Downregulation of miR‑214-3p attenuates mesangial hypercellularity by targeting PTEN‑mediated JNK/c-Jun signaling in IgA nephropathy

Mesangial cell (MC) proliferation and matrix expansion are basic pathological characteristics of IgA nephropathy (IgAN). However, the stepwise mechanism of MC proliferation and the exact set of related signaling molecules remain largely unclear. In this study, we found a significant upregulation of miR-214-3p in the renal cortex of IgAN mice by miRNA sequencing. In situ hybridization analysis showed that miR-214-3p expression was obviously elevated in MCs in the renal cortex in IgAN. Functionally, knockdown of miR-214-3p alleviated mesangial hypercellularity and renal lesions in IgAN mice. In vitro, the inhibition of miR-214-3p suppressed MC proliferation and arrested G1-S cell cycle pSrogression in IgAN. Mechanistically, a luciferase reporter assay verified PTEN as a direct target of miR-214-3p. Downregulation of miR-214-3p increased PTEN expression and reduced p-JNK and p-c-Jun levels, thereby inhibiting MC proliferation and ameliorating renal lesions in IgAN. Moreover, these changes could be attenuated by co-transfection with PTEN siRNA. Collectively, these results illustrated that miR-214-3p accelerated MC proliferation in IgAN by directly targeting PTEN to modulate JNK/c-Jun signaling. Therefore, miR-214-3p may represent a novel therapeutic target for IgAN.     

Mechanistic role of microRNA-146a in endotoxin-induced differential cross-regulation of TLR signaling

Human TLRs are critical sensors for microbial components leading to the production of proinflammatory cytokines that are controlled by various mechanisms. Monocytes pretreated with LPS exhibit a state of hyporesponsiveness, referred to as cross-tolerance, to both homologous and heterologous ligands, which play a broader role in innate immunity. To date, LPS-induced cross-tolerance has not been examined regarding microRNA expression kinetics. In this study, THP-1 monocytes treated with various inflammatory ligands showed a continuous amplification of microRNA (miR)-146a over 24 h that is inversely correlated to TNF-α production. In contrast, inhibition of miR-146a showed a reciprocal effect. Thus, the characteristic upregulation of miR-146a in LPS-exposed THP-1 monocytes was studied for cross-tolerance. Strikingly, in LPS-tolerized THP-1 monocytes, only miR-146a showed a continuous overexpression, suggesting its crucial role in cross-tolerance. Similarly, peptidoglycan-primed THP-1 cells showed homologous tolerance associated with miR-146a upregulation. Subsequently, interchangeable differential cross-regulation was observed among non-LPS ligands. TLR2 and TLR5 ligands showed both homologous and heterologous tolerance correlated to miR-146a overexpression. More importantly, inflammatory responses to TLR4, TLR2, and TLR5 ligands were reduced due to knockdown of miR-146a targets IL-1R-associated kinase 1 or TNFR-associated factor 6, suggesting the regulatory effect of miR-146a on these TLRs signaling. Transfection of miR-146a into THP-1 cells caused reduction of TNF-α production, mimicking LPS-induced cross-tolerance. Aside from individual ligands, a whole bacterial challenge in LPS-primed THP-1 monocytes was accompanied by less TNF-α production, which is conversely correlated to miR-146a expression. Our studies have thus demonstrated that miR-146a plays a crucial role for in vitro monocytic cell-based endotoxin-induced cross-tolerance.     

MicroRNA Cargo of Extracellular Vesicles from Alcohol-exposed Monocytes Signals Naive Monocytes to Differentiate into M2 Macrophages

