Overexpression Of miR-21 Research Articles

MicroRNA-206 improves alcohol-mediated decreased myoblast differentiation

Background: With recent advances in antiretroviral therapy (ART), people living with HIV (PLWH) have a near-normal life expectancy but have higher risk for aging-related comorbidities, such as metabolic disorders and frailty earlier in life than the general population. At-risk alcohol use is twice as likely in PLWH compared to the general population and alcohol-related myopathy occurs in 40- 60% of people with alcohol use disorder. Previous studies demonstrated that in vivo chronic binge alcohol (CBA) administration in simian immunodeficiency virus (SIV)-infected male rhesus macaques decreased myoblast differentiation and downregulated microRNA-206 (miR-206) expression in skeletal muscle. We hypothesized that increasing miR-206 expression would increase differentiation in CBA-derived primary macaque myoblasts. Methods: Eight SIV-infected female rhesus macaques received either water (VEH) or CBA (13- 14 g/kg/week) for 14.5 months. Three months into either VEH or CBA administration, animals were vaginally infected with SIVmac251, and ART was started 2.5 months later. Animals were sacrificed after 9 months, and primary myoblasts isolated from vastus lateralis muscle 24 hours after the last dose of alcohol (blood alcohol=0 mM). Myoblasts at passage 4 (VEH & CBA) were proliferated for three days. Cells were then transfected with either miR-206 mimic or control (miR scramble). Four experimental groups were studied at day 5 of differentiation: VEH+scramble, CBA+scramble, VEH+miR-206, and CBA+miR-206. Expression of miR-206 was determined by qPCR. Myoblast differentiation was determined by myotube density, calculated as the average number of myotubes per frame, and fusion index calculated as the percentage of myonuclei that fused into myotubes. Results: In non-transfected CBA-derived cells, myotube density and miR-206 expression was lower compared to VEH-derived myotubes (p<0.001 and p<0.05, respectively). miR-206 transfection resulted in over 150-fold higher miR-206 expression compared to VEH-scramble (p<0.05) in CBA- and VEH-derived cells. miR-206 overexpression significantly increased myotube fusion index (p<0.05) compared to myoblasts transfected with miR scramble in CBA-derived cells. Conclusions: Data show significant improvement of alcohol-mediated decreased myoblast differentiation with miR-206 overexpression. These findings suggest that increasing muscle miR-206 can potentially be used as a therapeutic strategy to improve regenerative capacity in response to muscle injury or atrophy. (Supported by: T35AA021097, F30AA029358, P60AA009803, K01AA024494) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

MiR-210 regulates lung adenocarcinoma by targeting HIF-1α

ObjectThis study sought to elucidate the role of microRNA-210 (miR-210) in the occurrence and development of lung adenocarcinoma (LUAD). MethodsThe levels of lncRNA miR-210HG and miR-210 in LUAD tissues and corresponding normal tissues were analyzed by real-time quantitative PCR. The expression of the anti-hypoxia factor hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) were measured by qRT-PCR and Western blot. The target of miR-210 on HIF-1α was confirmed using TCGA, Western blot and luciferase reporter assay. The regulatory role of miR-210 on HIF-1α and VEGF in LUAD was investigated. The correlation of genes with clinical prognosis was analyzed using bioinformatics methods. The effect of miR-210 on LUAD cells was verified through apoptosis assays. ResultsThe expression of miR-210 and miR-210HG was significantly higher in LUAD tissues than in normal tissues. The expression of hypoxia-related indicators HIF-1α and VEGF was also significantly higher in LUAD tissues. MiR-210 suppressed HIF-1α expression by targeting site 113 of HIF-1α, thereby affecting VEGF expression. Overexpression of miR-210 inhibited HIF-1 expression by targeting the 113 site of HIF-1, thereby affecting VEGF expression. Conversely, inhibition of miR-210 resulted in a significant increase in HIF-1α and VEGF expression in LUAD cells. In TCGA-LUAD cohorts, the expression of VEGF-c and VEGF-d genes in LUAD tissues was significantly lower than in normal tissues, while overall survival was worse in LUAD patients with high expression of HIF-1α, VEGF-c and VEGF-d. Apoptosis was significantly lower in H1650 cells after miR-210 inhibition. ConclusionThis study reveals that miR-210 exerts an inhibitory effect on VEGF expression by down-regulating HIF-1α expression in LUAD. Conversely, inhibition of miR-210 significantly reduced H1650 apoptosis and led to worse patient survival by upregulating HIF-1α and VEGF. These results suggest that miR-210 could serve as a potential therapeutic target for the treatment of LUAD.

