Endoplasmic reticulum stress related factor IRE1α regulates TXNIP/NLRP3-mediated pyroptosis in diabetic nephropathy.

The nod-like receptor protein-3 (NLRP3)-mediated pyroptosis is involved in kidney diseases. Thioredoxin interacting protein (TXNIP) directly interacts with NLRP3. This study aimed to probe the mechanism of TXNIP and NLRP3 pathway in diabetic nephropathy (DN). Marker detection and histological staining indicated that in DN rats, the renal function was destroyed, and the TXNIP/NLRP3 axis was activated to induce inflammatory generation and pyroptosis. The protein levels of TXNIP, NLRP3 inflammatory components and endoplasmic reticulum stress (ERS)-related factors (ATF4, CHOP and IRE1α) were measured. DN rats were injected with LV-TXNIP-shRNA or IRE1α RNase specific inhibitor (STF-083010) to examine ERS- and pyroptosis-related proteins, and renal injury. Silencing TXNIP inhibited the NLRP3 axis and reduced renal damage in DN rats. ERS was activated in DN rats, and miR-200a expression was degraded by IRE1α. miR-200a bound to TXNIP. NRK-52E cells were induced by high glucose (HG) to simulate DN in vitro. The damage and pyroptosis of NRK-52E cells were analyzed. After inhibiting IRE1α, miR-200a expression increased and TXNIP expression decreased. miR-200a inhibition in HG-induced NRK-52E cells partially reversed the reduced pyroptosis by STF-083010. Overall, IRE1α upregulates miR-200a degradation in DN rats, and stimulates the TXINP/NLRP3 pathway-mediated pyroptosis and renal damage.

Upregulation of microRNA-200a in bone marrow mesenchymal stem cells enhances the repair of spinal cord injury in rats by reducing oxidative stress and regulating Keap1/Nrf2 pathway.

Spinal cord injury (SCI) is a common disease with high incidence, disability rate and treatment cost. microRNA (miR)-200a is reported to inhibit Keap1 to activate Nrf2 signaling. This study aimed to explore the effects of lentivirus-mediated miR-200a gene-modified bone marrow mesenchymal stem cells (BMSCs) transplantation on the repair of SCI in a rat model. BMSCs were isolated from the bone marrow of Sprague-Dawley rats. MiR-200a targeting to Keap1 was identified by luciferase reporter gene assay. The expressions of Keap1, nuclear factor erythroid 2-related factor 2 (Nrf2), NAD(P)H-dependent quinone oxidoreductase 1 (NQO-1), heme oxygenase-1 (HO-1) and glutamate-cysteine ligase catalytic subunit (GCLC) were detected by Western blotting in SCI rats. The locomotor capacity of the rats was evaluated using the Basso, Beattie, and Bresnahan scale. The levels of malondialdehyde (MDA), activities of superoxide dismutase (SOD), and catalase (CAT) were measured. miR-200a inhibited Keap-1 3' UTR activity in BMSCs. Transplantation of BMSCs with overexpression of miR-200a or si-Keap1 increased locomotor function recovery of rats after SCI, while decreased MDA level, increased SOD, CAT activities, and Nrf2 expression together with its downstream HO-1, NQO1, GCLC protein expressions in SCI rat. These results indicated that overexpressed miR-200a in BMSCs promoted SCI repair, which may be through regulating antioxidative signaling pathway.

miR-200a inhibits proliferation rate in drug-induced gingival overgrowth through targeting ZEB2.

BACKGROUND/PURPOSE: Gingival overgrowth can occur as a result of poor oral hygiene or a side effect of taking certain medications, such as cyclosporine A (CsA). It has been shown that this immunosuppressant drug induces epithelial-to-mesenchymal transition (EMT) in the gingival epithelium but the associated molecular mechanism remains to be elucidated. METHODS: We first assessed the relative expression of microRNA-200a (miR-200a) in response to the CsA treatment using qRT-PCR. Next, luciferase reporter assay was applied to examine whether miR-200a was able to regulate ZEB2 and Western blot was utilized to measure the expression of ZEB2 in normal human gingival fibroblasts (HGFs). To confirm the significance of miR-200a and ZEB2 in the CsA-induced gingival overgrowth, miR-200a inhibitor and shRNA mediated knockdown of ZEB2 were used and cell proliferation in HGFs was assessed by MTT assay. RESULTS: The expression of miR-200a was dose-dependently downregulated following the CsA treatment. Luciferase reporter assay confirmed that ZEB2 was a direct downstream target regulated by miR-200a and ZEB2 was indeed increased after the administration of CsA. We demonstrated that knockdown of ZEB2 hampered the CsA-induced HGFs proliferation and the elevated cell proliferation due to inhibition of miR-200a was reversed by repression of ZEB2. CONCLUSION: Our results showed that insufficient miR-200a in HGFs caused by CsA administration may lead to gingival enlargement mediated by the upregulation of ZEB2. This finding supported that CsA-induced EMT contributed to the adverse effect of using CsA and miR-200a may serve as an upstream target to prevent the overgrowth of the gingiva.