Membrane-coated extracellular vesicles (EVs) released by cells can serve as vehicles for delivery of biological materials and signals. Recently, we demonstrated that alcohol-treated hepatocytes cross-talk with immune cells via exosomes containing microRNA (miRNAs). Here, we hypothesized that alcohol-exposed monocytes can communicate with naive monocytes via EVs. We observed increased numbers of EVs, mostly exosomes, secreted by primary human monocytes and THP-1 monocytic cells in the presence of alcohol in a concentration- and time-dependent manner. EVs derived from alcohol-treated monocytes stimulated naive monocytes to polarize into M2 macrophages as indicated by increased surface expression of CD68 (macrophage marker), M2 markers (CD206 (mannose receptor) and CD163 (scavenger receptor)), secretion of IL-10, and TGFβ and increased phagocytic activity. miRNA profiling of the EVs derived from alcohol-treated THP-1 monocytes revealed high expression of the M2-polarizing miRNA, miR-27a. Treatment of naive monocytes with control EVs overexpressing miR-27a reproduced the effect of EVs from alcohol-treated monocytes on naive monocytes and induced M2 polarization, suggesting that the effect of alcohol EVs was mediated by miR-27a. We found that miR-27a modulated the process of phagocytosis by targeting CD206 expression on monocytes. Importantly, analysis of circulating EVs from plasma of alcoholic hepatitis patients revealed increased numbers of EVs that contained high levels of miR-27a as compared with healthy controls. Our results demonstrate the following: first, alcohol increases EV production in monocytes; second, alcohol-exposed monocytes communicate with naive monocytes via EVs; and third, miR-27a cargo in monocyte-derived EVs can program naive monocytes to polarize into M2 macrophages.     

Wolbachia heat shock protein 60 induces pro-inflammatory cytokines and apoptosis in monocytes in vitro

Recombinant Wolbachia heat shock protein 60 (rWmhsp60) induces gene expression of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α in human monocytic cell line THP-1. In addition, it inhibits the phagocytic activity and does not alter the nitric oxide production by differentiated THP-1 macrophages, which corroborates with no significant change in inducible nitric oxide synthase gene expression in rWmhsp60 treated THP-1 monocytes. Further, 24 h stimulation of peripheral blood mononuclear cells from normal individuals by rWmhsp60 reveals that monocytes enter the late apoptotic stage, while lymphocytes do not show apoptosis. Thus these findings suggest that rWmhsp60 may contribute to inflammation mediated monocyte dysfunction in filarial pathogenesis.     

Cooperative miRNA-dependent PTEN regulation drives resistance to BTK inhibition in B-cell lymphoid malignancies

Aberrant microRNA (miR) expression plays an important role in pathogenesis of different types of cancers, including B-cell lymphoid malignancies and in the development of chemo-sensitivity or -resistance in chronic lymphocytic leukemia (CLL) as well as diffuse large B-cell lymphoma (DLBCL). Ibrutinib is a first-in class, oral, covalent Bruton’s tyrosine kinase (BTK) inhibitor (BTKi) that has shown impressive clinical activity, yet many ibrutinib-treated patients relapse or develop resistance over time. We have reported that acquired resistance to ibrutinib is associated with downregulation of tumor suppressor protein PTEN and activation of the PI3K/AKT pathway. Yet how PTEN mediates chemoresistance in B-cell malignancies is not clear. We now show that the BTKi ibrutinib and a second-generation compound, acalabrutinib downregulate miRNAs located in the 14q32 miRNA cluster region, including miR-494, miR-495, and miR-543. BTKi-resistant CLL and DLBCL cells had striking overexpression of miR-494, miR-495, miR-543, and reduced PTEN expression, indicating further regulation of the PI3K/AKT/mTOR pathway in acquired BTKi resistance. Additionally, unlike ibrutinib-sensitive CLL patient samples, those with resistance to ibrutinib treatment, demonstrated upregulation of 14q32 cluster miRNAs, including miR-494, miR-495, and miR-543 and decreased pten mRNA expression. Luciferase reporter gene assay showed that miR-494 directly targeted and suppressed PTEN expression by recognizing two conserved binding sites in the PTEN 3′-UTR, and subsequently activated AKT^Ser473. Importantly, overexpression of a miR-494 mimic abrogated both PTEN mRNA and protein levels, further indicating regulation of apoptosis by PTEN/AKT/mTOR. Conversely, overexpression of a miR-494 inhibitor in BTKi-resistant cells restored PTEN mRNA and protein levels, thereby sensitizing cells to BTKi-induced apoptosis. Inhibition of miR-494 and miR-495 sensitized cells by cooperative targeting of pten, with additional miRNAs in the 14q32 cluster that target pten able to contribute to its regulation. Therefore, targeting 14q32 cluster miRNAs may have therapeutic value in acquired BTK-resistant patients via regulation of the PTEN/AKT/mTOR signaling axis.Subject terms: Cancer, Chronic lymphocytic leukaemia     

