Untranslated Region Luciferase Assay Research Articles

MicroRNA-409-3p/BTG2 signaling axis improves impaired angiogenesis and wound healing in obese mice.

Wound healing is facilitated by neoangiogenesis, a complex process that is essential to tissue repair in response to injury. MicroRNAs are small, noncoding RNAs that can regulate the wound healing process including stimulation of impaired angiogenesis that is associated with type-2 diabetes (T2D). Expression of miR-409-3p was significantly increased in the nonhealing skin wounds of patients with T2D compared to the non-wounded normal skin, and in the skin of a murine model with T2D. In response to high glucose, neutralization of miR-409-3p markedly improved EC growth and migration in human umbilical vein endothelial cells (HUVECs), promoted wound closure and angiogenesis as measured by increased CD31 in human skin organoids, while overexpression attenuated EC angiogenic responses. Bulk mRNA-Seq transcriptomic profiling revealed BTG2 as a target of miR-409-3p, where overexpression of miR-409-3p significantly decreased BTG2 mRNA and protein expression. A 3' untranslated region (3'-UTR) luciferase assay of BTG2 revealed decreased luciferase activity with overexpression of miR-409-3p, while inhibition had opposite effects. Mechanistically, in response to high glucose, miR-409-3p deficiency in ECs resulted in increased mTOR phosphorylation, meanwhile BTG-anti-proliferation factor 2 (BTG2) silencing significantly decreased mTOR phosphorylation. Endothelial-specific and tamoxifen-inducible miR-409-3p knockout mice (MiR-409IndECKO ) with hyperglycemia that underwent dorsal skin wounding showed significant improvement of wound closure, increased blood flow, granulation tissue thickness (GTT), and CD31 that correlated with increased BTG2 expression. Taken together, our results show that miR-409-3p is a critical mediator of impaired angiogenesis in diabetic skin wound healing.

MiR-199a and miR-199b facilitate diffuse gastric cancer progression by targeting Frizzled-6

Pathological markers that can monitor the progression of gastric cancer (GC) may facilitate the diagnosis and treatment of patients with diffuse GC (DGC). To identify microRNAs (miRNAs) that can differentiate between early and advanced DGC in the gastric mucosa, miRNA expression profiling was performed using the NanoString nCounter method in human DGC tumors. Ectopic expression of miR-199a and miR-199b (miR-199a/b) in SNU601 human GC cells accelerated the growth rate, viability, and motility of cancer cells and increased the tumor volume and weight in a mouse xenograft model. To study their clinicopathological roles in patients with GC, miR-199a/b levels were measured in human GC tumor samples using in situ hybridization. High miR-199a/b expression level was associated with enhanced lymphovascular invasion, advanced T stage, and lymph-node metastasis. Using the 3′-untranslated region (UTR) luciferase assay, Frizzled-6 (FZD6) was confirmed to be a direct target of miR-199a/b in GC cells. siRNA-mediated depletion of FZD6 enhanced the motility of SNU601 cells, and addback of FZD6 restored cancer cell motility stimulated by miR-199a/b. In conclusion, miR-199a/b promotes DGC progression by targeting FZD6, implying that miR-199a/b can be used as prognostic and diagnostic biomarkers for the disease.

Liver X Receptor Alpha Activation Inhibits Autophagy and Lipophagy in Hepatocytes by Dysregulating Autophagy-Related 4B Cysteine Peptidase and Rab-8B, Reducing Mitochondrial Fuel Oxidation.

Fat accumulation results from increased fat absorption and/or defective fat metabolism. Currently, the lipid-sensing nuclear receptor that controls fat utilization in hepatocytes is elusive. Liver X receptor alpha (LXRα) promotes accumulation of lipids through the induction of several lipogenic genes. However, its effect on lipid degradation is open for study. Here, we investigated the inhibitory role of LXRα in autophagy/lipophagy in hepatocytes and the underlying basis. In LXRα knockout mice fed a high-fat diet, or cell models, LXRα activation suppressed the function of mitochondria by inhibiting autophagy/lipophagy and induced hepatic steatosis. Gene sets associated with "autophagy" were enriched in hepatic transcriptome data. Autophagy flux was markedly augmented in the LXRα knockout mouse liver and primary hepatocytes. Mechanistically, LXRα suppressed autophagy-related 4B cysteine peptidase (ATG4B) and Rab-8B, responsible for autophagosome and -lysosome formation, by inducing let-7a and microRNA (miR)-34a. Chromatin immunoprecipitation assay enabled us to find LXRα as a transcription factor of let-7a and miR-34a. Moreover, 3' untranslated region luciferase assay substantiated the direct inhibitory effects of let-7a and miR-34a on ATG4B and Rab-8B. Consistently, either LXRα activation or the let-7a/miR-34a transfection lowered mitochondrial oxygen consumption rate and mitochondrial transmembrane potential and increased fat levels. In obese animals or nonalcoholic fatty liver disease (NAFLD) patients, let-7a and miR-34a levels were elevated with simultaneous decreases in ATG4B and Rab-8B levels. LXRα inhibits autophagy in hepatocytes through down-regulating ATG4B and Rab-8B by transcriptionally activating microRNA let-7a-2 and microRNA 34a genes and suppresses mitochondrial biogenesis and fuel consumption. This highlights a function of LXRα that culminates in the progression of liver steatosis and steatohepatitis, and the identified targets may be applied for a therapeutic strategy in the treatment of NAFLD.