Inhibition of microRNA-200a Upregulates the Expression of Striatal Dopamine Receptor D2 to Repress Apoptosis of Striatum via the cAMP/PKA Signaling Pathway in Rats with Parkinson's Disease.

BACKGROUND/AIMS: Parkinson's disease (PD) is a neurodegenerative movement disease with a high annual incidence. Accumulating evidence demonstrates that microRNAs play important roles in the pathogenesis of multiple neurological disorders, including PD. This study aims to investigate how microRNA-200a (miR-200a) regulates striatal dopamine receptor D2 (DRD2) to affect apoptosis of striatum in rats with PD and to explore the associated mechanism. METHODS: After successfully establishing a PD model by 6-hydroxydopamine injections, PD rats were mainly treated with miR-200a mimics, inhibitors, Forskolin or a combination of miR-200a inhibitors and Forskolin. High-performance liquid chromatography-electrochemical detection (HPLC-ECD) was employed to detect the levels of dopamine, 3, 4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and chemistry colorimetric methods were applied to detect the levels of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). A TUNEL assay and immunocytochemical staining were performed to observe apoptosis and tyrosine hydroxylase (TH)-positive cells in the striatum. The expression of miR-200a, DRD2, Bad, Bax, Bcl-2, cAMP and PKA was determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot assays. RESULTS: In the cellular experiments, after transfection with the inhibitor of miR-200a, decreased levels of Bax, GSH-Px, SOD, dopamine, DOPAC and HVA but increased levels of MDA and Bcl-2 were found along with a reduced apoptosis rate and increased TH-positive cell number. In addition, downregulating miR-200a resulted in lower expression of AKT, cAMP and PKA but higher expression of DRD2 and CREB, indicating that the downregulation of miR-200a increases DRD2 expression, which blocks the cAMP/PKA signaling pathway. CONCLUSION: This study provides evidence that the inhibition of miR-200a can repress apoptosis in the striatum via inhibition of the cAMP/PKA signaling pathway by upregulating DRD2 expression in PD rats.

MicroRNA-200a Affects the Proliferation of Airway Smooth Muscle Cells and Airway Remodeling by Targeting FOXC1 via the PI3K/AKT Signaling Pathway in Ovalbumin-Induced Asthmatic Mice.

BACKGROUND/AIMS: The etiology of asthma, which is a complicated disorder with various symptoms, including wheezing, coughing, and airflow obstruction, remains poorly understood. In addition, the effects of microRNAs (miRs) have not been explored. This study explored the effect of microRNA-200a (miR-200a) on airway smooth muscle cells (ASMCs) and airway remodeling in asthmatic mice. Furthermore, we speculated that miR-200a achieves its effect by targeting FOXC1 via the PI3K/AKT signaling pathway based on differentially expressed gene screening, target miR predictions and a bioinformatics analysis. METHODS: Eighty mice were assigned to a saline group or an ovalbumin (OVA) group, and the OVA group was transfected with a series of inhibitors, activators, and siRNAs to test the established mouse model. Airway reactivity and the ratio of eosinophils (EOSs) to leukocytes were detected. An ELISA was adopted to measure the levels of interleukin (IL)-4, IL-6, IL-8, tumor necrosis factor (TNF)-α, and immunoglobulin E (IgE). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting were used to determine the expression of FOXC1, PI3K, AKT, NF-κB, cyclin D1, TGF-ß1 and p-AKT in ASMCs. Finally, CCK-8 assays were performed to detect cell proliferation and flow cytometry to detect apoptosis and cell cycle entry. RESULTS: The bioinformatics analysis indicated that miR-200a mediated the PI3K/AKT signaling pathway by targeting FOXC1. In addition, mouse models of asthma were established. An elevated expression of miR-200a, a decreased mRNA and protein expression of FOXC1, PI3K, AKT, NF-κB, cyclin D1 and TGF-ß1, a decreased expression of p-AKT, suppressed cell proliferation, accelerated apoptosis, and an increased number of cells at the G0/G1 phase were observed following the upregulation of miR-200a and downregulation of FOXC1. CONCLUSION: The overexpression of miR-200a may downregulate FOXC1, thereby inhibiting the activation of the PI3K/AKT signaling pathway and ultimately suppressing ASMC proliferation and airway remodeling in asthmatic mice. This evidence supports the potential that miR-200a represents a new approach to treating asthma.