miR-29b regulates migration of human breast cancer cells

microRNAs (miRNAs) are short non-coding RNAs that regulate gene expression by targeting mRNAs, inhibiting the expression of the associated proteins. Although a role for aberrant miRNA expression in cancer has been postulated, the pathophysiologic role and relevance of aberrantly expressed miRNAs in tumor biology has not been established. We evaluated the expression pattern of miRNAs in human breast cancer cells by qPCR, finding out an up-regulated miRNA miR-29b and studying its biological effect by migration assay. We defined a target gene PTEN by bioinformatics approach and western blot. In breast cancer cell line MDA-MB-231 cell, which migrate faster than MCF-7, we observed that miR-29b was highly over-expressed. Inhibition of miR-29b in cultured cells increased the expression of the phosphatase and tensin homolog (PTEN) tumor suppressor, promoting apoptosis, decreasing migration, and decreasing invasion. In contrast, enhanced miR-29b expression by transfection with pre-miR-29b decreased the expression of PTEN and impaired apoptosis, increasing tumor cell migration and invasion. Moreover, PTEN was shown to be a direct target of miR-29b and was also shown to contribute to the miR-29b-mediated effects on cell invasion. Modulation of miR-29b altered the role of PTEN involved in cell migration and invasion. Aberrant expression of miR-29b, which modulates PTEN expression, can contribute to migration, invasion, and anti-apoptosis.     

Long noncoding RNA PTENP1 affects the recovery of spinal cord injury by regulating the expression of miR-19b and miR-21

Exosomes derived from differentiated P12 cells and MSCs were proved to suppress apoptosis of neuron cells, and phosphatase and tensin homolog pseudogene 1 (PTENP1) was reported to inhibit cell proliferation. In this study, we aimed to investigate the role of PTENP1 in the process of post-spinal cord injury (SCI) recovery, so as to evaluate the therapeutic effects of exosomes derived from MSCs transfected with PTENP1 short hairpin RNA (shRNA), as a type of novel biomarkers in the treatment of SCI. Electron microscopy was used to observe the morphology of different exosomes. Real-time polymerase chain reaction and western blot, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, flow cytometry, Nissl staining, immunohistochemistry assay, and terminal deoxynucleotidyl transferase dUTP nick end labeling assay were conducted to investigate and validate the underlying molecular signaling pathway. PTENP1-shRNA downregulated PTENP1 and PTEN while upregulating miR-21 and miR-19b. PTENP1-shRNA also accelerated cell apoptosis and reduced cell viability. In addition, PTENP1 reduced the miR-21 and miR-19b expression by directly targeting miR-21 and miR-19b. Meanwhile, both miR-21 and miR-19b reduced the expression of PTEN by directly targeting the 3′-untranslated region of PTEN. Furthermore, PTEN level and apoptosis index of neuron cells was the highest in the SCI group, while the treatment with exosomes+PTENP1-shRNA reduced the PTEN expression to a level similar to that in the sham group. Finally, PTENP1 inhibited miR-21 and miR-19b expression but upregulated PTEN expression. The upregulation of miR-21/miR-19b also suppressed the apoptosis of neuron cells by downregulating the PTEN expression. PTENP1 is involved in the recovery of SCI by regulating the expression of miR-19b and miR-21, and exosomes from PTENP1-shRNA-transfected cells may be used as a novel biomarker in SCI treatment.     

