Decreased expression of microRNA‐21 correlates with the imbalance of Th17 and Treg cells in patients with rheumatoid arthritis

The imbalance of Th17/Treg cell populations has been suggested to be involved in the regulation of rheumatoid arthritis (RA) pathogenesis; however, the mechanism behind this phenomenon remains unclear. Recent studies have shown how microRNAs (miRNAs) are important regulators of immune responses and are involved in the development of a variety of inflammatory diseases, including RA. In this study, we demonstrated that the frequencies of CD3⁺CD4⁺IL‐17⁺Th17 cells were significantly higher, and CD4⁺CD25⁺FOXP3⁺ Treg cells significantly lower in peripheral blood mononuclear cells from RA patients. Detection of cytokines from RA patients revealed an elevated panel of pro‐inflammatory cytokines, including IL‐17, IL‐6, IL‐1β, TNF‐α and IL‐22, which carry the inflammatory signature of RA and are crucial in the differentiation and maintenance of pathogenic Th17 cells and dysfunction of Treg cells. However, the level of miR‐21 was significantly lower in RA patients, accompanied by the increase in STAT3 expression and activation, and decrease in STAT5/pSTAT5 protein and Foxp3 mRNA levels. Furthermore, lipopolysaccharide stimulation up‐regulated miR‐21 expression from healthy controls, but down‐regulated miR‐21 expression from RA patients. Therefore, we speculate that miR‐21 may be part of a negative feedback loop in the normal setting. However, miR‐21 levels decrease significantly in RA patients, suggesting that this feedback loop is dysregulated and may contribute to the imbalance of Th17 and Treg cells. MiR‐21 may thus serve as a novel regulator in T‐cell differentiation and homoeostasis, and provides a new therapeutic target for the treatment of RA.     

Regulatory role of miR‐2909 in cell‐mediated immune response

Keeping in view the micromanagement of immune response by micro RNAs, the present study was directed to explore the role of miR‐2909 in the differentiation and maturation of T‐lymphocytes within the population of normal human peripheral blood mononuclear cells maintained in in vitro culture. The results of such a study revealed that miR‐2909 had the inherent capacity to significantly increase Treg (CD4+CD25+Foxp3+) cell population and dominant Th1‐type cytokine (especially with decrease in IL‐4 level and higher levels of INF‐β and INF‐γ) profile. Based upon these results, we propose that miR‐2909 may modulate native immunity in general and help in providing protective immunity against viral infections in particular. Copyright © 2012 John Wiley & Sons, Ltd.     

Down‐expression of miR‐154 suppresses tumourigenesis in CD133+ glioblastoma stem cells

Glioblastoma multiforme (GBM) is the most common and aggressive form of brain cancer. Evidences have suggested that CD133 is a marker for a subset of glioblastoma cancer stem cells. However, whether miRNA plays a critical role in CD133⁺ GBM is poorly understood. Here, we identified that miR‐154 was upregulated in CD133⁺ GBM cell lines. Knockdown of miR‐154 remarkably suppressed proliferation and migration of CD133⁺ GBM cells. Further study found that PRPS1 was a direct target of miR‐154 in CD133⁺ GBM cells. Overexpression of PRPS1 exhibited similar effects as miR‐154 knockdown in CD133⁺ GBMs. Our study identified miR‐154 as a previously unrecognized positive regulator of proliferation and migration in CD133⁺ GBM cells and a potentially therapeutic target of GBMs. Copyright © 2016 John Wiley & Sons, Ltd.     

miR‐200c suppresses stemness and increases cellular sensitivity to trastuzumab in HER2+ breast cancer