MiR-890 inhibits proliferation and invasion and induces apoptosis in triple-negative breast cancer cells by targeting CD147

BackgroundTriple-negative breast cancer (TNBC) is a type of breast cancer with a high degree of malignancy. Because of the remarkable biological characteristics of high invasion, metastasis and recurrence, TNBC is often accompanied by a poor prognosis. As a molecular characteristic of TNBC, high expression of CD147 has been confirmed by a large number of studies. However, the mechanism of CD147 expression regulation in TNBC remains elusive. In this study, we investigated the roles of miR-890 in inhibiting CD147.MethodsQuantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) was used to detect CD147 mRNA and miR-890 level, and western blotting was used to detect CD147 protein. Bioinformatics screening and 3′-Untranslated Region (3′-UTR) luciferase assays were used to analyze the microRNAs (miRNA) binding site. Cell proliferation, apoptosis and invasion were assessed by using CCK-8, flow cytometry and transwell assays.ResultsThe upregulation of miR-890 inhibited cell proliferation and invasion, induced apoptosis in MDA-MB-231 and HCC-70 TNBC cells by negatively regulating its target gene, CD147, and the upregulation of CD147 rescued the inhibitory effects of miR-890. miR-890 targeted CD147 by binding to its 3′-UTR. Further results showed that the upregulation of miR-890 also inhibited the expression of MMPs, the downstream genes of CD147, and promoted the cleavage of Caspase-3. The CD147 recovery experiment was further confirmed by the activity changes in the downstream MMPs of CD147. In addition, it was confirmed that the effect of CD147 in promoting TNBC cell proliferation and invasion, inhibiting apoptosis was related to the change in caspase-3 activity.ConclusionThe downregulation of miR-890 is the potential cause of high CD147 expression in TNBC, which can promote the malignant transformation of TNBC.

Inhibition of miR-24 suppresses malignancy of human non-small cell lung cancer cells by targeting WWOX in vitro and in vivo.

BackgroundWe investigated the effect of micro‐RNA 24 (miR‐24) and WWOX on non‐small cell lung cancer (NSCLC) cell proliferation and migration in vitro and in vivo.MethodsWe performed bioinformatics analysis and 3′ untranslated region luciferase assay to investigate the direct target of miR‐24. Proliferation, apoptosis, and transwell invasion assays were employed to evaluate the effect of WWOX overexpression with pcDNA3‐WWOX and knocking down miR‐24 with miR‐24 small interfering RNA. Quantitative real‐time PCR, Western blot, and immunohistochemistry were also used to investigate miR‐24 and c‐Kit expression, and apoptosis and invasion‐related proteins. Finally, we constructed a tumor xenograft model in nude mice to confirm the effect of miR‐24 on NSCLC cell proliferation in vivo.ResultsAccording to our experimental data, miR‐24 inhibition could induce apoptosis by activating caspase 3 and suppress the viability and proliferation of NSCLC cells in vitro and in vivo. MiR‐24 downregulation could reduce the invasive ability of NSCLC cells by downregulating MMP9. WWOX was identified as a functional target of miR‐24. WWOX overexpression generated the same effect with antagonizing miR‐24, while blocking WWOX counteracted the tumor suppressive effect caused by miR‐24 inhibition. MiR‐24 may function as an oncogene and play an important role in the cell growth and migration of NSCLC.ConclusionsOur findings enhance understanding of the miR‐24 regulatory network and the molecular mechanism that underlies the oncogenesis and development of NSCLC. Suppressing the effect of miR‐24 on cancer cells using a miR‐24 inhibitor may be an attractive therapeutic strategy against NSCLC.

MiR-194-3p Represses the Progesterone Receptor and Decidualization in Eutopic Endometrium From Women With Endometriosis.