MicroRNA 200a inhibits erythroid differentiation by targeting PDCD4 and THRB.

Previous studies on erythropoiesis revealed that microRNAs (miRNAs) play a critical role in erythroid differentiation. Given the abundance of identified miRNAs and the limited understanding of erythroid miRNAs, additional examination is required. Here, two sets of erythroid differentiation miRNome data were analysed to screen for novel erythroid-inhibiting miRNAs. MIR200A was selected based on its pattern of downregulated expression in the miRNome datasets during induction of erythroid differentiation. Overexpression of MIR200A in K562 and TF-1 cells confirmed its inhibitory role in erythroid differentiation. Further in vivo study indicated that overexpression of mir200a inhibited primitive erythropoiesis of zebrafish. Transcriptome analyses after MIR200A overexpression in TF-1 cells indicated a significant role in regulating erythroid function and revealed potential regulation networks. Additionally, bioinformatics and experimental analyses confirmed that PDCD4 (programmed cell death 4) and THRB (thyroid hormone receptor, beta) are both targets of MIR200A-3p. Gain- and loss-of-function studies of PDCD4 and THRB revealed that the two targets were capable of promoting erythroid gene expression. Overall, our results revealed that microRNA 200a inhibits erythroid differentiation by targeting PDCD4 and THRB.

miR-200a controls hepatic stellate cell activation and fibrosis via SIRT1/Notch1 signal pathway.

OBJECTIVES: miR-200a has been established as a key regulator of HSC activation processes in liver fibrosis. Epigenetic silencing of miR-200a contributing to SIRT1 over-expression has been discussed in breast cancer; however, whether miR-200a controls SIRT1 gene expression in hepatic fibrosis is still unknown. METHODS AND MATERIALS: We analyzed miR-200a regulation of SIRT1 expression in CCl4-induced liver fibrosis and TGF-ß1-mediated activation of HSC. miR-200a, SIRT1, α-SMA, Col1A1, Notch1 and NICD expression were estimated by Western blotting, qRT-PCR and Immunohistochemistry. HSCs were transfected with miR-200a mimic, miR-200a inhibitor and SIRT1-RNAi. Luciferase reporter assays further confirmed the interaction between miR-200a and the SIRT1 mRNA 3'-UTR. Cell proliferation ability was assessed by MTT and cell cycle. RESULTS: We found that treatment activated HSC with miR-200a mimics, restored miR-200a expression and reduced SIRT1 levels. Conversely, treatment activated HSC with miR-200a inhibitors, decreased miR-200a expression and up-regulated SIRT1 levels. Restoration of miR-200a or the knockdown of SIRT1 prevented HSC activation and proliferation. We have established the SIRT1 transcript as subject to regulation by miR-200a, through miR-200a targeting of SIRT1 3'-UTR. Finally, HSC transfected with SIRT1-siRNA increased the levels of Notch1 protein and mRNA expression. CONCLUSIONS: Our study demonstrated that miR-200a regulates SIRT1/Notch1 expression during HSC activation and fibrosis.

Shear-Sensitive circRNA-LONP2 Promotes Endothelial Inflammation and Atherosclerosis by Targeting NRF2/HO1 Signaling.