Identification of candidate microRNA biomarkers in renal fibrosis: a meta-analysis of profiling studies

Abstract

The prognostic, diagnostic and therapeutic value of microRNA (miRNA) expression aberrations in renal fibrosis has been studied in recent years. However, the miRNA expression profiling efforts have led to inconsistent results between the studies. The aim of this study was to perform a meta-analysis on the renal fibrosis miRNA expression profiling studies to identify candidate diagnostic biomarkers. We performed comprehensive literature searches in several databases to identify miRNA expression studies of renal fibrosis in animal models and humans. The miRNAs expression data were extracted from 20 included studies, and both miRNA vote-counting strategy and Robust Rank Aggregation method were utilized to identify significant miRNA meta-signatures. The predicted and validated targets of miRNA meta-signature were obtained by using MultiMiR package in 11 databases. Then a gene set enrichment analysis (KEGG, PANTHER pathways and GO processes) were carried out with GeneCodis web tool to recognize pathways that are most strongly influenced by modified expressions of these miRNAs. We recognized in both meta-analysis approaches a significant miRNA meta-signature of five up-regulated (miR-142-3p, miR-223-3p, miR-21-5p, miR-142-5p and miR-214-3p) and two down-regulated (miR-29c-3p and miR-200a-3p) miRNAs. Enrichment analysis confirmed that miRNA meta-signature cooperatively target functionally related genes in signalling and developmental pathways in renal fibrosis. This meta-analysis identified seven highly significant and consistently dysregulated miRNAs from 20 datasets, as the focus of future investigations to discover their potential influence to renal fibrosis and their clinical utility as biomarkers and/or as therapeutic mediators against chronic kidney disease..     

Down-regulation of miR-20a-5p triggers cell apoptosis to facilitate mycobacterial clearance through targeting JNK2 in human macrophages

Induction of cell apoptosis is one of the major host defense mechanisms through which macrophages control Mycobacterium tuberculosis (Mtb) infection. However, the mechanisms underlying macrophage apoptosis triggered by Mtb infection are still largely unknown. In this study, a microarray profiling survey revealed 14 miRNAs were down-regulated in CD14+ monocytes from active pulmonary tuberculosis patients, and only the reduction of miR-20a-5p could be reversed after successful anti-tuberculosis treatment. Validation of miR-20a-5p expression was confirmed using real time qPCR. Moreover, miR-20a-5p expression also decreased in differentiated THP-1 macrophages after mycobacterial infection in vitro. Functional assays through forced or inhibited expression of miR-20a-5p in THP-1 macrophages demonstrated that miR-20a-5p functioned as a negative regulator of mycobacterial-triggered apoptosis. Importantly, inhibition of miR-20a-5p expression resulted in more efficient mycobacterial clearance from infected THP-1 macrophages while miR-20a-5p overexpression promoted mycobacterial survival. Mechanistically, miR-20a-5p was demonstrated to regulate Bim expression in a JNK2-dependent manner, unlike Bcl2, and luciferase assay showed JNK2 was a novel direct target of miR-20a-5p. Together, our findings indicate that downregulation of miR-20a-5p triggers macrophage apoptosis as a novel mechanism for host defense against mycobacterial infection.     

Modulation of monocyte function by activated protein C, a natural anticoagulant

Activated protein C is the first effective biological therapy for the treatment of severe sepsis. Although activated protein C is well established as a physiological anticoagulant, emerging data suggest that it also exerts anti-inflammatory and antiapoptotic effects. In this study, we investigated the ability of activated protein C to modulate monocyte apoptosis, inflammation, phagocytosis, and adhesion. Using the immortalized human monocytic cell line THP-1, we demonstrated that activated protein C inhibited camptothecin-induced apoptosis in a dose-dependent manner. The antiapoptotic effect of activated protein C requires its serine protease domain and is dependent on the endothelial cell protein C receptor and protease-activated receptor-1. In primary blood monocytes from healthy individuals, activated protein C inhibited spontaneous apoptosis. With respect to inflammation, activated protein C inhibited the production of TNF, IL-1beta, IL-6, and IL-8 by LPS-stimulated THP-1 cells. Activated protein C did not influence the phagocytic internalization of Gram-negative and Gram-positive bioparticles by THP-1 cells or by primary blood monocytes. Activated protein C also did not affect the expression of adhesion molecules by LPS-stimulated blood monocytes nor the ability of monocytes to adhere to LPS-stimulated endothelial cells. We hypothesize that the protective effect of activated protein C in sepsis reflects, in part, its ability to prolong monocyte survival in a manner that selectively inhibits inflammatory cytokine production while maintaining phagocytosis and adherence capabilities, thereby promoting antimicrobial properties while limiting tissue damage.     