Progesterone resistance in the eutopic endometrium (EuE) is suggested to be a critical factor for decreased endometrial receptivity and implantation failure in reproductive-aged women with endometriosis. Altered expression of miRNAs has been reported to play an important role in the pathophysiology of endometriosis-associated infertility. However, the underlying mechanisms of aberrant progesterone receptor (PR) and deficient decidualization regulated by miRNAs in endometriosis have not been thoroughly elucidated. The goal of this study was to explore the regulation and roles of miR-194-3p in aberrant PR expression and impaired decidualization in endometrial stromal cells (ESCs) from the EuE of women with mild or minimal endometriosis. Using a series of studies, we observed decreased PR mRNA expression and an increasing PR-A/PR-B mRNA ratio trend in the midsecretory phase of the EuE of women with minimal or mild endometriosis (n = 19) compared with controls (n = 14); the increased expression of miR-194-3p in the endometriosis group was consistent with previous microarray analysis. We also found that PR protein levels were inhibited by the transfection of ESCs with an miR-194-3p mimic and upregulated by miR-194-3p inhibition. As predicted by the bioinformatic analysis, the 3'-untranslated region luciferase assay indicated the direct regulation of PR expression by miR-194-3p. Furthermore, miR-194-3p overexpression inhibited the in vitro decidualization of ESCs via both cellular morphological changes and prolactin levels. Therefore, our study demonstrated that miR-194-3p contributes to progesterone resistance in endometriosis, which hinders fertility by repressing the levels of PR and decidualization in the EuE. Thus, miR-194-3p regulation is a future therapeutic strategy for endometriosis.

Abstract 5726: A novel tumor-promoting role for miR-4516 in glioblastoma

Abstract Background: Glioblastomas are the most aggressive high-grade brain tumors, which are often life-threatening due to their location and rapid growth. Although survival rates differ depending on a variety of genetic and environmental factors, the average survival rate for a glioblastoma (GBM) patient is less than 15 months. Although the mechanisms of tumorigenesis are still being elucidated, miRNAs are promising candidates to explore as novel and prognostic biomarkers in GBM. Here we demonstrate a novel role for miR-4516 in promoting growth and migration of GBM and establish the molecular mechanisms mediating these functions. Methods: Formalin-fixed, paraffin-embedded tissue blocks (n=268) were collected for all patients and total RNA was isolated. miRNAs were analyzed simultaneously using the nCounter human miRNA v2 assay (NanoString Technologies; Seattle,WA). Functional characterization studies were conducted in vitro and in vivo. The effect of miR-4516 on GBM cell growth and motility were evaluated by cell proliferation assay, migration and invasion assay, and Annexin-V assay. Realtime PCR, immunoblotting, and 3’ untranslated region luciferase assays were used to analyze miR-4516 targets and signaling pathways. Intracranial injection will be performed to investigate the role of miR-4516 in tumor growth in vivo. Results: Univariate analysis showed that miR-4516 expression in GBM patients was inversely correlated with overall survival (FDR=0.002, p=1.02E-05). Knockdown of miR-4516 blocked tumor growth and induced cell apoptosis. Tumor cell growth, migration and invasion were induced in both transient miR-4516 overexpressed GBM cells (LN229, LN18, and U87) and stable miR-4516 overexpressed GBM cells (U87-EGFRvIII). These miR-4516 tumor-promoting effects were mediated in part via direct targeting PTPN14 and CDKN1A. Investigation of the other miR-4516 targets and in vivo functional study are in process. Conclusion: Taken together, these results suggest that miR-4516 acts as a prognostic biomarker for GBM patients. Funding Information: 1R01CA169368 (PI: Houghton; Co-I:Chakravarti); 1R01CA11522358 Multiple-PI R01: Chakravarti (PI); Xia (PI); 1R01CA1145128 Baroukhim (PI); Chakravarti (Co-PI) 7/2015-6/2020; R01CA108633 (PI:Chakravarti); 1RC2CA148190 (Scientific PI: Chakravarti) Citation Format: Tiantian Cui, Ashley Gray, Ziyan Liu, Marjolein Geurts, Pierre Robe, Joseph McElroy, Erica Hlavin Bell, Arnab Chakravarti. A novel tumor-promoting role for miR-4516 in glioblastoma [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 5726. doi:10.1158/1538-7445.AM2017-5726

Upregulation of miRNA-4776 in Influenza Virus Infected Bronchial Epithelial Cells Is Associated with Downregulation of NFKBIB and Increased Viral Survival.