Hemodynamic shear stress is a frictional force that acts on vascular endothelial cells and is essential for endothelial homeostasis. Physiological laminar shear stress (LSS) suppresses endothelial inflammation and protects arteries from atherosclerosis. Herein, we screened differentially expressed circular RNAs (circRNAs) that were significantly altered in LSS-stimulated endothelial cells and found that circRNA-LONP2 was involved in modulating the flow-dependent inflammatory response. Furthermore, endothelial circRNA-LONP2 overexpression promoted endothelial inflammation and atherosclerosis in vitro and in vivo. Mechanistically, circRNA-LONP2 competitively sponged miR-200a-3p and subsequently promoted Kelch-like ECH-associated protein 1, Yes-associated protein 1, and enhancer of zeste homolog 2 expression, thereby inactivating nuclear factor erythroid 2-related factor 2/heme oxygenase-1 signaling, promoting oxidative stress and endothelial inflammation, and accelerating atherosclerosis. LSS-induced down-regulation of circRNA-LONP2 suppresses endothelial inflammation, at least in part, by activating the miR-200a-3p-mediated nuclear factor erythroid 2-related factor 2/heme oxygenase-1 signaling pathway. CircRNA-LONP2 may serve as a new therapeutic target for atherosclerosis.

The implication of miR-200a and miR-132 expression and their correlations with NF-κB/TNF-alpha signaling in adults with diabetic nephropathy.

Aim: The primary factor causing chronic renal failure is diabetic nephropathy (DN) worldwide. However, the current biomarkers for DN have limited diagnostic utility. Thus, this work aimed to clarify the implications of microRNA-200a (miR-200a) and microRNA-132 (miR-132) and their correlation with NF-κB (nuclear factor- kappa beta), and, TNF-α (tumor necrosis factor -alpha) signaling to identify biomarkers able to distinguish late-stage from early- stage DN. Methods: Fifty healthy controls, and 271 type 2 diabetic (T2D) patients (166 male plus 105 female) were enrolled. Participants were stratified into seven groups according to along with the estimated glomerular filtration rate (eGFR), glycated hemoglobin (HbA1c%), healthy controls, diabetes without DN (G1), diabetes with mild renal impairment (G2), and four DN grades (G3a, G3b, G4, and G5). Results: Compared to healthy controls, the DN groups exhibited linear increases in serum miR-200a, TNF-α, NF-κB, matrix metalloproteinase (MMP-9) and interleukin-6 (IL-6) levels and reductions in miR-132 serum expression. Among the patients, NF-κB and TNF-α produced a negative correlation with miR-132, while, positive correlation has been discovered with miR-200-a. The operating characteristic of the receiver curve (ROC), proved that, miR-200a also miR-132 had good diagnostic performance in distinguishing early from advanced DN. Conclusion: MiR-200a as well as miR-132 expression levels, and their correlations with NF-κB/TNF-alpha signaling, were able to differentiate between DN patients with lower eGFR, suggesting their utility as diagnostic and prognostic biomarkers.

MicroRNA 200a as a histologically independent marker for meningioma recurrence: Results of a four microRNA panel analysis in meningiomas.

INTRODUCTION: Meningiomas are mostly benign neoplasms of the central nervous system. Nevertheless there are recurrences in about 20% after surgical resection. Previous studies could reveal several predictors of meningioma recurrence. Tumor progression often is associated with a specific pattern of chromosome losses. Our study investigated the potential function of selected microRNAs as markers of tumor progression. METHODS: By real-time polymerase chain reaction the expressions of microRNA 21-3p, 34a-3p, 200a-3p, and 409-3p were analyzed in solid tumor and in blood samples of 51 meningioma patients as well as in blood samples of 20 healthy individuals. Additionally, aberrations of parts of chromosomes 1, 14, 18, and 22 were analyzed by FISH. Tumor and blood samples were statistically analyzed, using Spearman's rank correlation coefficient as well as Mann-Whitney U- and Kruskal-Wallis-Test. RESULTS: MicroRNA 200a showed significantly lower expressions in recurrent meningiomas than in newly diagnosed ones. MicroRNA 409 in meningiomas was correlated significantly with tumor volume and showed a significant negative correlation with patient age. Significance was found between the expression patterns of microRNAs 34a and 200a with the respective aberrations of chromosome 1p and the microRNA 409 with aberration of chromosome 14. In the male cohort the expression of microRNA 200a in blood was significantly upregulated in patients compared to healthy volunteers. By our research the function of microRNA 200a was proved to detect meningioma patients by liquid biopsy. CONCLUSION: We detected microRNA 200a as a new biomarker to indicate meningioma recurrences. Future transferability to blood could be important for patient follow-up.

MicroRNA-200a regulates skin pigmentation by targeting WNT5A and FZD4 in Cashmere goats.