MiR-22-enriched breast cancer cells display repressed glycolytic metabolism, increased glycogen synthesis, and reduced survival in low glucose conditions.

Breast cancer (BC) is the second leading cause of cancer-related mortality among women. Beyond the established tumourigenic role of genetic mutations, metabolic reprogramming is another key cancer hallmark. Glucose metabolism in particular is known to be prominently altered in tumours, in order to support biomass accumulation and cancer cell survival. The tumor suppressor microRNA (miRNA) miR-22 has been previously associated with a plethora of BC phenotypes such as growth, invasion-metastasis, and regulation of metabolic phenotypes such as lipid and folate metabolism. In this study, we aimed to investigate the role of miR-22 in the regulation of glucose metabolism in BC cells. Here we examined how miR-22 affects glucose metabolism in the MCF-7 BC cells. We found that over-expression of miR-22 caused a reduced glycolytic rate in these cells. Moreover, the miRNA also rendered MCF-7 cells more sensitive to lower glucose levels. We next unbiasedly screened the transcript levels of 84 genes relevant to glucose metabolism using the Human Glucose RT2 Profiler PCR Array. Interestingly, the strongest effect identified by this screen was the upregulation of genes involved in glycogen synthesis and the repression of gene involved in glycogen catabolism. Examination of publicly available transcriptomic datasets confirmed the correlations between expression of miR-22 and these glycogen metabolism genes in BC cells. This study has generated evidence for a regulatory role of miR-22 in glucose and glycogen metabolism, expanding the involvement of this miRNA in BC metabolic reprogramming.

MicroRNA-31 induced by Fusobacterium nucleatum infection promotes colorectal cancer tumorigenesis

Persistent Fusobacterium nucleatum infection is associated with the development of human colorectal cancer (CRC) and promotes tumorigenicity, but the underlying mechanisms remain unclear. Here, we reported that F.nucleatum promoted the tumorigenicity of CRC, which was associated with F.nucleatum-induced microRNA-31 (miR-31) expression in CRC tissues and cells. F.nucleatum infection inhibited autophagic flux by miR-31 through inhibiting syntaxin-12 (STX12) and was associated with the increased intracellular survival of F.nucleatum. Overexpression of miR-31 in CRC cells promoted their tumorigenicity by targeting eukaryotic initiation factor 4F-binding protein 1/2 (eIF4EBP1/2), whereas miR-31 knockout mice were resistant to the formation of colorectal tumors. In conclusion, F.nucleatum, miR-31, and STX12 form a closed loop in the autophagy pathway, and continuous F.nucleatum-induced miR-31 expression promotes the tumorigenicity of CRC cells by targeting eIF4EBP1/2. These findings reveal miR-31 as a potential diagnostic biomarker and therapeutic target in CRC patients with F.nucleatum infection.

Tumor suppressor miR-218 directly targets epidermal growth factor receptor (EGFR) expression in triple-negative breast cancer, sensitizing cells to irradiation

PurposeMicroRNA-218 (miR-218) is a key regulator of numerous processes relevant to tumor progression. In the present study, we aimed to characterize the relationship between miR-218 and the Epidermal Growth Factor Receptor (EGFR) as well as to understand downstream effects in triple-negative breast cancer (TNBC).MethodsWe assessed miR-218 and EGFR expression in cell lines and publicly available primary breast cancer gene expression data. We then overexpressed miR-218 in two TNBC cell lines and investigated effects on EGFR and downstream mitogen-activated protein (MAP) kinase signaling. Luciferase reporter assay was used to characterize a direct binding interaction between miR-218 and EGFR mRNA. Digital holographic microscopy helped investigate cell migration and dry mass after miR-218 overexpression. Cell division and invasion were assessed microscopically, while radiation response after miR-218 overexpression alone or combined with additional EGFR knockdown was investigated via clonogenic assays.ResultsWe found an inverse correlation between EGFR expression and miR-218 levels in cell lines and primary breast cancer tissues. MiR-218 overexpression resulted in a downregulation of EGFR via direct binding of the mRNA. Activation of EGFR and downstream p44/42 MAPK signaling were reduced after pre-miR-218 transfection. Cell proliferation, motility and invasiveness were inhibited whereas cell death and mitotic catastrophe were upregulated in miR-218 overexpressing cells compared to controls. MiR-218 overexpressing and EGFR siRNA-treated cells were sensitized to irradiation, more than miR-218 overexpressing cells alone.ConclusionThis study characterizes the antagonistic relationship between miR-218 and EGFR. It also demonstrates downstream functional effects of miR-218 overexpression, leading to anti-tumorigenic cellular changes.

