Knockdown Of miR-21 Research Articles

The long non-coding RNA ENST00000547547 reduces 5-fluorouracil resistance of colorectal cancer cells via competitive binding to microRNA-31.

Colorectal cancer (CRC) is one of the most common cancers and the third leading cause of cancer-related deaths due to its rapid progression and poor prognosis. 5-Fluorouracil (5-FU)-based chemotherapies are the standard treatment for locally advanced CRC. However, a considerable percentage of CRCs have inherent or acquired 5-FU resistance, which critically impedes clinical outcomes. In the present study, we reported that the expression level ENST00000547547 was downregulated in 5-FU-resistant CRC cells in comparison with the parental cells, While rising with the treatment of 5-FU in parental cells. Overexpression of ENST00000547547 promoted 5-FU-induced cell apoptosis and reduced the chemoresistance of 5-FU invitro. Moreover, we found that ENST00000547547 was a target of miR-31, as confirmed by dual-luciferase reporter assay and RNA immunoprecipitation assay. Notably, miR-31 was upregulated in 5-FU-resistant CRC cells, and knockdown of miR-31 increased the chemosensitivity of 5-FU-resistant CRC cells. Furthermore, we demonstrated that ENST00000547547 reduced the chemoresistance of 5-FU via competitive binding to miR-31 in 5-FU-resistant CRC cell lines. Collectively, our findings revealed that ENST00000547547 reduced chemoresistance in 5-FU of 5-FU-resistant CRC cells through competitive binding to miR-31 and has the potential to serve as a therapeutic target in CRC patients.

MicroRNA-126 deficiency enhanced the activation and function of CD4+ T cells by elevating IRS-1 pathway.

Recent evidence has shown that microRNA-126 (miR-126) has been involved in the development and function of immune cells, which contributed to the pathogenesis of related clinical diseases. However, the potential role of miR-126 in the development and function of CD4+ T cells remains largely unknown. Here we first found that the activation and proliferation, as well as the expression of interferon (IFN)-γ, of CD4+ T cells from miR-126 knock-down (KD) mice using the miRNA-sponge technique were enhanced significantly in vitro, compared with those in CD4+ T cells from wild-type (WT) mice. To monitor further the possible effect of miR-126 deficiency on the function of CD4+ T cells in vivo, we used dextran sulphate sodium (DSS)-induced murine model of acute autoimmune colitis and found that miR-126 deficiency could elevate the pathology of colitis. Importantly, the proportion of CD4+ T cells in splenocytes increased significantly in miR-126KD mice. Moreover, the expression levels of CD69 and CD44 on CD4+ T cells increased significantly and the expression level of CD62L decreased significantly. Of note, adoptive cell transfer assay showed that the pathology of colitis was more serious in carboxyfluorescein succinimidyl ester (CFSE)-labelled miR-126KD CD4+ T cell-transferred group, compared with that in the CFSE-labelled WT CD4+ T cells transferred group. Consistently, the expression levels of CD69 and CD44 on CFSE+ cells increased significantly. Furthermore, both the proliferation and IFN-γ secretion of CFSE+ cells also increased significantly in the CFSE-labelled miR-126KD CD4+ T cell-transferred group. Mechanistic evidence showed that the expression of insulin receptor substrate 1 (IRS-1), as a functional target of miR-126, was elevated in CD4+ T cells from miR-126KD mice, accompanied by altered transduction of the extracellular regulated kinase, protein B (AKT) and nuclear factor kappa B (NF-κB) pathway. Our data revealed a novel role in which miR-126 was an intrinsic regulator in the function of CD4+ T cells, which provided preliminary basis for exploring further the role of miR-126 in the development, function of CD4+ T cells and related clinical diseases.

MiR-221/222 promote chemoresistance to cisplatin in ovarian cancer cells by targeting PTEN/PI3K/AKT signaling pathway.