MicroRNAs are small, non-coding RNAs that regulate the expression of target genes. Previous research has demonstrated that microRNA-200a regulates cell apoptosis, tumor progression, and autoimmune disease. Preliminary studies found that microRNA-200a was differently expressed in the skin of Cashmere goats of various coat colors. However, the role of microRNA-200a in skin pigmentation remained poorly understood. In the current study, we investigated the effect of microRNA-200a on pigmentation in Cashmere goats. The expression of target genes was detected by real-time quantitative PCR, western blot, and immunohistochemistry staining both in vivo and in vitro. Luciferase reporter assays were used to demonstrate the relationship between microRNA-200a and its target genes Wnt family member 5A and frizzled class receptor 4 (WNT5A and FZD4) in HEK293T cells. BALB/c mice were injected with antagomiR-200a to detect melanin content and the expression of microRNA-200a and its target genes. The results demonstrated that the expression of microRNA-200a was significantly higher in brown tissue. Luciferase reporter assays confirmed that microRNA-200a targeted WNT5A and FZD4. The expression of WNT5A and FZD4 in the skin of brown Cashmere goats was significantly lower than that in white Cashmere goats by the detection of mRNA and protein levels. Overexpression/inhibition of microRNA-200a in keratinocytes decreased/increased the mRNA and protein expression of WNT5A and FZD4, respectively. In addition, the expression of WNT5A and FZD4 increased in the skin of BALB/c mice injected with antagomiR-200a, but the melanin content decreased. In summary, this study indicated that microRNA-200a regulates skin pigmentation by targeting WNT5A and FZD4 in Cashmere goats.

MicroRNA-200a-3p and GATA6 are abnormally expressed in patients with non-small cell lung cancer and exhibit high clinical diagnostic efficacy.

Lung cancer is one of the main threats to human health. Survival of patients with lung cancer depends on timely detection and diagnosis. Among the genetic irregularities that control cancer development and progression, there are microRNAs (miRNAs/miRs). The present study aimed to investigate the expression patterns of miR-200a-3p and transcription factor GATA-6 (GATA6) in peripheral blood of patients with non-small cell lung cancer (NSCLC) and their clinical significance. The expression patterns of miR-200a-3p and GATA6 in the peripheral blood of patients with NSCLC and healthy subjects were measured via reverse transcription-quantitative PCR. The correlation between GATA6/miR-200a-3p expression and their diagnostic efficacy were analyzed by receiver operating characteristic curve analysis. The association between miR-200a-3p/GATA6 expression with the patient clinicopathological characteristics, and their correlation with carcinoembryonic antigen (CEA), neuron specific enolase (NSE) and squamous cell carcinoma antigen (SCCAg) were evaluated. The cumulative survival rate was examined, and whether miR-200a-3p and GATA6 expression levels were independently correlated with the prognosis of NSCLC was analyzed using multivariate logistic regression model. The results demonstrated that the expression of miR-200a-3p was high and that of GATA6 was low in the peripheral blood of patients with NSCLC, and both exhibited high clinical diagnostic efficacy. miR-200a-3p was revealed to target GATA6 by dual-luciferase assay. miR-200a-3p in the peripheral blood was correlated with TNM stage, lymph node metastasis and distal metastasis, while GATA6 in the peripheral blood was correlated with TNM stage and lymph node metastasis. miR-200a-3p and GATA6 were positively correlated with CEA and SCCAg, but not with NSE. High expression of miR-200a-3p and low expression of GATA6 predicted poor prognosis in patients with NSCLC. After adjusting for TNM stage, lymph node metastasis, distance metastasis, GATA6, CEA, NSE and SCCAg in the logistic regression model, it was indicated that the high expression of miR-200a-3p increased the risk of death in patients with NSCLC. Collectively, it was revealed that miR-200a-3p and GATA6 were abnormally expressed in the peripheral blood of patients with NSCLC. Serum levels of miR-200a-3p >1.475 and GATA6 <1.195 may assist the early diagnosis of NSCLC. GATA6 may function in NSCLC as a miR-200a-3p target, which may provide a future reference for NSCLC early diagnosis and treatment.

MicroRNA-200a and microRNA-141 have a synergetic effect on the suppression of epithelial-mesenchymal transition in liver cancer by targeting STAT4.