Total saponins from Panax japonicus reduce inflammation in adipocytes through the miR155/SOCS1/NFκB signaling pathway

BackgroundThe rising incidence of metabolic diseases due to chronic inflammation in the adipose tissue has been attributed to factors such as high fat diet (HFD). Previous studies have demonstrated that the total saponins from Panax japonicus (TSPJ) can reduce HFD-induced adipocyte inflammation, but the underlying mechanism remains unclear. In this work, we explored the molecular mechanism by which TSPJ reduces inflammation response in adipocytes. MethodsWe first established C57BL/6 mouse and 3T3-L1 adipocyte models. Lentiviruses packaged with the plasmids were injected into mice through the tail vein or into adipocytes to generate the in vivo and in vitro models with miR155 knockdown and overexpression. The mice were fed with HFD to trigger inflammation and administered TSPJ (25 mg/kg∙d and 75 mg/kg∙d) by gavage. The adipocytes were treated with palmitic acid (PA) to trigger inflammation response, then treated with TSPJ (25 μg/ml and 50 μg/ml). Finally, the expression of miR155, inflammatory factors, SOCS1, and NFκB pathway-related proteins was explored. ResultsTSPJ significantly inhibited the expression of inflammation-related genes and the miR155 expression in adipocytes both in vitro and in vivo. The dual luciferase reporter gene assay revealed that miR155 mediated the downregulation of SOCS1. TSPJ significantly inhibited and upregulated the phosphorylation of the NFκB protein and the SOCS1 proteins, respectively. ConclusionTSPJ inhibits miR155 to upregulate the SOCS1 expression, which subsequently inhibits the NFκB signaling pathway, thereby mitigating the inflammatory response in the adipocytes of HFD mice.

Potential involvement of miR-183/96/182 cluster-gene target interactions in transdifferentiation of human retinal pigment epithelial cells into retinal neurons

miR-183/96/182 cluster plays a critical role in the developing retina by regulating many target genes involved in signaling pathways. This study aimed to survey the miR-183/96/182 cluster-target interactions that, potentially contribute to human retinal pigmented epithelial (hRPE) cell differentiation into photoreceptors. Target genes of the miR-183/96/182 cluster were obtained from miRNA-target databases and applied to construct miRNA-target networks. Gene ontology and KEGG pathway analysis was performed. miR-183/96/182 cluster sequence was cloned into an eGFP-intron splicing cassette in an AAV2 vector and overexpressed in hRPE cells. The expression level of target genes including HES1, PAX6, SOX2, CCNJ, and RORΒ was evaluated using qPCR. Our results showed that miR-183, miR-96, and miR-182 share 136 target genes that are involved in cell proliferation pathways such as PI3K/AKT and MAPK pathway. qPCR data indicated a 22-, 7-, and 4-fold overexpression of miR-183, miR-96, and miR-182, respectively, in infected hRPE cells. Consequently, the downregulation of several key targets such as PAX6, CCND2, CDK5R1, and CCNJ and upregulation of a few retina-specific neural markers such as Rhodopsin, red opsin, and CRX was detected. Our findings suggest that the miR-183/96/182 cluster may induce hRPE transdifferentiation by targeting key genes that involve in the cell cycle and proliferation pathways.