MiR-205 determines the radioresistance of human nasopharyngeal carcinoma by directly targeting PTEN

Radiotherapy is the primary treatment for nasopharyngeal carcinoma (NPC), but radioresistance severely reduces NPC radiocurability. Here, we have established a radio-resistant NPC cell line, CNE-2R, and investigate the role of miRNAs in radioresistance. The miRNAs microarray assay reveals that miRNAs are differentially expressed between CNE-2R and its parental cell line CNE-2. We find that miR-205 is elevated in CNE-2R. A target prediction algorithm suggests that miR-205 regulates expression of PTEN, a tumor-suppressor. Introducing miR-205 into CNE-2 cells suppresses PTEN protein expression, followed by activation of AKT, increased number of foci formation and reduction of cell apoptosis post-irradiation. On the other hand, knocking down miR-205 in CNE-2R cells compromises the inhibition of PTEN and increases cell apoptosis. Significantly, immunohistochemistry studies demonstrate that PTEN is downregulated at late stages of NPC, and that miR-205 is significantly elevated followed the radiotherapy. Our data conclude that miR-205 contributes to radioresistance of NPC by directly targeting PTEN. Both miR-205 and PTEN are potential predictive biomarkers for radiosensitivity of NPC and may serve as targets for achieve successful radiotherapy in NPC.Key words: nasopharyngeal carcinoma, PTEN, miRNA, miR-205, radioresistance     

Inhibition of the receptor for advanced glycation endproducts (RAGE) protects pancreatic β-cells

Advanced glycation endproducts (AGEs) and the receptor for AGEs (RAGE) have been linked to the pathogenesis of diabetic complications, such as retinopathy, neuropathy, and nephropathy. AGEs may induce β-cell dysfunction and apoptosis, another complication of diabetes. However, the role of AGE-RAGE interaction in AGE-induced pancreatic β-cell failure has not been fully elucidated. In this study, we investigated whether AGE–RAGE interaction could mediate β-cell failure. We explored the potential mechanisms in insulin secreting (INS-1) cells from a pancreatic β-cell line, as well as primary rat islets. We found that glycated serum (GS) induced apoptosis in pancreatic β-cells in a dose- and time-dependent manner. Treatment with GS increased RAGE protein production in cultured INS-1 cells. GS treatment also decreased bcl-2 gene expression, followed by mitochondrial swelling, increased cytochrome c release, and caspase activation. RAGE antibody and knockdown of RAGE reversed the β-cell apoptosis and bcl-2 expression. Inhibition of RAGE prevented AGE-induced pancreatic β-cell apoptosis, but could not restore the function of glucose stimulated insulin secretion (GSIS) in rat islets. In summary, the results of the present study demonstrate that AGEs are integrally involved in RAGE-mediated apoptosis and impaired GSIS dysfunction in pancreatic β-cells. Inhibition of RAGE can effectively protect β-cells against AGE-induced apoptosis, but cannot reverse islet dysfunction in GSIS.     

Inhibition of PTEN Gene Expression by Oncogenic miR-23b-3p in Renal Cancer

Background

miR-23b is located on chromosome number 9 and plays different roles in different organs especially with regards to cancer development. However, the functional significance of miR-23b-3p in renal cell carcinoma (RCC) has not been reported.

Methods and Results

We measured miR-23b-3p levels in 29 pairs of renal cell carcinoma and their normal matched tissues using real-time PCR. The expression level of miR-23b-3p was correlated with the 5 year survival rate of renal cancer patients. In 15 cases (52%), miR-23b-3p expression was found to be high. All patients with moderate to low miR-23b-3p expression survived 5 years, while those with high miR-23b-3p expression, only 50% survived. After knocking down miRNA-23b-3p expression in RCC cell lines, there was an induction of apoptosis and reduced invasive capabilities. MiR-23b-3p was shown to directly target PTEN gene through 3′UTR reporter assays. Inhibition of miR-23b-3p induces PTEN gene expression with a concomitant reduction in PI3-kinase, total Akt and IL-32. Immunohistochemistry showed the lack of PTEN protein expression in cancerous regions of tissue samples where the expression of miR-23b-3p was high. We studied the in vitro effects of the dietary chemo preventive agent genistein on miR-23b-3p expression and found that it inhibited expression of miR-23b-3p in RCC cell lines.