MicroRNAs (miRNAs or miRs) are non-coding small RNAs that target specific messenger RNAs to inhibit protein translation. miR-200a and miR-141 function as tumor suppressors by targeting STAT4. These two miRNAs belong to the same family, and their expression is often decreased in various cancer types, but are located on different chromosomes of the human genome. The present study showed that the expression levels of miR-141 and miR-200a in serum and cells of liver cancer are significantly downregulated. The expression levels of miR-141 and miR-200a are closely associated with clinicopathological features of liver cancer, especially metastasis and invasion. It is first reported that STAT4 is the new common target gene of miR-141 and miR-200a. In the present study, miR-141 and miR-200a were confirmed to inhibit the expression of E-cadherin and vimentin synergistically during epithelial-mesenchymal transition to regulate the proliferation, migration and invasion of liver cancer cells by targeting STAT4. Simultaneous overexpression of miR-200a and miR-141 resulted in stronger effects compared with each miRNA alone. In addition, overexpression of STAT4 significantly reversed the tumor suppressive roles of miR-200a and miR-141 in liver cancer cells. These findings enrich the tumor suppressor mechanisms of the miR-200 family, and may also provide new experimental and theoretical basis for the use of miRNAs for early diagnosis, prognosis and thorough treatment of liver cancer.

Upregulated LINC00319 aggravates neuronal injury induced by oxygen-glucose deprivation via modulating miR-200a-3p.

Ischemic stroke is one of the main causes of physical disability and mortality worldwide. Long non-coding RNAs (lncRNAs) are reported to be dysregulated in various biological progressions and serve important roles in pathological processes of cerebral ischemia. However, their biological actions and potential mechanisms in the progression of ischemic stroke remain unknown. The present study aimed to investigate the functions of LINC00319 on ischemic brain injury. It was identified that LINC00319 was significantly upregulated in the Gene Expression Omnibus profile of ischemic stroke. Furthermore, LINC00319 overexpression elevated caspase-3 activity and increased the apoptotic rate of neuronal cells, as well as decreased cell viability and glucose uptake. It was also demonstrated that LINC00319 participated in oxygen-glucose deprivation (OGD)-induced cerebral ischemic injury. LINC00319 could competitively bind with microRNA (miR)-200a-3p and decrease its expression. Moreover, miR-200a-3p could partly offset the negative effects of LINC00319 overexpression on neuronal injury caused by OGD. Collectively, the present results suggested that LINC00319 promoted apoptosis and aggravated neuronal injury induced by OGD by regulating miR-200a-3p, which may be important for ischemic stroke treatment.

Protective Role of microRNA-200a in Diabetic Retinopathy Through Downregulation of PDLIM1.

BACKGROUND: Diabetic retinopathy (DR) is a most common microvascular complication and regarded as the leading cause of blindness in the working age population. The involvement of miR-200a in various disorders has become recognized, and the objective of this study was to identify the protective effect of miR-200a in the development of DR. METHODS: The contents of miR-200a and its potential target gene, PDZ and LIM domain protein 1 (PDLIM1), were detected in both in-vivo and in-vitro DR models. Retinal leakage and inflammatory factor concentrations were detected after vitreous injections of miR-200a/PDLIM1 vectors in mice. The cellular viability, apoptosis and cellular migration were investigated using trypan blue staining, flow cytometry and transwell assay with human retinal microvascular endothelial cells (HRMECs). Besides, the prediction and confirmation of miR-200a targeting PDLIM1 were conducted with bioinformation analyses and dual-luciferase reporter assay. RESULTS: Lower miR-200a and higher PDLIM1 levels were detected in both in-vivo and in-vitro DR models. Besides, it was found that miR-200a treatment would significantly inhibit retinal permeability and inflammatory factors. Through targeting PDLIM1, it was found that miR-200a could improve cellular viability, remit apoptotic status and reduce cellular migration significantly in high glucose-treated HRMECs. CONCLUSION: Our results demonstrated that miR-200a could be used as a potential therapy target through down-regulating PDLIM1 in DR.

MicroRNA-200a represses myocardial infarction-related cell death and inflammation by targeting the Keap1/Nrf2 and ß-catenin pathways.