Influenza A virus (IAV) infection remains a significant cause of morbidity and mortality worldwide. One key transcription factor that is activated upon IAV infection is nuclear factor Kappa B (NF-κB). NF-κB regulation involves the inhibitor proteins NF-κB inhibitor beta (NFKBIB), (also known as IκB β), which form complexes with NF-κB to sequester it in the cytoplasm. In this study, microarray data showed differential expression of several microRNAs (miRNAs) on exposure to IAV. Target scan analysis revealed that miR-4776, miR-4514 and miR-4742 potentially target NFKBIB messenger RNA (mRNA). Time-course analysis of primary bronchial epithelial cells (HBEpCs) showed that miR-4776 expression is increased within 1 h of infection, followed by its downregulation 4 h post-exposure to IAV. NFKBIB upregulation of miR-4776 correlated with a decrease in NFKBIB expression within 1 h of infection and a subsequent increase in NFKBIB expression 4 h post-infection. In addition, miRNA ago-immunoprecipitation studies and the three prime untranslated region (3’ UTR) luciferase assay confirmed that miR-4776 targets NFKBIB mRNA. Furthermore, uninfected HBEpCs transfected with miR-4776 mimic showed decreased expression of NFKBIB mRNA. Overexpression of NFKBIB protein in IAV infected cells led to lower levels of IAV. Taken together, our data suggest that miRNA-4776 modulates IAV production in infected cells through NFKBIB expression, possibly through the modulation of NF-κB.

A MicroRNA93-Interferon Regulatory Factor-9-Immunoresponsive Gene-1-Itaconic Acid Pathway Modulates M2-Like Macrophage Polarization to Revascularize Ischemic Muscle.

Currently, no therapies exist for treating and improving outcomes in patients with severe peripheral artery disease (PAD). MicroRNA93 (miR93) has been shown to favorably modulate angiogenesis and to reduce tissue loss in genetic PAD models. However, the cell-specific function, downstream mechanisms, or signaling involved in miR93-mediated ischemic muscle neovascularization is not clear. Macrophages were best known to modulate arteriogenic response in PAD, and the extent of arteriogenic response induced by macrophages is dependent on greater M2 to M1 activation/polarization state. In the present study, we identified a novel mechanism by which miR93 regulates macrophage polarization to promote angiogenesis and arteriogenesis to revascularize ischemic muscle in experimental PAD. In vitro (macrophages, endothelial cells, skeletal muscle cells under normal and hypoxia serum starvation conditions) and in vivo experiments in preclinical PAD models (unilateral femoral artery ligation and resection) were conducted to examine the role of miR93-interferon regulatory factor-9-immunoresponsive gene-1 (IRG1)-itaconic acid pathway in macrophage polarization, angiogenesis, arteriogenesis, and perfusion recovery. In vivo, compared with wild-type controls, miR106b-93-25 cluster-deficient mice (miR106b-93-25-/-) showed decreased angiogenesis and arteriogenesis correlating with increased M1-like macrophages after experimental PAD. Intramuscular delivery of miR93 in miR106b-93-25-/- PAD mice increased angiogenesis, arteriogenesis, and the extent of perfusion, which correlated with more M2-like macrophages in the proximal and distal hind-limb muscles. In vitro, miR93 promotes and sustains M2-like polarization even under M1-like polarizing conditions (hypoxia serum starvation). Delivery of bone marrow-derived macrophages from miR106b-93-25-/- to wild-type ischemic muscle decreased angiogenesis, arteriogenesis, and perfusion, whereas transfer of wild-type macrophages to miR106b-93-25-/- had the opposite effect. Systematic analysis of top differentially upregulated genes from RNA sequencing between miR106b-93-25-/- and wild-type ischemic muscle showed that miR93 regulates IRG1 function to modulate itaconic acid production and macrophage polarization. The 3' untranslated region luciferase assays performed to determine whether IRG1 is a direct target of miR93 revealed that IRG1 is not an miR93 target but that interferon regulatory factor-9, which can regulate IRG1 expression, is an miR93 target. In vitro, increased expression of interferon regulatory factor-9 and IRG1 and itaconic acid treatment significantly decreased endothelial angiogenic potential. miR93 inhibits interferon regulatory factor-9 to decrease IRG1-itaconic acid production to induce M2-like polarization in ischemic muscle to enhance angiogenesis, arteriogenesis, and perfusion recovery in experimental PAD.

MicroRNA-145 modulates epithelial-mesenchymal transition and suppresses proliferation, migration and invasion by targeting SIP1 in human cervical cancer cells.