Conclusions

The current study shows that miR-23b-3p is an oncogenic miRNA and inhibits PTEN tumor suppressor gene in RCC. Therefore, inhibition of miR-23b-3p may be a useful therapeutic target for the treatment of renal cell carcinoma.     

Retracted: Upregulated microRNA-132 rescues cardiac fibrosis and restores cardiocyte proliferation in dilated cardiomyopathy through the phosphatase and tensin homolog–mediated PI3K/Akt signal transduction pathway

Cardiac fibrosis is known to be present in dilated cardiomyopathy (DCM) and it predicts the occurrence of sudden death and congestive heart failure. The aim of our study is to investigate the expression of microRNA-132 (miR-132) and its effect on cardiocyte proliferation, apoptosis, and cardiac fibrosis by binding to phosphatase and tensin homolog (PTEN) through the phosphateidylinositol 3-kinase (PI3K)/protein kinase (Akt) signal transduction pathway in DCM rats. DCM rat models induced by doxorubicin were established and confirmed by an ultrasonic cardiogram. Epithelial cells were treated with inhibitors, activators, and small interfering RNAs to identify the mechanisms by which miR-132 controls cardiocyte activity and cardiac fibrosis. Angiotensin II (Ang II) and aldosterone (ALD) expressions were detected by an enzyme-linked immunosorbent assay. The relationship between PTEN and miR-132 was verified by a dual-luciferase reporter assay. Cell proliferation and apoptosis were tested by the MTT assay and flow cytometry. PTEN was determined to be the target gene of miR-132. Rat models of DCM exhibited a lower level of miR-132, PI3K, Akt, B-cell lymphoma 2, collagen I, and collagen III, but a higher level of PTEN, Bcl-2–associated X protein, and proliferating cell nuclear antigen as well as inflammatory response (Ang II and ALD), accompanied by declined cardiocyte proliferation and elevated apoptosis and cardiac fibrosis. Upregulated miR-132 or silenced PTEN activated the PI3K/Akt pathway, thus facilitating cardiocyte proliferation and repressing cardiocyte apoptosis and cardiac fibrosis, as well as inflammatory responses. Downregulated miR-132 reversed this tendency. These findings indicate that miR-132 activates the PI3K/Akt pathway by inhibiting PTEN expression, thus facilitating cardiocyte proliferation and inhibiting apoptosis and cardiac fibrosis in DCM rats.     

Knockdown of differentiation antagonizing non-protein coding RNA exerts anti-tumor effect by up-regulating miR-214 in endometrial carcinoma

Differentiation antagonizing non-protein coding RNA (DANCR) is a valuable long noncoding RNA (lncRNA) that involves in the progress of various cancers. However, the functions of DANCR in endometrial carcinoma (EC) have not been validated. In the present study, we aimed to evaluate the roles of DANCR in EC and explore the underlying mechanism. Expression patterns of DANCR in EC specimens and normal control specimens were determined using qRT-PCR. DANCR was knocked down in EC cell lines (AN3CA and HEC-1B) through transfection with small interfering RNA (siRNA) targeting DANCR (si-DANCR). Cell proliferation was examined using the cell counting kit-8 (CCK-8) assay. Cell apoptosis was measured by flow cytometry. Online software starBase was used to predict the target gene of DANCR. Luciferase reporter assay was carried out to confirm the association between DANCR and the predicted target microRNA (miRNA). DANCR expression was up-regulated in EC tissues as compared to the normal control tissues. Knockdown of DANCR in AN3CA and HEC-1B cells markedly suppressed cell proliferation and induced cell apoptosis. miR-214 was found to be a target miRNA of DANCR and its expression was significantly decreased in EC tissues. Suppression of miR-214 abolished the effects of si-DANCR on cell proliferation and apoptosis in AN3CA and HEC-1B cells. DANCR played an important role in promoting tumorigenesis of EC via sponging miR-214. DANCR might serve as a therapeutic target for the treatment of EC.