BACKGROUND: Acute myocardial infarction (MI) is a main cause of emergency death in the world. MicroRNAs (miRs/miRNAs) are a series of small non-coding RNA molecules, which regulate cardiovascular disorders that involve MI. In this study, we explored the function of miR-200a in MI treatment. METHODS: We observed down-regulation of miR-200a levels and up-regulation of Keap1 and ß-catenin levels in H2O2-treated newborn murine ventricular cardiomyocytes (NMVCs) and the infarcted heart tissues of MI mouse models, compared to the non-treated NMVCs and normal heart tissues of healthy mice. RESULTS: CCK-8 and colony formation assays indicated the reduction in NMVC vitality due to H2O2 treatment and the recovery of cell vitality due to miR-200a overexpression, respectively. Flow cytometry with Annexin and PI staining indicated the inhibition of H2O2-triggered cell apoptosis through ectopically expressed miR-200a. Western blotting and ELISA analyses that detected pro-inflammatory cell factors [interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α] confirmed that miR-200a prevented H2O2-induced NMVC inflammation. Moreover, miR-200a inhibited up-regulation of Keap1 and ß-catenin expression in H2O2-treated NMVCs by directly binding with the 3'-UTR regions of both Keap1 and ß-catenin. Furthermore, overexpression of Keap1 and ß-cateninin in H2O2-treated NMVCs with recovered miR-200a elevated inflammation and apoptosis, respectively. CONCLUSION: The results showed that miR-200a expression was inhibited in murine cardiomyocytes due to H2O2 stress in MI cardiac tissues and overexpressed miR-200a could protect the cells from death by regulating the Keap1/Nrf2 and ß-catenin signal transduction pathways.

miR-200a-3p facilitates bladder cancer cell proliferation by targeting the A20 gene.

BACKGROUND: MicroRNAs (miRs) are endogenous, single-stranded, noncoding RNAs that are involved in various physiological processes, and the development and the progression of various types of cancer. Specifically, the role of miR-200a-3p has been implicated in various types of cancer in contributing to a diverse array of cancer types has been previously reported. The present study aimed to investigate the expression levels of miR-200a-3p in human bladder cancer, as well as its potential role in disease pathogenesis. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect the expression of has-mir-200a-3p and tumor necrosis factor α (TNF-α) induced protein 3 (A20) in tumor tissues and cell lines. Dual-luciferase reporter assay and combination with the expression intervention of hsa-mir-200a-3p and A20 in bladder cancer cell lines to clarify the binding relationship between hsa-mir-200a-3p and A20.After the expression intervention of hsa-mir-200a-3p and A20 in bladder cancer cells, the changes of cell proliferation, cell apoptosis, cell cycle, wound-healing ability and migration ability were detected by CCK8, flow cytometry, wound-healing and Transwell methods. Xenograft transplantation model was performed subcutaneously in nude mice by implantation of J82 and T24 cells, and then the bladder cancer growth curve was calculated from mice exposed to has-mir-200a-3p minic or minic-NC. RESULTS: Bladder cancer tissues demonstrated significantly upregulated miR-200a-3p expression levels. Moreover, increased miR-200a-3p expression was significantly associated with distant metastasis and advanced stage. In addition, compared with the miR-control (Ctr) group, miR-200a-3p overexpression promoted bladder cancer cell proliferation, migration, invasion, cell cycle, and release of inflammatory cytokines, but inhibited cell apoptosis. Mechanistically, A20 was identified as a target gene of miR-200a-3p in bladder cancer cell lines. Moreover, compared with the miR-Ctr group, the miR-200a-3p overexpression group exhibited significantly promoted tumor growth in vivo, and A20 overexpression blocked the promoting effect of miR-200a-3p on bladder cancer. CONCLUSIONS: The results of the present study indicated that miR-200a-3p might serve act as an oncogene in human bladder cancer by targeting a novel the gene A20 gene; therefore, miR-200a-3p and A20 might serve could serve as novel therapeutic targets for bladder cancer.

Downregulation of miR-200a Protects Mouse Leydig Cells Against Triptolide by Triggering Autophagy.

BACKGROUND: MicroRNAs play important roles in testicular development and spermatogenesis. Previous research has indicated that the level of miR-200a was significantly upregulated in patients with different spermatogenic impairments. However, the mechanism by which miR-200a regulated spermatogenic impairments remains unclear. METHODS: Leydig cells were treated with triptolide (TP) to mimic spermatogenic impairments. CCK-8 and flow cytometry were used to detect the proliferation and apoptosis in Leydig cells, respectively. In addition, Western blot assay was used to examine ATG7, ATG5, p62 protein levels in MLTC-1 cells. RESULTS: TP dose-dependently upregulated the expression of miR-200a in MLTC-1 cells. In addition, TP inhibited the proliferation of MLTC-1 cells via inducing apoptosis and oxidative stress; however, these phenomena were notably reversed by miR-200a antagomir. Furthermore, luciferase reporter assay identified that ATG7 was the direct binding target of miR-200a. TP treatment markedly inhibited the activation of autophagy in MLTC-1 cells via inhibition of ATG7. Conversely, downregulation of miR-200a significantly induced autophagy in TP-treated MLTC-1 cells by activation of ATG7. Meanwhile, the cell protective effects of miR-200a against TP were reversed by autophagy inhibitor 3MA, indicating that autophagy plays an important role. CONCLUSION: These results indicated that downregulation of miR-200a could protect MLTC-1 cells against TP by inducing autophagy. Therefore, miR-200a might serve as a new therapeutic target for the treatment of male hypogonadism.