Previously, it has been reported that microRNA-145 (miR-145) is lowly expressed in human cervical cancers and that its putative tumour suppressive role may be attributed to epithelial-mesenchymal transition (EMT) regulation. Here, we aimed to assess whether miR-145 may affect EMT-associated markers/genes and suppress cervical cancer growth and motility, and to provide a mechanistic basis for these phenomena. The identification of the SMAD-interacting protein 1 (SIP1) mRNA as putative miR-145 target was investigated usinga 3' untranslated region (3'UTR) luciferase assay and Western blotting, respectively. The functional effects of exogenous miR-145 expression, miR-145 suppression or siRNA-mediated SIP1 expression down-regulation in cervical cancer-derived C33A and SiHa cells were analysed using Western blotting, BrdU incorporation (proliferation), transwell migration and invasion assays. In addition, the expression levels of miR-145 and SIP1 were determined in primary human cervical cancer and non-cancer tissue samples using qRT-PCR. We found that miR-145 binds to the wild-type 3'UTR of SIP1, but not to its mutant counterpart, and that, through this binding, miR-145 can effectively down-regulate SIP1 expression. In addition, we found that exogenous miR-145 expression or siRNA-mediated down-regulation of SIP1 expression attenuates the proliferation, migration and invasion of C33A and SiHa cells and alters the expression of the EMT-associated markers CDH1, VIM and SNAI1, whereas inhibition of endogenous miR-145 expression elicited the opposite effects. The expression of miR-145 in cervical cancer tissue samples was found to be low, while that of SIP1 was found to be high compared to non-cancerous cervical tissues. An inverse expression correlation between the two was substantiated through the anlaysis of data deposited in the TCGA database. Our data indicate that low miR-145 expression levels in conjunction with elevated SIP1 expression levels may contribute to cervical cancer development. MiR-145-mediated regulation of SIP1 provides a novel mechanistic basis for its tumour suppressive mode of action in human cervical cancer cells.

Leukocyte response is regulated by microRNA let7i in patients with acute ischemic stroke.

To evaluate microRNA let7i in ischemic stroke and its regulation of leukocytes. A total of 212 patients were studied: 106 with acute ischemic stroke and 106 controls matched for risk factors. RNA from circulating leukocytes was isolated from blood collected in PAXgene tubes. Let7i microRNA expression was assessed using TaqMan quantitative reverse transcription PCR. To assess let7i regulation of gene expression in stroke, messenger RNA (mRNA) from leukocytes was measured by whole-genome Human Transcriptome Array Affymetrix microarray. Given microRNAs act to destabilize and degrade their target mRNA, mRNAs that inversely correlated with let7i were identified. To demonstrate let7i posttranscriptional regulation of target genes, a 3' untranslated region luciferase assay was performed. Target protein expression was assessed using ELISA. Let7i was decreased in patients with acute ischemic stroke (fold change -1.70, p < 0.00001). A modest inverse correlation between let7i and NIH Stroke Scale score at admission (r = -0.32, p = 0.02), infarct volume (r = -0.21, p = 0.04), and plasma MMP9 (r = -0.46, p = 0.01) was identified. The decrease in let7i was associated with increased expression of several of its mRNA targets, including CD86, CXCL8, and HMGB1. In vitro studies confirm let7i posttranscriptional regulation of target genes CD86, CXCL8, and HMGB1. Functional analysis predicted let7i regulates pathways involved in leukocyte activation, recruitment, and proliferation including canonical pathways of CD86 signaling in T helper cells, HMGB1 signaling, and CXCL8 signaling. Let7i is decreased in circulating leukocytes of patients with acute ischemic stroke. Mechanisms by which let7i regulates inflammatory response post stroke include targeting CD86, CXCL8, and HMGB1.

MicroRNA-214 suppresses growth, migration and invasion through a novel target, high mobility group AT-hook 1, in human cervical and colorectal cancer cells.

Background:MicroRNA-214 (miR-214) has been shown to act as a tumour suppressor in human cervical and colorectal cancer cells. The aim of this study was to experimentally validate high mobility group AT-hook 1 as a novel target for miR-214-mediated suppression of growth and motility.Methods:HMGA1 and miR-214 expression levels were estimated in cervical and colorectal clinical specimens using qPCR. HMGA1 3′ untranslated region luciferase assays were performed to validate HMGA1 as a target of miR-214. Effect of altering the expression of miR-214 or HMGA1 on proliferation, migration and invasion of human cervical and colorectal cancer cells was investigated.Results:miR-214 expression was poor while that of HMGA1 was high in cervical and colorectal cancer tissues. miR-214-re-expression or HMGA1 downregulation inhibited proliferation, migration and invasion of cancer cells while miR-214 inhibition had opposite effects. miR-214 was demonstrated to bind to the wild-type 3′ untranslated region of HMGA1 but not with its mutant.Conclusions:Low expression of miR-214 concurrent with elevated levels of HMGA1 may contribute to cervical and colorectal cancer progression. miR-214-mediated regulation of HMGA1 is a novel mechanism for its tumour-suppressive actions in human cervical and colorectal cancer cells and opens up avenues for novel therapeutic strategies for these two cancers.

Altered expression of microRNA-451 in eutopic endometrium of baboons (Papio anubis) with endometriosis.