Cisplatin resistance is one of the main limitations in the treatment of ovarian cancer, and its mechanism has not been fully understood. The objectives of this study were to determine the role of miR-221/222 and its underlying mechanism in chemoresistance of ovarian cancer. We demonstrated that miR-221/222 expression levels were higher in A2780/CP cells compared with A2780 S cells. An in vitro cell viability assay showed that downregulation of miR-221/222 sensitized A2780/CP cells to cisplatin-induced cytotoxicity. Moreover, we found that knockdown of miR-221/222 by its specific inhibitors promoted the cisplatin-induced apoptosis in A2780/CP cells. Using bioinformatic analysis and luciferase reporter assay, miR-221/222 were found to directly target PTEN. Moreover, knockdown of miR-221/222 in A2780/CP cells significantly upregulated PTEN and downregulated PI3KCA and p-Akt expression. In conclusion, our results demonstrated that miR-221/222 induced cisplatin resistance by targeting PTEN mediated PI3K/Akt pathway in A2780/CP cells, suggesting that miR-221/222/PTEN/PI3K/Akt may be a promising prognostic and therapeutic target to overcome cisplatin resistance and treat ovarian cancer in the future.

Propofol Attenuates Inflammatory Response in LPS-Activated Microglia by Regulating the miR-155/SOCS1 Pathway.

Propofol is a widely used intravenous anesthetic agent with potential neuroprotective effect in diverse models of neuronal injury, including ischemic stroke and traumatic brain injury. However, few studies have been carried out to determine the effects and molecular mechanisms of propofol in classic microglial activation (M1 activation) related to neuronal injury. This study explored the anti-inflammatory effects of propofol in LPS-activated BV2 microglia. Propofol potently decreased the pro-inflammatory mediators, such as nitric oxide, TNF-α, and IL-6, at both the transcriptional and translational levels. Furthermore, propofol suppressed the expression of miR-155 in LPS-activated cells. Knockdown of miR-155 attenuated the anti-inflammatory effect of propofol in cells after LPS exposure. miR-155 was also confirmed as a negative regulator of SOCS1 expression. The inhibitory effect of propofol on LPS-induced inflammation involved the upregulation of SOCS1. Overall, these results suggest that propofol can suppress the neuroinflammatory response of microglia to LPS through the regulation of the miR-155/SOCS1 pathway.

Propofol-induced neurotoxicity in hESCs involved in activation of miR-206/PUMA signal pathway

Studies in developing animals have demonstrated that when anesthetic agents, such as propofol, are early administered in life, it can lead to neuronal cell death and learning disabilities. However, the mechanisms causing these effectsremains unknown. A recent report found that propofol could significantly upregulat miR-206 expression in the human ASCs. miR-206 could also induce apoptosis in human malignant cancers. Therefore, in this study, we hypothesized that propofol induces neurotoxicity in human embryonic stem cells (hESCs). hESCs were exposed to propofol (50μM) for 6hr and cell death was assessed using TUNEL staining, and cleaved caspase-3 expression. miR-206 was knocked down using antagomir. PUMA was knocked down using a small interfering RNA. microRNA-206 (miR-206) expression was assessed using quantitative reverse transcription polymerase chain reaction (qRT-PCR). PUMA protein expression was detected using western blot assay. hESCs exposed to propofol showed a significant increase in TUNEL positive cells and cleaved caspase-3 expression, followed by the upregulation of miR-206 and PUMA expression. Targeting PUMA inhibits propofol-induced cell apoptosis; miR-206 knockdown decreased propofol-induced cell apoptosis, cleaved caspase-3 and PUMA expression. Propofol induce s cell death in hESC-derived neurons via activation of miR-206/PUMA signal pathway.

Knockdown of miR-155 protects microglia against LPS-induced inflammatory injury via targeting RACK1: a novel research for intracranial infection

BackgroundIntracranial infection, one of the complications of traumatic brain injury, is usually associated with inflammation. Several microRNAs (miRNAs), including miR-155, have been reported to be critical modulators in peripheral and central nervous system inflammation. In this study, we investigated the role of miR-155 in lipopolysaccharide (LPS)-induced inflammatory injury in mouse microglia BV2 cells.ResultsThe expression level of miR-155 was significantly up-regulated after LPS stimulation in BV2 cells. LPS administration decreased BV2 cell viability, promoted apoptosis and increased the release of pro-inflammatory cytokines; while miR-155 knockdown rescued BV2 cell from LPS-induced injury. RACK1 was a directly target of miR-155. Interestingly, miR-155 knockdown did not attenuate LPS-induced inflammatory injury when RACK1 was knocked down. The mechanistic study indicated that miR-155 knockdown deactivated MAPK/NF-κB and mTOR signaling pathways under LPS-treated conditions.ConclusionsKnockdown of miR-155 protected mouse microglia BV2 cells from LPS-induced inflammatory injury via targeting RACK1 and deactivating MAPK/NF-κB and mTOR signaling pathways.