MicroRNA-200a Inhibits Inflammation and Atherosclerotic Lesion Formation by Disrupting EZH2-Mediated Methylation of STAT3.

Endothelial inflammation and dysfunction are critical to the process of atherosclerosis. Emerging evidence demonstrates that upregulation of miR-200a reduces VCAM-1 expression and prevents monocytic cell adhesion onto the aortic endothelium. However, limited information is available about the role of microRNA-200a (miR-200a) in facilitating atherosclerotic lesion formation. We investigated the anti-inflammatory and anti-atherosclerotic actions of miR-200a. Human umbilical vein endothelial cells (HUVECs) were cultured in the presence of oxidized low-density lipoprotein (ox-LDL), and their viability and apoptosis were evaluated using CCK-8 assays and flow cytometric analysis. The enhancer of zeste homolog 2 (EZH2) promoter activity was evaluated in the presence of miR-200a by dual luciferase reporter gene assay. EZH2-mediated methylation of signal transducer and activator of transcription 3 (STAT3) was validated by ChIP and IP assays. ApoE-/- mice were given a 12-week high-fat diet and developed as in vivo atherosclerotic models. miR-200a was downregulated but EZH2 and HMGB1 were upregulated in ox-LDL-treated HUVECs and the aorta tissues of atherosclerotic mouse models. Elevated miR-200a was shown to protect HUVECs against ox-LDL-induced apoptosis and inflammation. EZH2 was verified as a target of miR-200a. The protective effects of miR-200a were abrogated upon an elevation of EZH2. EZH2 methylated STAT3 and enhanced STAT3 activity by increased tyrosine phosphorylation of STAT3, thereby increasing apoptosis and release of pro-inflammatory cytokines in ox-LDL-treated HUVECs. An anti-atherosclerotic role of miR-200a was also demonstrated in atherosclerotic mouse models. Our study demonstrates that miR-200a has anti-inflammatory and anti-atherosclerotic activities dependent on the EZH2/STAT3 signaling cascade.

Gli3 is a novel downstream target of miR­200a with an anti­ï¬brotic role for progression of liver fibrosis in vivo and in vitro.

GLI family zinc finger 3 (Gli3), as the upstream transcriptional activator of hedgehog signaling, has previously been demonstrated to participate in the process of liver fibrosis. The present study aimed to investigate the potential functions of microRNA (miR)­200a and Gli3 in the progression of liver fibrosis. The expression levels of miR­200a and Gli3 in cells and tissues were determined by PCR and western blotting; the interaction of Gli3 and miR­200a was evaluated by bioinformatics analysis and dual­luciferase reporter assay. miR­200a was significantly reduced in serum samples from clinical patients, liver tissues of a carbon tetrachloride (CCl4)­induced rat model and activated LX2 cells. The expression of α­smooth muscle actin (α­SMA) and albumin at the mRNA and protein levels was increased and decreased in LX2 cells, respectively. However, the expression levels of α­SMA and albumin were reversed and Gli3 expression was markedly decreased in LX2 cells when transfected with miR­200a mimics. In addition, the dual­-luciferase reporter assay confirmed the target interaction between miR­200a and Gli3. Finally, following the administration of miR­200a mimics to CCl4­induced rats, it was revealed that the alterations of α­SMA, albumin and Gli3 presented a similar trend to that in LX2 cells with miR­200a mimics transfection. Taken together, these results indicated that downregulation of miR­200a might enhance the formation of liver fibrosis, probably by targeting Gli3, and elevated miR­200a may attenuate the progression of liver fibrosis by suppressing Gli3. These findings suggested that miR­200a may function as a novel anti­ï¬brotic agent in liver fibrosis via inhibition of the expression of Gli3.