Are microRNAs (miRs) altered in the eutopic endometrium (EuE) of baboons following the induction of endometriosis? Induction of endometriosis causes significant changes in the expression of eight miRs, including miR-451, in the baboon endometrium as early as 3 months following induction of the disease. Endometriosis is one of the most common gynecological disorders and causes chronic pelvic pain and infertility in women of reproductive age. Altered expression of miRs has been reported in women and has been suggested to play an important role in the pathophysiology of several gynecological disorders including endometriosis. EuE was obtained from the same group of baboons before and 3 months after the induction of endometriosis. The altered expression of miR-451 was validated in the eutopic and ectopic endometrium of additional baboons between 3 and 15 months following disease induction. Timed endometrial biopsies from women with and without endometriosis were also used to validate the expression of miR-451. Total RNA was extracted from EuE samples before and after the induction of endometriosis, and miRNA expression was analyzed using a 8 × 15 K miR microarray. Microarray signal data were preprocessed by AgiMiRna software, and an empirical Bayes model was used to estimate the changes. The present study focused on quantitative RT-PCR validation of the microarray data, specifically on miR-451 and its target genes in both baboons (n = 3) and women [control (n = 7) and endometriosis (n = 19)]. Descriptive and correlative analysis of miR-451 and target gene expression was conducted using in situ hybridization and immunohistochemistry, while functional analysis utilized an in vitro 3' untranslated region (UTR) luciferase assay and overexpression of miR-451 in human endometrial and endometriotic cell lines. Induction of endometriosis results in the altered expression of miR-451, -141, -29c, -21, -424, -19b, -200a and -181a in the baboon endometrium. In the baboon, induction of endometriosis significantly decreased the expression of miR-451 at 3 months (P < 0.001), which was also associated with increased expression of its target gene YWHAZ (14.3.3ζ). A similar significant (P < 0.0001) decrease in miR-451 expression was observed in women with endometriosis. The 3' UTR luciferase assay confirmed the regulation of YWHAZ expression by miR-451. Furthermore, overexpression of miR-451 in 12Z cells (immortalized human endometriotic epithelial cell line) led to the decreased expression of its target YWHAZ and this was correlated with decreased cell proliferation. The study focused only on miR-451 and one of its targets, namely YWHAZ. A single miR could target number of genes and a single gene could also be regulated by number of miRs; hence, it is possible that other miRs and their regulated genes may contribute to the pathophysiology of endometriosis. Our data suggest that the presence of ectopic lesions in baboon causes changes in EuE miR expression as early as 3 months postinduction of the disease, and some of these changes may persist throughout the course of the disease. We propose that the marked down-regulation of miR-451 in both baboons and women with endometriosis increases the expression of multiple target genes. Increased expression of one of the target genes, YWHAZ, increases proliferation, likely contributing to the pathophysiology of the disease.

1α,25-DihydroxyvitaminD3 prevents the differentiation of human lung fibroblasts via microRNA-27b targeting the vitaminD receptor.

Pulmonary fibroblasts have key roles in the formation and maintenance of lung structure and function, and are involved in tissue repair and remodeling. Transforming growth factor-β1 (TGF-β1) induces differentiation of fibroblasts into myofibroblasts, the key effector cells in fibrotic states, which are characterized by the expression of α-smooth muscle actin (α-SMA) markers. 1α,25-Dihydroxyvitamin D3 [1,25(OH)2D3] has been implicated in regulating differentiation, and the vitamin D receptor (VDR) may be a regulator of TGF-β signaling. In addition, there is presently only limited information regarding microRNA (miRNA) regulation of lung fibroblast differentiation. To determine the role of 1,25(OH)2D3 in regulating the differentiation of fibroblasts induced by TGF-β1 and the functional importance of miR-27b, cell culture systems, cell transfection and the 3′ untranslated region (3′UTR) luciferase assay were employed. 1,25(OH)2D3 inhibited differentiation and downregulated miR-27b expression in human lung fibroblasts induced by TGF-β1. In addition, human lung fibroblasts were transfected with miR-27b mimic or miR-27b inhibitor, and demonstrated that the overexpression of miR-27b decreased the VDR protein expression and increased the expression of α-SMA, while reducing levels of miR-27b had opposing effects. Finally, the luciferase reporter assays were performed to confirm that miR-27b directly targeted VDR 3′UTR. Taken together, these results suggest that 1,25(OH)2D3 inhibits lung fibroblast differentiation induced by TGF-β1 via miR-27b targeting VDR 3′UTR, which may be used as a novel treatment strategy in differentiation pathways.

