Involvement of microRNA-155 in the mechanism of electroacupuncture treatment effects on experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a neurodegenerative and demyelinating autoimmune disease mediated by autoreactive T cells that affects the central nervous system (CNS). Electroacupuncture (EA) has emerged as an alternative or supplemental treatment for MS, but the mechanism by which EA may alleviate MS symptoms is unresolved. Here, we examined the effects of EA at the Zusanli (ST36) acupoint on mice with experimental autoimmune encephalomyelitis (EAE), the predominant animal model of MS. The effects of EA on EAE emergence, inflammatory cell levels, proinflammatory cytokines, and spinal cord pathology were examined. EA treatment attenuated the EAE clinical score and associated spinal cord demyelination, while reducing the presence of proinflammatory cytokines in mononuclear cells (MNCs), downregulating microRNA (miR)-155, and upregulating the opioid peptide precursor proopiomelanocortin (POMC) in the CNS. Experiments in which cultured neurons were transfected with a miR-155 mimic or a miR-155 inhibitor further showed that the direct modulation of miR-155 levels could regulate POMC levels in neurons. In conclusion, the alleviation of EAE by EA is characterized by reduced proportions of Th1/Th17 cells and increased proportions of Th2 cells, POMC upregulation, and miR-155 downregulation, while miR-155 itself can suppress POMC expression. These results, support the hypothesis that the effects of EA on EAE may involve the downregulation of miR-155.

Piceatannol inhibits pyroptosis and suppresses oxLDL-induced lipid storage in macrophages by regulating miR-200a/Nrf2/GSDMD axis.

As a major bioactive compound from grapes, piceatannol (PIC) has been reported to exert anti-atherosclerotic activity in various studies. Nevertheless, the mechanism underlying the effect of piceatannol against atherosclerosis (AS) is elusive. Our study identified miR-200a/Nrf2/GSDMD signaling pathway as critical mediators in the effect of piceatannol on macrophages. In the present study, we confirmed that treatment of piceatannol repressed the oxLDL-induced lipid storage in macrophages. Compared with control group, piceatannol inhibited TG storage and the activity of caspase1. It is noting that in response to oxLDL challenge, piceatannol abated the pyroptosis in RAW264.7 cells, with a decreased expression of caspase1, gasdermin D (GSDMD), IL-18, IL-1ß and NLRP3. Moreover, we investigated the role of microRNA (miR)-200a/Nrf2 signaling pathway in the effect of piceatannol. The results declared that after transfection of si-miR-200a or si-Nrf2 plasmids, the effects of piceatannol on macrophages were converted, including lipid storage and pyroptosis. Importantly, si-miR-200a plasmid reduced the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), indicating that miR-200a acted as an enhancer of Nrf2 in macrophages. Collectively, our findings demonstrate that piceatannol exerts anti-atherosclerotic activity on RAW264.7 cells by regulating miR-200a/Nrf2/GSDMD signaling. The present study is the first time to identify miR-200a as a candidate target in AS and declared an association between miR-200a and pyroptosis, which provides a novel therapy for the treatment of AS.

Let-7b-5p promotes electroacupuncture tolerance by downregulating Penk1 gene in CFA-induced inflammatory nociception rats.

BACKGROUND: Studies showed that increased let-7b-5p microRNA during repeated electroacupuncture (EA) treatment was associated the formation of EA tolerance, which manifested as gradually decreased nociceptive threshold. Proenkephalin (PENK) is the precursor of enkephalin which is a pivot neuropeptide responsible for the decreased nociceptive threshold in EA. The aim of this study was to evaluate the relationship between let-7b-5p and PENK in EA tolerance. METHODS: The target gene of let-7b-5p microRNA was determined through the dual-luciferase reporter assay in cortical neurons. Seventy-two Sprague Dawley rats received a combination of EA and intracerebroventricular injection of microRNA (let-7b-5p agomir, antagomir or their controls). The nociceptive thresholds were assessed with radiant heat tail-flick method. PENK and let-7b-5p were measured with Western Blot and qPCR, respectively, after administration of let-7b-5p agomir, antagomir, and their controls at day 1, 4 and 7. RESULTS: Let-7b-5p targeted the 3' untranslated region of Penk1. The nociceptive thresholds in Let-7b-5p agomir + EA group were decreased (p < 0.05) compared with those in Let-7b-5p antagomir + EA group at day 1 to 7. Compared with Let-7b-5p agomir + EA group, the expression level of PENK in Let-7b-5p antagomir + EA group was increased at days 1, 4, and 7 (p < 0.05) CONCLUSION: Let-7b-5p may be a new potential target for decreasing the EA tolerance effect and facilitating the application of EA in treating chronic nociception of patients.

miR-191 promotes radiation resistance of prostate cancer through interaction with RXRA.