Resistance to trastuzumab remains a major obstacle in HER2‐overexpressing breast cancer treatment. miR‐200c is important for many functions in cancer stem cells (CSCs), including tumour recurrence, metastasis and resistance. We hypothesized that miR‐200c contributes to trastuzumab resistance and stemness maintenance in HER2‐overexpressing breast cancer. In this study, we used HER2‐positive SKBR3, HER2‐negative MCF‐7, and their CD44⁺CD24^? phenotype mammospheres SKBR3‐S and MCF‐7‐S to verify. Our results demonstrated that miR‐200c was weakly expressed in breast cancer cell lines and cell line stem cells. Overexpression of miR‐200c resulted in a significant reduction in the number of tumour spheres formed and the population of CD44⁺CD24^? phenotype mammospheres in SKBR3‐S. Combining miR‐200c with trastuzumab can significantly reduce proliferation and increase apoptosis of SKBR3 and SKBR3‐S. Overexpression of miR‐200c also eliminated its downstream target genes. These genes were highly expressed and positively related in breast cancer patients. Overexpression of miR‐200c also improved the malignant progression of SKBR3‐S and SKBR3 in vivo. miR‐200c plays an important role in the maintenance of the CSC‐like phenotype and increases drug sensitivity to trastuzumab in HER2+ cells and stem cells.     

Altered expression and editing of miRNA-100 regulates iTreg differentiation

RNA editing of miRNAs, especially in the seed region, adds another layer to miRNA mediated gene regulation which can modify its targets, altering cellular signaling involved in important processes such as differentiation. In this study, we have explored the role of miRNA editing in CD4⁺ T cell differentiation. CD4⁺ T cells are an integral component of the adaptive immune system. Naïve CD4⁺ T cells, on encountering an antigen, get differentiated either into inflammatory subtypes like Th1, Th2 or Th17, or into immunosuppressive subtype Treg, depending on the cytokine milieu. We found C-to-U editing at fifth position of mature miR-100, specifically in Treg. The C-to-U editing of miR-100 is functionally associated with at least one biologically relevant target change, from MTOR to SMAD2. Treg cell polarization by TGFβ1 was reduced by both edited and unedited miR-100 mimics, but percentage of Treg in PBMCs was only reduced by edited miR-100 mimics, suggesting a model in which de-repression of MTOR due to loss of unedited mir-100, promotes tolerogenic signaling, while gain of edited miR-100 represses SMAD2, thereby limiting the Treg. Such delicately counterbalanced systems are a hallmark of immune plasticity and we propose that miR-100 editing is a novel mechanism toward this end.     

Targeting prohibitins at the cell surface prevents Th17‐mediated autoimmunity

T helper (Th)17 cells represent a unique subset of CD4⁺ T cells and are vital for clearance of extracellular pathogens including bacteria and fungi. However, Th17 cells are also involved in orchestrating autoimmunity. By employing quantitative surface proteomics, we found that the evolutionarily conserved prohibitins (PHB1/2) are highly expressed on the surface of both murine and human Th17 cells. Increased expression of PHBs at the cell surface contributed to enhanced CRAF/MAPK activation in Th17 cells. Targeting surface‐expressed PHBs on Th17 cells with ligands such as Vi polysaccharide (Typhim vaccine) inhibited CRAF‐MAPK pathway, reduced interleukin (IL)‐17 expression and ameliorated disease pathology with an increase in FOXP3⁺‐expressing Tregs in an animal model for multiple sclerosis (MS). Interestingly, we detected a CD4⁺ T cell population with high PHB1 surface expression in blood samples from MS patients in comparison with age‐ and sex‐matched healthy subjects. Our observations suggest a pivotal role for the PHB‐CRAF‐MAPK signalling axis in regulating the polarization and pathogenicity of Th17 cells and unveil druggable targets in autoimmune disorders such as MS.     

MiR‐16 regulates mouse peritoneal macrophage polarization and affects T‐cell activation

MiR‐16 is a tumour suppressor that is down‐regulated in certain human cancers. However, little is known on its activity in other cell types. In this study, we examined the biological significance and underlying mechanisms of miR‐16 on macrophage polarization and subsequent T‐cell activation. Mouse peritoneal macrophages were isolated and induced to undergo either M1 polarization with 100 ng/ml of interferon‐γ and 20 ng/ml of lipopolysaccharide, or M2 polarization with 20 ng/ml of interleukin (IL)‐4. The identity of polarized macrophages was determined by profiling cell‐surface markers by flow cytometry and cytokine production by ELISA. Macrophages were infected with lentivirus‐expressing miR‐16 to assess the effects of miR‐16. Effects on macrophage–T cell interactions were analysed by co‐culturing purified CD4⁺ T cells with miR‐16‐expressing peritoneal macrophages, and measuring activation marker CD69 by flow cytometry and cytokine secretion by ELISA. Bioinformatics analysis was applied to search for potential miR‐16 targets and understand its underlying mechanisms. MiR‐16‐induced M1 differentiation of mouse peritoneal macrophages from either the basal M0‐ or M2‐polarized state is indicated by the significant up‐regulation of M1 marker CD16/32, repression of M2 marker CD206 and Dectin‐1, and increased secretion of M1 cytokine IL‐12 and nitric oxide. Consistently, miR‐16‐expressing macrophages stimulate the activation of purified CD4⁺ T cells. Mechanistically, miR‐16 significantly down‐regulates the expression of PD‐L1, a critical immune suppressor that controls macrophage–T cell interaction and T‐cell activation. MiR‐16 plays an important role in shifting macrophage polarization from M2 to M1 status, and functionally activating CD4⁺ T cells. This effect is potentially mediated through the down‐regulation of immune suppressor PD‐L1.     