Regulation of NF-κB signaling by oxidized glycerophospholipid and IL-1β induced miRs-21-3p and -27a-5p in human aortic endothelial cells

Exposure of endothelial cells (ECs) to agents such as oxidized glycerophospholipids (oxGPs) and cytokines, known to accumulate in atherosclerotic lesions, perturbs the expression of hundreds of genes in ECs involved in inflammatory and other biological processes. We hypothesized that microRNAs (miRNAs) are involved in regulating the inflammatory response in human aortic endothelial cells (HAECs) in response to oxGPs and interleukin 1β (IL-1β). Using next-generation sequencing and RT-quantitative PCR, we characterized the profile of expressed miRNAs in HAECs pre- and postexposure to oxGPs. Using this data, we identified miR-21-3p and miR-27a-5p to be induced 3- to 4-fold in response to oxGP and IL-1β treatment compared with control treatment. Transient overexpression of miR-21-3p and miR-27a-5p resulted in the downregulation of 1,253 genes with 922 genes overlapping between the two miRNAs. Gene Ontology functional enrichment analysis predicted that the two miRNAs were involved in the regulation of nuclear factor κB (NF-κB) signaling. Overexpression of these two miRNAs leads to changes in p65 nuclear translocation. Using 3' untranslated region luciferase assay, we identified 20 genes within the NF-κB signaling cascade as putative targets of miRs-21-3p and -27a-5p, implicating these two miRNAs as modulators of NF-κB signaling in ECs.

Investigating the Role of the Post-transcriptional Gene Regulator MiR-24-3p in the Proliferation, Migration and Apoptosis of Human Arterial Smooth Muscle Cells in Arteriosclerosis Obliterans

Aims: To explore the expression of miR-24-3p in human arteries with arteriosclerosis obliterans (ASO) as well as the role of miR-24-3p in the pathogenesis of ASO. Methods: We used quantitative real-time PCR (qRT-PCR) and in situ hybridization to monitor miR-24-3p expression in human arteries. To investigate the effect of miR-24-3p on human arterial smooth muscle cells (HASMCs), we applied cell counting and EdU assays to monitor proliferation and transwell and wound healing assays to investigate migration and flow cytometry to investigate apoptosis. Furthermore, we applied 3'-untranslated region (3'-UTR) luciferase assays to investigate the role of miR-24-3p in targeting platelet-derived growth factor receptor B (PDGFRB) and c-Myc. Results: MiR-24-3p was mainly located in the media of arteries and was downregulated in ASO arteries compared with normal arteries. Platelet-derived growth factor BB (PDGF-BB) treatment reduced the expression of miR-24-3p in primary cultured HASMCs. MiR-24-3p mimic oligos inhibited the proliferation and migration, and promotes apoptosis of HASMCs. Our 3'-UTR luciferase assays confirmed that PDGFRB and c-Myc were targets of miR-24-3p. Conclusion: The results suggest that miR-24-3p regulates the proliferation and migration of HASMCs by targeting PDGFRB and c-Myc. The PDGF/miR-24-3p/PDGFRB and PDGF/miR-24-3p/c-Myc pathways may play critical roles in the pathogenesis of ASO. These findings highlight the potential for new therapeutic targets for ASO.

MiR-328 promotes glioma cell invasion via SFRP1-dependent Wnt-signaling activation

Background Diffusely infiltrative growth of human astrocytic gliomas is one of the major obstacles to successful tumor therapy. Thorough insights into the molecules and pathways signaling glioma cell invasion thus appear of major relevance for the development of targeted and individualized therapies. By miRNA expression profiling of microdissected human tumor biopsy specimens we identified miR-328 as one of the main miRNAs upregulated in invading glioma cells in vivo and further investigated its role in glioma pathogenesis. Methods We employed miRNA mimics and inhibitors to functionally characterize miR-328, 3' untranslated region luciferase assays, and T-cell factor/lymphoid enhancer factor reporter assays to pinpoint miR-328 targets and signaling pathways, and analyzed miR-328 expression in a large panel of gliomas. Results First, we corroborated the invasion-promoting role of miR-328 in A172 and TP365MG glioma cells. Secreted Frizzled-related protein 1 (SFRP1), an inhibitor of Wnt signaling, was then pinpointed as a direct miR-328 target. SFRP1 expression is of prognostic relevance in gliomas with reduced expression, being associated with significantly lower overall patient survival in both the Repository of Molecular Brain Neoplasia Data (REMBRANDT) and The Cancer Genome Atlas. Of note, miR-328 regulated both SFRP1 protein expression levels and Wnt signaling pathway activity. Finally, in human glioma tissues miR-328 appeared to account for the downregulation of SFRP1 preferentially in lower-grade astrocytic gliomas and was inversely related to SFRP1 promoter hypermethylation. Conclusion Taken together, we report on a novel molecular miR-328-dependent mechanism that via SFRP1 inhibition and Wnt activation contributes to the infiltrative glioma phenotype at already early stages of glioma progression, with unfavorable prognostic implications for the final outcome of the disease.