Radiation therapy is a common treatment for prostate cancer, however recurrence remains a problem. MicroRNA expression is altered in prostate cancer and may promote therapy resistance. Through bioinformatic analyses of TCGA and CPC-GENE patient cohorts, we identified higher miR-191 expression in tumor versus normal tissue, and increased expression in higher Gleason scores. In vitro and in vivo experiments demonstrated that miR-191 overexpression promotes radiation survival, and contributes to a more aggressive phenotype. Retinoid X receptor alpha, RXRA, was discovered to be a novel target of miR-191, and knockdown recapitulated radioresistance. Furthermore, treatment of prostate cancer cells with the RXRA agonist 9-cis-retinoic acid restored radiosensitivity. Supporting this relationship, patients with high miR-191 and low RXRA abundance experienced quicker biochemical recurrence. Reduced RXRA translated to a higher risk of distant failure after radiotherapy. Notably, this miR-191/RXRA interaction was conserved in a novel primary cell line derived from radiorecurrent prostate cancer. Together, our findings demonstrate that miR-191 promotes prostate cancer survival after radiotherapy, and highlights retinoids as a potential option to improve radiotherapy response.

microRNA-431 as a Chemosensitizer and Potentiator of Drug Activity in Adrenocortical Carcinoma.

BACKGROUND: Adrenocortical carcinoma (ACC) is a rare endocrine cancer with treatments limited in efficacy for metastatic disease. New molecular targeted therapies have yet to improve patient outcomes. In contrast, established treatment regimens of adrenolytics and chemotherapy have demonstrated treatment benefit, although admittedly in a minority of patients. Identification of microRNAs (miRNAs) in patients responsive to adjuvant therapy may offer a means to sensitize patients with progressive disease to existing adjuvant regimens. MATERIALS AND METHODS: Samples from primary ACC tumors of 10 Stage IV patients were examined for differentially expressed miRNAs between a "sensitive" and "resistant" cohort. Candidate microRNAs were restored via transfection in two functional ACC cell lines. Gain of function and effects on apoptosis and cell cycle were assessed. RESULTS: microRNA-431 (miR-431) was underexpressed in patients with ACC with progressive disease undergoing adjuvant therapy. Restoration of miR-431 in vitro decreased the half maximal inhibitory concentrations of doxorubicin and mitotane, with markedly increased apoptosis. We found that a reversal of epithelial-mesenchymal transition underlies the action of miR-431 with doxorubicin treatment, with Zinc Finger E-Box Binding Homeobox 1 implicated as the molecular target of miR-431 in ACC. CONCLUSION: This is the first report of the potential of miRNA therapy to sensitize ACC to current established adjuvant therapy regimens, which may mitigate the resistance underlying treatment failure in patients with advanced ACC. Effective and well-studied methods of targeted miRNA delivery in existence hints at the imminent translatability of these findings. IMPLICATIONS FOR PRACTICE: Adrenocortical carcinoma (ACC) is a rare endocrine cancer with outcomes not improving despite extensive research and new targeted therapies. Mitotane and etoposide/doxorubicin/cisplatin chemotherapy is trial validated for improved recurrence-free survival. However, a minority of patients experience sustained benefit. Significant side effects exist for this regimen, with patients often unable to attain target drug doses shown to give survival benefit. This preclinical study examines the role of microRNAs in sensitizing ACC to doxorubicin or mitotane. This study offers an important bridge between new and existing cancer treatments, offering an imminently translatable approach to the treatment of adrenocortical carcinoma.

Total flavonoids from Smilax glabra Roxb blocks epithelial-mesenchymal transition and inhibits renal interstitial fibrosis by targeting miR-21/PTEN signaling.

