in non-small cell lung cancer (NSCLC).

Results

qRT-PCR was used to detect miR-30d expression in NSCLC tissues and cell lines. miR-30d was frequently down-regulated in NSCLC and its expression was associated with clinicopathological features of NSCLCC patients. Over-expression of miR-30d notably inhibited cell migration and invasion in NSCLC cell lines. miR-30d could negatively regulate Nuclear factor I B (NFIB) by directly targeting its 3′-UTR, which was confirmed by luciferase assay. NFIB also reversed miR-30d-mediated suppression on the migration and invasion in NSCLC cell lines.

Conclusion

miR-30d suppressed cell migration and invasion by directly targeting NFIB in NSCLC, and NFIB could partially abrogated the inhibition of biological functions by miR-30d.

     

Sex differences in the expression of lupus-associated miRNAs in splenocytes from lupus-prone NZB/W<Subscript>F1</Subscript> mice

Background

A majority of autoimmune diseases, including systemic lupus erythematosus (SLE), occur predominantly in females. Recent studies have identified specific dysregulated microRNAs (miRNAs) in both human and murine lupus, implying an important contribution of these miRNAs to lupus pathogenesis. However, to date, there is no study that examined sex differences in miRNA expression in immune cells as a plausible basis for sex differences in autoimmune disease. This study addresses this aspect in NZB/W_F1 mice, a classical murine lupus model with marked female bias, and further investigates estrogen regulation of lupus-associated miRNAs.

Methods

The Taqman miRNA assay system was used to quantify the miRNA expression in splenocytes from male and female NZB/W_F1 mice at 17–18, 23, and 30 weeks (wks) of age. To evaluate potential estrogen's effect on lupus-associated miRNAs, 6-wk-old NZB/W_F1 male mice were orchidectomized and surgically implanted with empty (placebo) or estrogen implants for 4 and 26 wks, respectively. To assess the lupus status in the NZB/W_F1 mice, serum anti-dsDNA autoantibody levels, proteinuria, and renal histological changes were determined.

Results

The sex differences in the expression of lupus-associated miRNAs, including the miR-182-96-183 cluster, miR-155, miR-31, miR-148a, miR-127, and miR-379, were markedly evident after the onset of lupus, especially at 30 wks of age when female NZB/W_F1 mice manifested moderate to severe lupus when compared to their male counterparts. Our limited data also suggested that estrogen treatment increased the expression of aforementioned lupus-associated miRNAs, with the exception of miR-155, in orchidectomized male NZB/W_F1 mice to a similar level in age-matched intact female NZB/W_F1 mice. It is noteworthy that orchiectomy, itself, did not affect the expression of lupus-associated miRNAs.

Conclusion

To our knowledge, this is the first study that demonstrated sex differences in the expression of lupus-associated miRNAs in splenocytes, especially in the context of autoimmunity. The increased expression of lupus-associated miRNA in female NZB/W_F1 mice and conceivably in estrogen-treated orchidectomized male NZB/W_F1 mice was associated with lupus manifestation. The notable increase of lupus-associated miRNAs in diseased, female NZB/W_F1 mice may be a result of both lupus manifestation and the female gender.

     

miR-34a attenuates glioma cells progression and chemoresistance via targeting PD-L1

Objective

To investigate the roles of miR-34a in progression and chemoresistance of glioma cells.

Results

Quantitative real-time PCR analysis showed that miR-34a level was lower, but PD-L1 expression level was higher in glioma tissue specimens compared with normal brain tissues and their expression levels were negatively correlated. Ectopic expression of miR-34a inhibited glioma cell proliferation, promoted cell cycle arrest in G1/S phase and cell apoptosis. Additionally, miR-34a/PD-L1 axis was again confirmed and co-expression of PD-L1 with miR-34a mimics attenuated the effects of miR-34a on cell proliferation and apoptosis in glioma cells. Importantly, PD-L1 overexpression resulted in chemoresistance in glioma cells, this effect was attenuated by miR-34a overexpression.

Conclusions

miR-34a inhibits glioma cells progression and chemoresistance via targeting PD-L1.

     

miR-93 enhances hepatocellular carcinoma invasion and metastasis by EMT via targeting PDCD4

Objectives

To clarify the potential biological function of miR-93 and its related molecular mechanism underlying metastasis in human hepatocellular carcinoma (HCC).

Results

miR-93 was significantly up-regulated in HCC tissues and was associated with poor 5-year overall survival in HCC patients. Transwell assays showed that over-expression of miR-93 increased HCC cell migration and invasion in vitro. Programmed cell death 4 (PDCD4) was a target gene of miR-93 and miR-93 could down-regulate the expression of PDCD4 by directly targeting its 3′-UTR. The re-expression of PDCD4 could attenuate the HCC cell invasion and migration induced by miR-93, while the knockdown of PDCD4 would promote HCC cell migration and invasion via the epithelial-mesenchymal transition (EMT) pathway.

Conclusions

miR-93 provides new insight into the molecular mechanisms of pathogenesis and progression in HCC and offer a potential therapeutic target for the treatment of HCC patients.