miR‐181a and miR‐150 regulate dendritic cell immune inflammatory responses and cardiomyocyte apoptosis via targeting JAK1–STAT1/c‐Fos pathway

The immune inflammatory response plays a crucial role in many cardiac pathophysiological processes, including ischaemic cardiac injury and the post‐infarction repair process. MicroRNAs (miRNAs) regulate the development and function of dendritic cells (DCs), which are key players in the initiation and regulation of immune responses; however, the underlying regulatory mechanisms remain unclear. Here, we used the supernatants of necrotic primary cardiomyocytes (Necrotic‐S) to mimic the myocardial infarction (MI) microenvironment to investigate the role of miRNAs in the regulation of DC‐mediated inflammatory responses. Our results showed that Necrotic‐S up‐regulated the DC maturation markers CD40, CD83 and CD86 and increased the production of inflammatory cytokines, concomitant with the up‐regulation of miR‐181a and down‐regulation of miR‐150. Necrotic‐S stimulation activated the JAK/STAT pathway and promoted the nuclear translocation of c‐Fos and NF‐κB p65, and silencing of STAT1 or c‐Fos suppressed Necrotic‐S‐induced DC maturation and inflammatory cytokine production. The effects of Necrotic‐S on DC maturation and inflammatory responses, its activation of the JAK/STAT pathway and the induction of cardiomyocyte apoptosis under conditions of hypoxia were suppressed by miR‐181a or miR‐150 overexpression. Taken together, these data indicate that miR‐181a and miR‐150 attenuate DC immune inflammatory responses via JAK1–STAT1/c‐Fos signalling and protect cardiomyocytes from cell death under conditions of hypoxia.     

MiR‐139‐5p,miR‐940 and miR‐193a‐5p inhibit the growth of hepatocellular carcinoma by targeting SPOCK1

The study was aimed to screen out miRNAs with differential expression in hepatocellular carcinoma (HCC), and to explore the influence of the expressions of these miRNAs and their target gene on HCC cell proliferation, invasion and apoptosis. MiRNAs with differential expression in HCC were screened out by microarray analysis. The common target gene of these miRNAs (miR‐139‐5p, miR‐940 and miR‐193a‐5p) was screened out by analysing the target genes profile (acquired from Targetscan) of the three miRNAs. Expression levels of miRNAs and SPOCK1 were determined by quantitative real time polymerase chain reaction (qRT‐PCR). The target relationships were verified by dual luciferase reporter gene assay and RNA pull‐down assay. Through 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide,thiazolyl blue tetrazolium bromide (MTT) and transwell assays and flow cytometry, HCC cell viability, invasion and apoptosis were determined. In vivo experiment was conducted in nude mice to investigate the influence of three miRNAs on tumour growth. Down‐regulation of miR‐139‐5p, miR‐940 and miR‐193a‐5p was found in HCC. Overexpression of these miRNAs suppressed HCC cell viability and invasion, promoted apoptosis and inhibited tumour growth. SPOCK1, the common target gene of miR‐139‐5p, miR‐940 and miR‐193a‐5p, was overexpressed in HCC. SPOCK1 overexpression promoted proliferation and invasion, and restrained apoptosis of HCC cells. MiR‐139‐5p, miR‐940 and miR‐193a‐5p inhibited HCC development through targeting SPOCK1.     