MiR-21 modulates the effect of EZH2 on the biological behavior of human lung cancer stem cells in vitro.

Non-small-cell lung cancer has a high mortality rate and poor prognosis. Therefore, novel therapeutic approaches are urgently needed to enhance patient survival rates. In this study, we investigated the effects of miR-21 and EZH2 on the biological behavior of human lung cancer stem cells in vitro. We found increased expression of EZH2 and miR-21 in LCSCs, and miR-21 overexpression increased EZH2 levels in LCSCs. In addition, EZH2 and miR-21 knockdown increased the sensitivity of LCSCs to chemo- and radiation therapy, and exogenous EZH2 expression rescued the effects of anti-miR-21. Cell proliferation was reduced by 39.2% and 69.7% in the presence of radio- or chemotherapy combined with anti-miR-21 transfection, respectively. The downstream molecules included Cdc2, cyclin B1, and Bcl-2, which are involved in the regulation of cell cycle and apoptosis and which could themselves be reduced or enhanced by changes in miR-21 and EZH2 levels in LCSCs. This study demonstrates the direct relationship between miR-21 and EZH2 which was increased by 43% after the application of the miR-21 mimic. Above data indicates that these two molecules can influence the biological behavior of LCSCs by altering their corresponding targets. Our findings support the potential roles of miR-21 and EZH2 in improving the therapeutic efficacy of clinical lung cancer treatments.

Inhibition of LHX2 by miR-124 suppresses cellular migration and invasion in non-small cell lung cancer.

Downregulated microRNA (miR)-124 is common in numerous types of cancer, including non-small cell lung cancer (NSCLC). A previous study by the authors demonstrated that LIM-homeobox domain 2 (LHX2) was upregulated and promoted cell growth in NSCLC. However, whether LHX2 affects the migratory and invasive abilities of NSCLC cells and the association of LHX2 with miR-124 remains unclear. The present study revealed that miR-124 expression was frequently decreased in human NSCLC cells and tissues and negatively correlated with LHX2 expression, which was increased in NSCLC cells and tissues. Furthermore, the transfection of miR-124 mimic significantly inhibited endogenous expression of LHX2 mRNA and protein in A549 and H1299 cells, and miR-124 inhibitor promoted LHX2 expression. Of note, overexpression of miR-124 in A549 and H1299 cells attenuated cellular migratory and invasive abilities, and this was observed in LHX2-silenced A549 and H1299 cells. Knockdown of miR-124 augmented the migratory and invasive abilities in A549 and H1299 cells. The 3′-untranslated region of LHX2 transcript has also been identified to be a putative target of miR-124. Taken together, the results revealed that miR-124 may inhibit migration and invasion by repressing LHX2 expression in NSCLC cells. The findings of the present study suggested that overexpression of miR-124 or silencing of LHX2 may provide a therapeutic strategy for advanced NSCLC.

Abstract 3557: miR-155 positively regulates glucose metabolism via PI3K p85<alpha>-FOXO3a-cMYC axis in breast cancer