BACKGROUND: Smilax glabra Roxb, a traditional Chinese herb, has been widely used in folk medicine. The current study was performed to investigate the protective effect of S. glabra Roxb extract, pure total flavonoids from Smilax glabra Roxb (PTFS), on renal interstitial fibrosis (RIF) and its underlying mechanism. METHODS: First, a surgical model of unilateral ureteral obstruction was established in rats to induce RIF. Then, rats were grouped and treated with PTFS at different concentration. Second, HK-2 cells underwent an epithelial-mesenchymal transition (EMT) by the addition of transforming growth factor-ß1 (TGF-ß1). Additionally, HK-2 cells after inducing for EMT were transfected with microRNA-21 (miR-21) mimic or inhibitor. These HK-2 cells were grouped and treated with PTFS at different concentration. Finally, real-time polymerase chain reaction and Western blot analysis were performed to detect the expression of possible signaling factor involved in RIF in renal tissues or HK-2 cells after PTFS treatment. RESULTS: In vivo and in vitro experiments indicated that PTFS treatment could decrease the expression of α-smooth muscle actin (α-SMA; mesenchymal marker) and increase the expression of E-cadherin (epithelial marker) in both messenger RNA and protein level. Moreover, PTFS also attenuated the expression of TGF-ß1/Smad signaling in both renal tissues and HK-2 cells that underwent EMT. Overexpression or inhibition of miR-21 in HK-2 cells activated or blocked the PI3K/Akt signaling via targeting phosphatase and tension homolog (PTEN), and then promoted or suppressed the progress of TGF-ß1-induced EMT by regulating the expression of α-SMA and E-cadherin. Furthermore, PTFS treatment inhibited TGF-ß1-induced EMT progress by blocking miR-21/PTEN/PI3K/Akt signaling. CONCLUSION: PTFS has strong anti-EMT and antifibrosis effects both in vitro and in vivo. The mechanism underlying these effects may be related to inhibition of TGF-ß1/Smad, and their downstream miR-21/PTEN signaling, leading to blocks of EMT process during RIF.

BRM270 inhibits cancer stem cell maintenance via microRNA regulation in chemoresistant A549 lung adenocarcinoma cells.

Chemotherapy is a standard treatment for non-small-cell lung cancer (NSCLC). However, the dose-limiting toxicity of drugs and the development of chemoresistance are major clinical challenges to successful management of NSCLC. Asian traditional medicine is gaining global attention as a non-toxic alternative to chemotherapy. BRM270 is an extract formulated from seven Asian medicinal plants that has been shown to inhibit tumor cell proliferation in diverse cancer types. We previously demonstrated that BRM270 suppresses tumorigenesis by negatively regulating nuclear factor-κB signaling in multidrug-resistant cancer stem cells (CSCs). In this study we report that the growth, migration, and invasion of normal human lung adenocarcinoma cells and their chemoresistant derivatives was inhibited by BRM270 treatment. Notably, BRM270 was found to modulate CSC self-renewal and tumor-initiating capacity via positive regulation of the miRNA-128. Thus, combination therapy with miRNA-128 and BRM270 may be an effective treatment strategy for chemoresistant NSCLC.

Genipin alleviates high-fat diet-induced hyperlipidemia and hepatic lipid accumulation in mice via miR-142a-5p/SREBP-1c axis.

Hyperlipidemia is a chronic disorder which plays an important role in the development of cardiovascular diseases, type 2 diabetes, atherosclerosis, hypertension, and nonalcoholic fatty liver disease. Genipin (GNP) is a metabolite from genipioside, which is an active component of the traditional Chinese medicine Gardenia jasminoides Ellis, and has been recognized as a beneficial compound against metabolic disorders. However, whether it can correct overnutrition-induced dyslipidemia is still unknown. In this study, the effects of GNP on attenuating hyperlipidemia and hepatic lipid accumulation were investigated using normal and obese mice induced with a high-fat diet (HFD) and primary hepatocytes treated with free fatty acids. We also sought to identify potential targets of GNP to mediate its effects in the liver. We found that obese mice treated with GNP showed a decrease in the body weight, serum lipid levels, as well as hepatic lipid accumulation. Besides, GNP regulated hepatic expression levels of lipid metabolic genes, which are important in maintaining systemic lipid homeostasis. At the molecular level, GNP increased the expression levels of miR-142a-5p, which bound to 3' untranslated region of Srebp-1c, an important regulator of lipogenesis, which thus led to the inhibition of lipogenesis. Collectively, our data demonstrated that GNP effectively antagonized HFD-induced hyperlipidemia and hepatic lipid accumulation in mice. Such effects were achieved by regulating miR-142a-5p/SREBP-1c axis.

Curcumin and Lung Cancer: the Role of microRNAs.