A tumor microenvironment-stimuli responsive nano-prodrug for overcoming gemcitabine chemoresistance by co-delivered miRNA-21 modulator

Gemcitabine (Gem) has been recommended as a first-line clinical chemotherapeutics for pancreatic ductal adenocarcinoma (PDAC) treatment. Gem treatment could generate chemoresistance associated with abnormal expressions of multiple miRNAs. In the PDAC setting, miRNA-21 (miR-21) overexpression is an important contributing factor of inducing Gem chemoresistance. Inhibition of miR-21 can significantly increase Gem chemosensitivity, which requires an efficient delivery platform to conduct combinational Gem and miR-21 siRNA (miR-21i) therapy. Herein, we synthesized a tumor microenvironment (TME) stimuli-responsive poly(beta-amino ester)s (PBAE)-based polymer nano-prodrug (miR-21i@HA-Gem-SS-P12) that could co-deliver miR-21 siRNA and Gem. The disulfide linkages conjugating GEM onto PBAE can be triggered by elevated reduction stimulus in TME to release the cargo Gem. The hyaluronic acid (HA) fabrication further improved the drug accumulation at the tumor site. Benefiting from the multiple functional improvements and synergism between Gem and miR-21i, the miR-21i@HA-Gem-SS-P12 nano-prodrugs displayed superior tumor inhibition in PDAC in vitro and in vivo. This study established an effective stimuli-responsive nano-prodrug strategy for cooperative treatment with small molecule agents and nucleotide modulators in PDAC.

The role of regulatory micro-RNAs in inflammatory processes and production of tumor necrosis factor-alpha in patients with rheumatoid arthritis

Background. Micro-RNAs are fundamental agents of post-transcriptional control of gene expression. In recent years many works have appeared on the possible role of micro-RNAs in rheumatoid arthritis (RA). Studies of the role of micro-RNA and the relationship with the synthesis of tumor necrosis factor-α (TNF-α) are very promising for understanding the pathogenesis and treatment of RA and other autoimmune diseases. The purpose of the research was to study the role of regulatory micro-RNAs in inflammatory processes and the possible connection with the production of TNF-α in patients with RA. Materials and methods. 29 patients with active RA and 20 healthy individuals (control) were examined. All subjects were examined for 16 micro-RNAs. The choice of micro-RNA was based on previous studies and theoretical conclusions (according to the miRWalk database). Rheumatoid factor, the level of antibodies to cyclic peptides containing citrulline, C-reactive protein (СRP), levels of TNF-α (serum, spontaneous, and stimulated) were determined in the blood of patients. Results. Statistical analysis demonstrated significant overexpression of miR-221, miR-203, miR-146b, miR-132, miR-21 and miR-17-3p and inhibition of miR-223 synthesis in RA patients. The activation of TNF-α synthesis at rest and the increased production of TNF-α by mononuclear cells after stimulation in RA were shown. Differences in the levels of relative expression of some micro-RNAs between seropositive and seronegative groups of RA patients were found, but only hyperexpression of miR-155 was highly reliable. For the first time, a possible relationship between TNF-α production and miR-29 and miR-155 micro-RNAs, as well as a correlation between miR-16, miR-99b and miR-203 and CRP levels, was revealed. Conclusions. The obtained data on the profile of micro-RNAs in RA makes it possible to distinguish the most “interesting” micro-RNAs for further study of pathogenesis, their role in inflammation, to study the choice of TNF-α inhibitors, and predicting the effectiveness of that treatment.

MiR96- and miR182-driven regulation of cytoskeleton results in inhibition of glioblastoma motility.

Glioblastoma multiforme (GBM) is one of the most common forms of brain tumor. As an excessively invasive tumor type, GBM cannot be fully cured due to its invasion ability into healthy brain tissues. Therefore, molecular mechanisms behind GBM migration and invasion need to be deeply investigated for the development of effective GBM treatments. Cellular motility and invasion are strictly associated with the cytoskeleton, especially with actins and tubulins. Palladin, an actin-binding protein, tightly bundles actins during initial invadopodia and contraction fiber formations, which are essential for cellular motility. Spastin, a microtubule-binding protein, cuts microtubules into small pieces and acts on invadopodia elongation and cellular trafficking of invadopodia-associated proteins. Regulation of proteins such as spastin and palladin involved in dynamic reorganization of the cytoskeleton, are rapidly carried out by microRNAs at the posttranscriptional level. Therefore, determining possible regulatory miRNAs of spastin and palladin is critical to elucidate GBM motility. miR96 and miR182 down-regulate SPAST and PALLD at both transcript and protein levels. Over-expression of miR96 and miR182 resulted in inhibition of the motility. However, over-expression of spastin and palladin induced the motility. Spastin and palladin rescue of miR96- or miR182-transfected U251 MG cells resulted in diminished effects of the miRNAs and rescued the motility. Our results demonstrate that miR96 and miR182 over-expressions inhibit GBM motility by regulating cytoskeleton through spastin and palladin. These findings suggest that miR96 and miR182 should be investigated in more detail for their potential use in GBM therapy.