Abstract Breast cancer is the most common cancer for women worldwide. Although the oncogenic roles of miR-155 in cancer have been reported, the metabolic function of miR-155 in breast cancer has not been fully elucidated. In this study, we have investigated how the energy metabolic shift occurs after a genetic inactivation or knockdown of miR-155. To this end, we performed metabolic profiling of the miR-155ko/ko or miR-155+/ko primary cancer cells, obtained from mouse breast cancer model. We found, in the absence of miR-155, less glucose uptake and glycolysis in tumor cells. Mechanistically, we revealed PIK3R1(Phosphoinositide-3-Kinase, Regulatory Subunit1, p85<alpha>) and FOXO3a (Forkhead transcription factor of the O class 3a) are major targets that trigger the metabolic shift. Furthermore, we showed cMYC level was consequently decreased in the absence of miR-155 that results in the down-regulation of proteins playing in glucose uptake and Glycolysis. This finding is further confirmed in mouse allograft model. Finally, we show the miR-155 expression is positively correlates with either energy metabolite levels or standardized uptake values (SUV) of fluoro-deoxy glucose (FDG)-PET Images in triple negative breast tumors. Taken together, our study demonstrates a novel mechanism by which the miR-155 controls energy metabolism in breast cancer, through PI3K p85<alpha>-FOXO3a-cMYC axis. Citation Format: Sinae Kim, Eunji Lee, Suhwan Chang. miR-155 positively regulates glucose metabolism via PI3K p85<alpha>-FOXO3a-cMYC axis in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3557. doi:10.1158/1538-7445.AM2017-3557

Quantitative Proteomics Analysis Reveals Novel Targets of miR-21 in Zebrafish Embryos

MicroRNAs (miRNAs) are noncoding RNAs which control gene expression by the suppression of translation or the degradation of mRNAs. Dre-miR-21 (miR-21) has been reported to impact cardiac valvulogenesis in zebrafish embryos. However, the target genes of miR-21 are still largely unknown. Here a tandem isobaric mass tag (TMT)-based quantitative proteomic strategy was employed to identify the global profile of miR-21-regulated proteins. A total of 251 proteins were dysregulated after miR-21 knockdown, suggesting that they may be regulated by miR-21. Bioinformatics analysis indicated that these differentially expressed proteins (DEPs) participate in various biological processes, suggesting that miR-21 may be involved in diverse cellular pathways. Sixteen DEPs were also predicted to be miR-21 targets by at least two algorithms, and several candidate target genes were selected for further luciferase reporter analysis. The results showed that genes encoding tropomyosin 1 (tpm1) and poly(rC) binding protein 2 (pcbp2) are direct miR-21 targets. Taken together, our results not only reveal a large number of novel miR-21 regulated proteins that possess pleiotropic functions, but also provide novel insights into the molecular mechanisms of miR-21 regulation of zebrafish cardiac valvulogenesis and embryonic development.

MicroRNA-206 regulates vascular smooth muscle cell phenotypic switch and vascular neointimal formation.

MiR-206 has been found to play a critical role in skeletal muscle proliferation, differentiation, and regeneration. However, little is known about the function of miR-206 in vascular smooth muscle cells (VSMCs) biology. In this study, we will investigate its roles in phenotypic switching of VSMCs and neointimal lesion formation. First, we identified the expression of miR-206 in VSMCs treated with various concentrations of TGFβ1 and in rat carotid arteries after angioplasty by using qPCR. TGFβ1 inhibited the expression of miR-206 and TGFβ1 inhibitor induced miR-206 expression. In VSMCs of injured vascular walls, miR-206 expression was upregulated. Then, we overexpressed miR-206 using lentivirus Lv-rno-mir-206 and knocked down miR-206 using LV-rno-mir-206-inhibitor in rat carotid arteries after angioplasty. Overexpression of miR-206 resulted in decreasing SM22α expression in VSMCs in vitro and knockdown of miR-206 suppressed neointimal lesion formation in vivo. Finally, ZFP580 (zinc finger protein 580) was identified as the direct target of miR-206 in VSMCs by using luciferase report assay. The results indicate that miR-206 is involved in phenotypic switching of VSMCs and neointimal lesion formation after angioplasty through targeting ZFP580. These findings may provide a novel therapeutic target in post-angioplasty restenosis.

MiR-221 and miR-222 synergistically regulate hepatocyte growth factor activator inhibitor type 1 to promote cell proliferation and migration in gastric cancer.