BACKGROUND: Lung cancer is one of the most common types of cancer worldwide and is characterized by a poor prognosis, related to both late diagnosis and lack of effective treatments. In the last years, microRNAs (miRNAs) have been demonstrated to have an important role in tumor microenvironment and immune regulation. These RNAs can be categorized into tumor-suppressor genes, such as let-7 family and miR-34, and oncogenes such as miR-221 and miR-222. Curcumin is a bioactive polyphenol that is documented to have promising anticancer activity, and to be well tolerated in humans. METHODS: The present review aims to gather available evidence on the involvement of mRNAs in the therapeutic effects of curcumin against lung cancer. RESULTS: The anti-cancer properties of curcumin against lung cancer have been shown in both cellular and experimental models and are mediated by modulation of several molecular targets that regulate the expression of transcription factors, inflammatory cytokines, enzymes, growth factors, receptors, adhesion molecules, antiapoptotic proteins, and cell cycle proteins, leading to cell apoptosis, inhibition of cell proliferation and migration, and also chemo- and radio-sensitization of lung cancer cells. Recent studies have documented that pharmacological effects of curcumin in lung cancer are also mediated by modulation of several miRNAs, such as downregulation of oncogenic miR-21 and upregulation of oncosuppressive miR-192-5p and miR-215. CONCLUSION: Further studies are necessary to explore this very promising field and the link between regulation of oncogenic and tumor-suppressive miRNAs and putative anti-cancer properties of curcumin.

Increasing the miR-126 expression in the peripheral blood of patients with diabetic foot ulcers treated with maggot debridement therapy.

BACKGROUND: miR-126 may increase angiogenesis in patients with diabetic foot ulcers (DFUs) treated with maggot debridement therapy (MDT). METHODS: Real-time quantitative PCR was used to detect expression of miR-126 mRNA in the peripheral blood among the non-diabetic population, type 2 diabetes mellitus patients without DFU, and patients with DFUs of type 2 diabetes mellitus. The expression of miR-126 mRNA in the peripheral blood of patients with DFUs was observed before and after MDT. Finally, human umbilical vein endothelial cells (HUVEC) were utilized to explore miR-126 mRNA expression with maggot excretions/secretions (ES). RESULTS: In the patients with DFUs, the miR-126 mRNA expression level in the peripheral blood was less than that type 2 diabetes mellitus patients without DFU, and much lower than that in the non-diabetic population (P<0.001). The miR-126 expression level was significantly increased in those DFU patients treated with MDT (P<0.05). Finally, using HUVEC co-cultured with ES, we showed the ES increased miR-126 expression in vitro (P<0.001). CONCLUSION: MDT upregulates the miR-126 expression in the peripheral blood of patients with DFUs.

Tetrandrine induces microRNA differential expression in human hypertrophic scar fibroblasts in vitro.

MicroRNAs (miRNAs) have recently been shown to play a role in normal wound healing process. miRNAs may be linked to pathologic wound healing and closely related to the formation of hypertrophic scars. This study aimed to explore the effects of tetrandrine on the miRNA expression profile in human hypertrophic scar fibroblasts (HSFs) in vitro. HSFs were randomly divided into two groups: the tetrandrine treatment group and the control group. The experimental and control groups were collected and analyzed by miRNA array after a 48-h culture. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to confirm the array results. The targets of differentially expressed miRNA were functionally annotated using bioinformatic approaches. miRNA microarray analysis identified 193 differentially expressed miRNAs and the expression of 186 miRNAs in the experimental group decreased while that of 7 miRNAs increased compared to the control group. The most significantly downregulated miRNA was hsa-miR-1246, and hsa-miR-27b had the highest expression level. Significant differentially expressed miRNAs were predicted to be related to several important signaling pathways related to scar wound healing. The differential miRNA expression identified in this study provides the experimental basis for further understanding the anti-fibrosis effect of tetrandrine.

MicroRNAs as early toxicity signatures of doxorubicin in human-induced pluripotent stem cell-derived cardiomyocytes.