MicroRNA Deregulation in Right Ventricular Outflow Tract Myocardium in Nonsyndromic Tetralogy of Fallot

BackgroundTetralogy of Fallot (TOF) is 1 of the most common heart defects in children, and the underlying mechanisms remain largely elusive. MicroRNAs (miRNAs) are a class of regulators of gene expression and are increasingly recognized for their roles in heart development. MethodsTo identify miRNAs abnormally expressed in TOF, microarrays were used to analyze the miRNA expression profiles of 5 samples of myectomy tissues from right ventricular outflow tract (RVOT) obstruction of infants with nonsyndromic TOF and 3 age-matched normal RVOT tissues. ResultsIn total, 41 candidate miRNAs were identified. To further validate the microarray results, the 41 miRNAs were detected using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) in a larger independent population of tissue samples, including 21 from patients with TOF and 6 from normal controls; it was found that 18 miRNAs were expressed at significantly different levels. Bioinformatic analysis revealed that these miRNAs targeted a network of genes involved in heart development and human congenital heart diseases. Further in vitro studies indicated that upregulation of miR-424/424* promoted proliferation and inhibited migration of primary embryonic mouse cardiomyocytes, whereas miR-222 promoted cardiomyocyte proliferation and reduced the cardiomyogenic differentiation of P19 cells. The 3'UTR (3' untranslated region) luciferase assay revealed that miR-424/424* suppressed the expression of HAS2 and NF1, and their mRNAs were underexpressed in the RVOT myocardial tissues of TOF. ConclusionsEighteen miRNAs were identified as being deregulated in RVOT myocardial tissues from infants with nonsyndromic TOF, and in vitro experiments indicated that miR-424/424* and miR-222 are involved in cardiomyocyte proliferation and migration and the cardiomyogenic differentiation of P19 cells.

Targeting of syndecan-1 by micro-ribonucleic acid miR-10b modulates invasiveness of endometriotic cells via dysregulation of the proteolytic milieu and interleukin-6 secretion

To study the function of syndecan-1 (SDC1) and its potential regulator miR-10b in endometriosis. Experimental laboratory study. University medical center. Not applicable. The human endometriotic cell line 12Z was transiently transfected with SDC1 small interfering RNA or miR-10b precursors and investigated for changes in cell behavior and gene expression. 12Z and primary eutopic endometrial stroma cells of two American Society for Reproductive Medicine class III endometriosis patients were transfected with miR-10b precursors to investigate posttranscriptional regulation of SDC1. Quantitative polymerase chain reaction, Western blotting, flow cytometry, 3' untranslated region luciferase assays, and zymography were used to measure miR-10b-dependent targeting of SDC1 and SDC1-dependent expression changes of proteases and interleukin-6. Altered cell behavior was monitored by Matrigel invasion assays, cell viability assays, and mitogen-activated protein kinase activation blots. Knockdown of SDC1 inhibited Matrigel invasiveness by >60% but did not affect cell viability. Interleukin-6 secretion, matrix metalloproteinase-9 expression, and matrix metalloproteinase-2 activity were reduced, whereas plasminogen activator inhibitor-1 protein expression was up-regulated. miR-10b overexpression significantly down-regulated SDC1, reduced Matrigel invasiveness by 20% and cell viability by 14%, and decreased mitogen-activated protein kinase activation in response to hepatocyte growth factor. Syndecan-1, a target of miR-10b, inhibits epithelial endometriotic cell invasiveness through down-regulation of metalloproteinase activity and interleukin-6.

Cullin 4B Is Recruited to Tristetraprolin-Containing Messenger Ribonucleoproteins and Regulates TNF-α mRNA Polysome Loading

TNF-α is a central mediator of inflammation and critical for host response to infection and injury. TNF-α biosynthesis is controlled by transcriptional and posttranscriptional mechanisms allowing for rapid, transient production. Tristetraprolin (TTP) is an AU-rich element binding protein that regulates the stability of the TNF-α mRNA. Using a screen to identify TTP-interacting proteins, we identified Cullin 4B (Cul4B), a scaffolding component of the Cullin ring finger ligase family of ubiquitin E3 ligases. Short hairpin RNA knockdown of Cul4B results in a significant reduction in TNF-α protein and mRNA in LPS-stimulated mouse macrophage RAW264.7 cells as well as a reduction in TTP protein. TNF-α message t(1/2) was reduced from 69 to 33 min in LPS-stimulated cells. TNF-3' untranslated region luciferase assays utilizing wild-type and mutant TTP-AA (S52A, S178A) indicate that TTP function is enhanced in Cul4B short hairpin RNA cells. Importantly, the fold induction of TNF-α mRNA polysome loading in response to LPS stimulation is reduced by Cul4B knockdown. Cul4B is present on the polysomes and colocalizes with TTP to exosomes and processing bodies, which are sites of mRNA decay. We conclude that Cul4B licenses the TTP-containing TNF-α messenger ribonucleoprotein for loading onto polysomes, and reduction of Cul4B expression shunts the messenger ribonucleoproteins into the degradative pathway.