Gastric cancer is a common malignancy with limited treatment options and poor prognosis. Introduction of novel pathways of gastric cancer will provide candidates for target therapy. Hepatocyte growth factor activator inhibitor type 1 is an integral-membrane proteinase inhibitor. Hepatocyte growth factor activator inhibitor type 1 abnormality is found in various cancers and correlates with tumor progression and metastasis. However, the mechanisms underlying the dysregulation of hepatocyte growth factor activator inhibitor type 1 expression in gastric cancer remain unclear. Although microRNAs have been reported to be involved in the development of cancer, the roles of miR-221 and miR-222 in gastric cancer have not been reported yet. In this study, we showed that hepatocyte growth factor activator inhibitor type 1 protein was downregulated, while miR-221 and miR-222 were significantly increased in gastric cancer tissues. Bioinformatic predictions and luciferase assay verified that the 3'-untranslated region of the HAI-1 gene is a direct target site for miR-221 and miR-222. Overexpression of miR-221 and miR-222 in MGC-803 cells leads to the inhibition of hepatocyte growth factor activator inhibitor type 1 protein expression, thus promoting cell proliferation and migration; whereas knockdown of miR-221 and miR-222 showed opposite effects. Moreover, we found that the expression level of hepatocyte growth factor activator protein was increased when hepatocyte growth factor activator inhibitor type 1 was knocked down in MGC-803 cells. Thus, gastric cancer is probably an autocrine tumor, and the antitumor mechanism of hepatocyte growth factor activator inhibitor type 1 in vitro might be mediated by regulating the expression of hepatocyte growth factor activator protein. Therefore, our data illustrated a novel pathway comprising miR-221and miR-222 and hepatocyte growth factor activator inhibitor type 1 in gastric cancer, which is a potential target for future clinical use.

MicroRNA-126 Inhibits Proliferation, Migration, Invasion, and EMT in Osteosarcoma by Targeting ZEB1.

Osteosarcoma (OS) is a tumor with rapid progression, high metastatic potential and poor clinical prognosis. This study was aimed to investigate the function of miR-126 in OS cells. The miR-126 expression in OS cell lines and OS tissues were explored by qRT-PCR. Then, the effects of miR-126 on proliferation, cycle, migration, invasion, and transforming growth factor (TGF)-β1 induced epithelial to mesenchymal transition (EMT) were assessed. Predicted by TargetScan, one of target genes for miR-126 was verified by luciferase activity assay. Meantime, the mRNA and protein expressions of ZEB1 were assessed by qRT-PCR and Western blot assay. Subsequently, the effect of ZEB1 silence on miR-126 down-regulated cells was also evaluated. Finally, the expressions of key kinases involved in c-Jun N-terminal kinase (JNK) and Janus-activated kinase (JAK)-1/signal transducer and activator of transcription (STAT)-3 pathways were detected by Western blot analysis. Result showed that miR-126 was down-regulated in OS tissues and cell lines. Overexpression of miR-126 significantly inhibited cell proliferation, migration, invasion, and TGF-β1 induced EMT. The effect of miR-126 knockdown was just the opposite. ZEB1 was predicted and verified as a target gene of miR-126. Meantime, the influence of miR-126 knockdown was abrogated by ZEB1 silence. Additionally, the phosphorylation levels of c-Jun, JNK, JAK1, and STAT3 were down-regulated in miR-126 over-expressed cells, and the effect of miR-126 knockdown was reversed by ZEB1 silence. In conclusion, miR-126 inhibits proliferation, migration, invasion and EMT in OS by targeting ZEB1 through inactivation of JNK and JAK1/STAT3 pathways. J. Cell. Biochem. 118: 3765-3774, 2017. © 2017 Wiley Periodicals, Inc.