An in depth investigation at the genomic level is needed to identify early human-relevant cardiotoxicity biomarkers that are induced by drugs and environmental toxicants. The main objective of this study was to investigate the role of microRNAs (miRNAs) as cardiotoxicity biomarkers using human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) that were exposed to doxorubicin (DOX) as a "gold standard" cardiotoxicant. hiPSC-CMs were exposed to 156 nM DOX for 2 days or for 6 days of repeated exposure, followed by drug washout and incubation in drug-free culture medium up to day 14 after the onset of exposure. The induced miRNAs were profiled using miRNA microarrays, and the analysis of the data was performed using the miRWalk 2.0 and DAVID bioinformatics tools. DOX induced early deregulation of 14 miRNAs (10 up-regulated and 4 down-regulated) and persistent up-regulation of 5 miRNAs during drug washout. Computational miRNA gene target predictions suggested that several DOX-responsive miRNAs might regulate the mRNA expression of genes involved in cardiac contractile function. The hiPSC-CMs exposed to DOX in a range from 39 to 156 nM did not show a significant release of the cytotoxicity marker lactate dehydrogenase (LDH) compared to controls. Quantitative real-time PCR analyses confirmed the early deregulation of miR-187-3p, miR-182-5p, miR-486-3p, miR-486-5p, miR-34a-3p, miR-4423-3p, miR-34c-3p, miR-34c-5p and miR-1303, and also the prolonged up-regulation of miR-182-5p, miR-4423-3p and miR-34c-5p. Thus, we identified and validated miRNAs showing differential DOX-responsive expression before the occurrence of cytotoxicity markers such as LDH, and these miRNAs also demonstrated the significant involvement in heart failure in patients and animal models. These results suggest that the DOX-induced deregulated miRNAs in human CMs may be used as early sensitive cardiotoxicity biomarkers for screening potential drugs and environmental cardiotoxicants with a similar mechanism of action.

Osthole suppresses the proliferation and accelerates the apoptosis of human glioma cells via the upregulation of microRNA-16 and downregulation of MMP-9.

Osthole (7-methoxy-8-isoamyl alkenyl coumarin) has been reported to exhibit marked anticancer effects on several types of cancer. The expression levels of matrix metalloproteinase-9 (MMP-9) are closely associated with the pathogenesis of glioma. Furthermore, it is reported that the upregulation of microRNA­16 (miR­16) by the MMP­9 signaling pathway can restrain the proliferation of cancer cells. To examine whether osthole increases the anticancer effect on human glioma cells in the present study, the common glioma cell line, U87, was treated with osthole at concentrations of 0, 50, 100 and 200 µΜ. The effects of osthole on cell viability were determined using a 3­(4,5­dimethylthiazol­2­thiazolyl)­2,5­diphenyl­tetrazolium bromide assay. The rate of cellular apoptosis was analyzed by measuring the activity of caspase­3 and using flow cytometry. The expression of MMP­9 was determined using gelatin zymography assays and the expression of miR­16 was determined using reverse transcription­quantitative polymerase chain reaction. The results demonstrated that osthole significantly suppressed the proliferation and accelerated the apoptosis of the U87 cells. Furthermore, increased expression levels of miR­16 and reduced protein expression levels of MMP­9 were found in the U87 cells. In addition, miR­16 was found to regulate the expression of MMP­9 in the U87 cells through transfection of miR­16 precursor and anti­miR­16 into the U87 cells. In conclusion, these observations indicated that osthole suppressed the proliferation and accelerated the apoptosis of human glioma cells through upregulation of the expression of miR­16 and downregulation of the expression of MMP-9.

Polyphenolic extracts from cowpea (Vigna unguiculata) protect colonic myofibroblasts (CCD18Co cells) from lipopolysaccharide (LPS)-induced inflammation--modulation of microRNA 126.

Cowpea (Vigna unguiculata) is a drought tolerant crop with several agronomic advantages over other legumes. This study evaluated varieties from four major cowpea phenotypes (black, red, light brown and white) containing different phenolic profiles for their anti-inflammatory property on non-malignant colonic myofibroblasts (CCD18Co) cells challenged with an endotoxin (lipopolysaccharide, LPS). Intracellular reactive oxygen species (ROS) assay on the LPS-stimulated cells revealed antioxidative potential of black and red cowpea varieties. Real-time qRT-PCR analysis in LPS-stimulated cells revealed down-regulation of proinflammatory cytokines (IL-8, TNF-α, VCAM-1), transcription factor NF-κB and modulation of microRNA-126 (specific post-transcriptional regulator of VCAM-1) by cowpea polyphenolics. The ability of cowpea polyphenols to modulate miR-126 signaling and its target gene VCAM-1 were studied in LPS-stimulated endothelial cells transfected with a specific inhibitor of miR-126, and treated with 10 mg GAE/L black cowpea extract where the extract in part reversed the effect of the miR-126 inhibitor. This suggests that cowpea may exert their anti-inflammatory activities at least in part through induction of miR-126 that then down-regulate VCAM-1 mRNA and protein expressions. Overall, Cowpea therefore is promising as an anti-inflammatory dietary component.