Anti-CD25 Treatment Depletes Treg Cells and Decreases Disease Severity in Susceptible and Resistant Mice Infected with Paracoccidioides brasiliensis

Regulatory T (Treg) cells are fundamental in the control of immunity and excessive tissue pathology. In paracoccidioidomycosis, an endemic mycosis of Latin America, the immunoregulatory mechanisms that control the progressive and regressive forms of this infection are poorly known. Due to its modulatory activity on Treg cells, we investigated the effects of anti-CD25 treatment over the course of pulmonary infection in resistant (A/J) and susceptible (B10.A) mice infected with Paracoccidioides brasiliensis. We verified that the resistant A/J mice developed higher numbers and more potent Treg cells than susceptible B10.A mice. Compared to B10.A cells, the CD4⁺CD25⁺Foxp3⁺ Treg cells of A/J mice expressed higher levels of CD25, CTLA4, GITR, Foxp3, LAP and intracellular IL-10 and TGF-β. In both resistant and susceptible mice, anti-CD25 treatment decreased the CD4⁺CD25⁺Foxp3⁺ Treg cell number, impaired indoleamine 2,3-dioxygenase expression and resulted in decreased fungal loads in the lungs, liver and spleen. In A/J mice, anti-CD25 treatment led to an early increase in T cell immunity, demonstrated by the augmented influx of activated CD4⁺ and CD8⁺ T cells, macrophages and dendritic cells to the lungs. At a later phase, the mild infection was associated with decreased inflammatory reactions and increased Th1/Th2/Th17 cytokine production. In B10.A mice, anti-CD25 treatment did not alter the inflammatory reactions but increased the fungicidal mechanisms and late secretion of Th1/Th2/Th17 cytokines. Importantly, in both mouse strains, the early depletion of CD25⁺ cells resulted in less severe tissue pathology and abolished the enhanced mortality observed in susceptible mice. In conclusion, this study is the first to demonstrate that anti-CD25 treatment is beneficial to the progressive and regressive forms of paracoccidioidomycosis, potentially due to the anti-CD25-mediated reduction of Treg cells, as these cells have suppressive effects on the early T cell response in resistant mice and the clearance mechanisms of fungal cells in susceptible mice.     

Endoplasmic reticulum stress‐induced exosomal miR‐27a‐3p promotes immune escape in breast cancer via regulating PD‐L1 expression in macrophages

Immune escape of breast cancer cells contributes to breast cancer pathogenesis. Tumour microenvironment stresses that disrupt protein homeostasis can produce endoplasmic reticulum (ER) stress. The miRNA‐mediated translational repression of mRNAs has been extensively studied in regulating immune escape and ER stress in human cancers. In this study, we identified a novel microRNA (miR)‐27a‐3p and investigated its mechanistic role in promoting immune evasion. The binding affinity between miR‐27a‐3p and MAGI2 was predicted using bioinformatic analysis and verified by dual‐luciferase reporter assay. Ectopic expression and inhibition of miR‐27a‐3p in breast cancer cells were achieved by transduction with mimics and inhibitors. Besides, artificial modulation of MAGI2 and PTEN was done to explore their function in ER stress and immune escape of cancer cells. Of note, exosomes were derived from cancer cells and co‐cultured with macrophages for mechanistic studies. The experimental data suggested that ER stress biomarkers including GRP78, PERK, ATF6, IRE1α and PD‐L1 were overexpressed in breast cancer tissues relative to paracancerous tissues. Endoplasmic reticulum stress promoted exosome secretion and elevated exosomal miR‐27a‐3p expression. Elevation of miR‐27a‐3p and PD‐L1 levels in macrophages was observed in response to exosomes‐overexpressing miR‐27a‐3p in vivo and in vitro. miR‐27a‐3p could target and negatively regulate MAGI2, while MAGI2 down‐regulated PD‐L1 by up‐regulating PTEN to inactivate PI3K/AKT signalling pathway. Less CD4⁺, CD8⁺ T cells and IL‐2, and T cells apoptosis were observed in response to co‐culture of macrophages and CD3⁺ T cells. Conjointly, exosomal miR‐27a‐3p promotes immune evasion by up‐regulating PD‐L1 via MAGI2/PTEN/PI3K axis in breast cancer.