Abstract 635: Role of Micro RNA-21 in Venous Neointimal Hyperplasia (VNH): Implications in Targeting MiR-21 For VNH Treatment

The exact molecular mechanisms involved in hemodialysis arteriovenous fistula (AVF) failure caused by venous neointimal hyperplasia (VNH) are not clear. It has been observed that there is an accumulation of extracellular matrix and up regulation of pro-fibrotic genes accompanied with presence of fibroblasts, smooth muscle cells, and inflammatory cells in the stenotic veins. Previous studies have demonstrated that adventitial and medial fibroblasts have a pivotal role(s) in VNH formation. MicroRNA-21 (miR-21) contributes to fibroblast to myofibroblast differentiation and dysregulation of miR-21 plays a pathological role in failure of coronary artery bypass grafts. The aim of the present study was to determine the role of miR-21 in VNH associated with AVF. We assessed miR-21 expression using qRT-PCR in the outflow veins of AVFs compared to control (contralateral jugular veins) veins in the C57BL/6J mice with chronic kidney disease (CKD). MiR-21 expression was upregulated accompanied with down regulation of miR-21 target genes; PPAR-α, PTEN and TIMP-3. In addition, gene expression of fibroblast specific protein (FSP) -1, TGF (transforming growth factor) -β1, matrix metalloproteinases (MMP)-2, -9, collagen-I, and IV were significantly increased at day 7 after AVF creation. Immunohistochemistry revealed that there was a significant increase in proliferating cell index (Ki-67) and fibroblast index (FSP-1) in the outflow veins of AVFs. Hypoxia has been shown to increase fibroblast to myofibroblast differentiation and this is predicted to be an early step in VNH formation. Therefore we assessed miR-21 expression in hypoxic (1%O 2 ) mouse pulmonary vein fibroblasts compared to normoxic cells in vitro and it was found that miR-21and TGF-β1 significantly elevated with down regulation of miR-21 target genes PTEN and TIMP-3. Furthermore, miR-21 knockdown in hypoxic fibroblasts attenuated TGF-β1 expression with a significant upregulation of genes targeted by miR-21 compared to controls. Together these results indicate that upregulation of miR-21 expression may result in fibroblast to myofibroblast differentiation resulting in VNH formation.

Precise Regulation of miR-210 is Critical for the Cellular Homeostasis Maintenance and Transplantation Efficacy Enhancement of Mesenchymal Stem Cells in Acute Liver Failure Therapy

Stem cell transplantation is a promising clinical strategy to cure acute liver failure. However, a low cell survival ratio after transplantation significantly impairs its therapeutic efficacy. This is partly due to insufficient resistance of transplanted stem cells to severe oxidative and inflammatory stress at the injury sites. In the current study, we demonstrated that a small molecule zeaxanthin dipalmitate (ZD) could enhance the defensive abilities against adverse stresses of human adipose-derived mesenchymal stem cells (hADMSCs) in vitro and increase their therapeutic outcomes of acute liver failure after transplantation in vivo. Treatment with ZD dramatically improved cell survival and suppressed apoptosis, inflammation, and reactive oxygen species (ROS) production of hADMSCs through the PKC/Raf-1/MAPK/NF-κB pathway to maintain a reasonably high expression level of microRNA-210 (miR-210). The regulation loop between miR-210 and cellular/mitochondrial ROS production was found to be linked by the ROS inhibitor iron–sulfur cluster assembly proteins (ISCU). Pretreatment with ZD and stable knockdown of miR-210 significantly improved and impaired the stem cell transplantation efficacy through the alteration of hepatic cell expansion and injury amelioration, respectively. Vehicle treatment with ZD did not pose any adverse effect on cell homeostasis or healthy animal. In conclusion, elevating endogenous antioxidant level of hADMSCs with ZD significantly enhances their hepatic tissue-repairing capabilities. Maintenance of a physiological level of miR-210 is critical for hADMSC homeostasis.

Role of microRNA-124 in cardiomyocyte hypertrophy inducedby angiotensin II

Cardiac hypertrophy is a crucial predictor of heart failure and is regulated by microRNAs. MicroRNA-124 (miR-124) is regarded as a prognostic indicator for outcomes after cardiac arrest. However, whether miR-124 participates in cardiac hypertrophy remains unclear. Therefore, our study aimed to determine the role of miR-124 in angiotensin II(AngII)-induced myocardial hypertrophy and the possible mechanism. Primary cultured rat neonatal cardiomyocytes(NCMs) were transfected with miR-124 mimics or inhibitor, followed by AngII stimulation. Quantitative RT-PCR, western blot analysis and determination of cell surface area of NCMs were used to detect the hypertrophic phenotypes. We observed that miR-124 was elevated in AngII-induced hypertrophic cardiomyocytes. Cell surface area of NCMs and mRNA expression of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC), indicators of myocardial hypertrophy, were higher in NCMs transfected with miR-124 mimics in the presence of AngII. On the contrary, knockdown of miR-124 by its specific inhibitor could restore these courses. Furthermore, downregulation of miR-124 alleviated the increased protein level of endoplasmic reticulum (ER) stress markers 78-kDa glucose-regulated protein (Grp78) and calreticulin(CRT) in AngII-induced NCMs. In conclusion, our study shows that inhibition of miR-124 effectively suppresses AngII-induced myocardial hypertrophy, which is associated with attenuation of ER stress.

MiR-155 promotes cell proliferation and inhibits apoptosis by PTEN signaling pathway in the psoriasis

MicroRNAs (miRNAs) have been demonstrated to contribute to malignant progression in psoriasis development. The purposes of the study was to evaluated the effects of miRNA-155 on cell proliferation, migration and apoptosis in psoriasis development via PTEN singaling pathway and identify its direct target protein. Quantitative real-time RT-PCR (qRT-PCR) was performed to examine the level of miR-155 in psoriasis cells, miR-155 was downregulated in a psoriasis cell line Hacat by transfected with small interfering RNA (siRNA), respectively. Cell survival was detected by the MTT assay and colony formation assay. Cell migration and invasion were measured via wound-healing assayand transwell assay. In addition, cell cycle and apoptosis about psoriasis cells was measured by flow cytometry.In this study, qRT-PCR assay showed that the expressions of miR-155 mRNA in psoriasis tissues were significantly higher than that in normal tissues. The assays about cell growth and proliferation showed that miR-155 knockdown led to a significant decrease in cell proliferation which was determined by MTT assay and colony formation assay compared to those of Lv-NC cells. Flow cytometry analysis showed that depletion of miR-155 could cause cell cycle change and the number of apoptotic cells was significantly increased in Lv-miR155 cells compared with control cells. In addition, the expression of several apoptosis-related factors were dramatically changed, such as PTEN, PIP3, AKT, p-AKT, Bax and Bcl-2. Our findings indicate that down-regulation of miR-155 significantly inhibits proliferation, migration, invasion and promotes apoptosis through PTEN singaling pathway in psoriasis cells. miR-155 might function as an oncogene miRNA in the progress of psoriasis.

WBP2 modulates G1/S transition in ER+ breast cancer cells and is a direct target of miR-206.

The mechanisms underlying the oncogenic properties of WW domain binding protein 2 (WBP2) in breast cancer have not been fully understood. In this study, we explored the role of WBP2 in cell cycle regulation in ER+ breast cancer cells and how it is regulated in the cancer cells. The association between WBP2 expression and prognosis in ER+ breast cancer was assessed by data mining in Breast Cancer Gene-Expression Miner v4.0. Cell cycle was assessed by PI staining and flow cytometry. EdU staining was applied to visualize cells in S phase. The binding between miR-206 and WBP2 were verified by dual luciferase assay. CCK-8 assay and flow cytometric analysis were applied to assess the functional role of WBP2 and miR-206 in the cancer cells. High WBP2 expression correlates with higher risk of any events (AE) and metastatic relapse (MR) and also indicates shorter AE-free survival and MR-free survival in ER+ breast cancer patients. In both MCF-7 and BT474 cells, WBP can influence the expression of G1/S-related cell cycle proteins, including p21, CDK4, and cyclin D1. In addition, WBP2 overexpression resulted in facilitated G1/S transition, while WBP2 knockdown impaired the transition. The 3'UTR of WBP2 has a conserved miR-206 binding site. Functionally, miR-206 knockdown decreased tamoxifen sensitivity in tamoxifen-sensitive (TamS) MCF-7 cells, while miR-206 overexpression and WBP2 knockdown enhanced the sensitivity in tamoxifen-resistant (TamR) MCF-7 cells. Based on these findings, we infer that the miR-206/WBP2 axis can modulate tamoxifen sensitivity via regulating G1/S progression in ER+ breast cancer.

Abstract WMP43: Neurorestorative Therapy of Stroke in T2DM Mice With Exosomes Derived From Brain Endothelial Cells

MicroRNA-126 is primarily expressed in endothelial cells (ECs), and is emerging as a key player in the pathogenesis of type two diabetes (T2DM) induced vascular damage. Stroke in T2DM increases vascular and white matter (WM) damage in the ischemic brain, and has a poor prognosis compared to non-DM stroke. In this study, we investigated the neurorestorative effects of exosomes derived from mouse brain ECs (EC-Exo) as a treatment of stroke in T2DM mice. Methods: Adult male BKS.Cg-m+/+Lepr db /J (T2DM) and db/+ (non-DM) control mice (3-4 m) were subjected to right extraluminal permanent distal middle cerebral artery occlusion (dMCAo), and intravenous injection at 3 days after dMCAo with: 1) PBS; 2) Liposome mimic as vehicle control (3X10 10 ); 3): EC-Exo (3x10 10 ); 4) Knockdown of miR-126 in EC-Exo (miR-126-/-EC-Exo, 3X10 10 ); 5) Knock-in miR-126 in EC-Exo (miR-126+/+EC-Exo, 3X10 10 ). Mice were sacrificed at 28 days after dMCAo. Results: 1) T2DM-dMCAo mice exhibit significantly decreased blood serum, brain tissue and brain-EC miR-126 expression compared to non-DM-dMCAo mice. 2) EC-Exo contain higher levels of miR-126 than exosomes derived from other types of cells (smooth muscle cells, neurons, astrocytes and marrow stromal cells); 3) EC-Exo treatment significantly increases axonal outgrowth and increases capillary tube formation in vitro. Compared to PBS or liposome mimic treatment groups, EC-Exo treatment of stroke in T2DM-dMCAo mice: 4) is safe and does not appear to induce adverse side effects in physiological parameters and does not decrease lesion volume, but significantly improves neurological outcome and cognitive function after dMCAo; 5) significantly increases axon, myelin, vessel density and increases artery diameter in the ischemic boundary zone; 6) significantly increases perfused vessel density measured by FITC-dextran injection. 7) miR-126-/-EC-Exo treatment significantly decreases, but miR-126+/+EC-Exo increases miR-126 expression compared to EC-Exo control. 8) miR-126+/+EC-Exo treatment significantly improves functional outcomes when compared to miR-126-/-EC-Exo, or PBS control. Conclusion: Treatment of stroke with EC-Exo increases miR-126 expression and promotes neurorestorative effects after stroke in T2DM mice.

Differential response of normal and transformed mammary epithelial cells to combined treatment of anti-miR-21 and radiation

Purpose: MicroRNA miR-21 has emerged as a therapeutic target in the treatment of breast cancer. This study was designed to compare the responses of breast cancer cells and non-transformed breast epithelial cells to a combined regimen of miR-21 inhibition and radiation.Materials and methods: The MDA-MB-361 (breast cancer) and MCF-10A (non-transformed mammary epithelial) cell lines were used for the comparison in this in vitro study. The stable knockdown of miR-21 was performed by using lentiviral approach. The response of the cells was monitored 4, 24 and 48 h after the irradiation with 0.25 and 2.5 Gy, using sham-irradiated cells as controls. The response of the cells was established by performing various functional assays – cell viability and cell attachment, clonogenic survival, cell cycle analysis and 3D microtissue formation.Results: The knockdown of miR-21 induced significant increase in apoptosis and growth delay in MDA-MB-361 cancer cells compared to non-transformed MCF-10A cells. After combined radiation and anti-miR-21 treatment, MDA-MB-361 cells show reduced cell growth and viability what is presented in their inability to form colonies. MCF-10A cells were not as sensitive to the combined treatment and that has also been confirmed with colony forming assay.Conclusions: Cellular response to a combined treatment of anti-miR-21 and radiation is different between cancer and non-cancer cells which highly support the idea of linking miR-21 inhibitor and radiation treatment in the future therapeutic approaches for breast cancer.