MiRNA-seq identification and clinical validation of CD138+ and circulating miR-25 in treatment response of multiple myeloma

BackgroundDespite significant advancements in multiple myeloma (MM) therapy, the highly heterogenous treatment response hinders reliable prognosis and tailored therapeutics. Herein, we have studied the clinical utility of miRNAs in ameliorating patients’ management.MethodsmiRNA-seq was performed in bone marrow CD138+ plasma cells (PCs) of 24 MM and smoldering MM (sMM) patients to analyze miRNAs profile. CD138+ and circulating miR-25 levels were quantified using in house RT-qPCR assays in our screening MM/sMM cohort (CD138+ plasma cells n = 167; subcohort of MM peripheral plasma samples n = 69). Two external datasets (Kryukov et al. cohort n = 149; MMRF CoMMpass study n = 760) served as institutional-independent validation cohorts. Patients’ mortality and disease progression were assessed as clinical endpoints. Internal validation was performed by bootstrap analysis. Clinical benefit was estimated by decision curve analysis.ResultsmiRNA-seq highlighted miR-25 of CD138+ plasma cells to be upregulated in MM vs. sMM, R-ISS II/III vs. R-ISS I, and in progressed compared to progression-free patients. The analysis of our screening cohort highlighted that CD138+ miR-25 levels were correlated with short-term progression (HR = 2.729; p = 0.009) and poor survival (HR = 4.581; p = 0.004) of the patients; which was confirmed by Kryukov et al. cohort (HR = 1.878; p = 0.005) and MMRF CoMMpass study (HR = 1.414; p = 0.039) validation cohorts. Moreover, multivariate miR-25-fitted models contributed to superior risk-stratification and clinical benefit in MM prognostication. Finally, elevated miR-25 circulating levels were correlated with poor survival of MM patients (HR = 5.435; p = 0.021), serving as a potent non-invasive molecular prognostic tool.ConclusionsOur study identified miR-25 overexpression as a powerful independent predictor of poor treatment outcome and post-treatment progression, aiding towards modern non-invasive disease prognosis and personalized treatment decisions.

Abstract 3760: MicroRNA-1 run down the growth and metastasis of small cell lung cancer

Abstract Small cell lung cancer (SCLC) is a highly aggressive and metastatic lung cancer subtype with universal relapse and poor prognosis. The lack of potential drug targets limits targeted therapies for SCLC patients. To track down the potential therapeutic molecules in SCLC, we performed micro-RNA sequencing from the serum samples of SCLC patients and compared with the bulk RNA-sequencing data from SCLC tumor tissues. A consistent downregulation of microRNA-1 (miR-1) was observed in the SCLC patient serum samples, cell lines, and tumor tissues compared to their matched normal control. Overexpression of miR-1 in SCLC cell lines decreased cell growth and oncogenic signaling. Metastatic studies using the intracardiac injection model of SCLC cell lines showed that miR-1 overexpression decreases distant organ metastasis. Interestingly, the loss of function studies using miR-1Zip/sponging showed increased tumorigenesis and metastasis in SCLC subcutaneous and intracardiac xenografts. Mechanistic investigations revealed the CXCR4/FOXM1/RRM2 axis as a unique downstream target of miR-1 in SCLC. We found that FOXM1 transcriptionally regulates the RRM2 expression by directly binding to its promoter site, and miR-1 modulates these interactions through CXCR4. The results of the present study provided a strong preclinical rationale that miR-1 has a high potential for developing innovative SCLC therapies. Citation Format: Parvez Khan, Jawed A. Siddiqui, Shailendra Kumar Maurya, Tamara Mirzapoiazova, Prakash G. Kshirsagar, Ramakanth Chirravuri Venkata, Sanjib Chaudhary, Ranjana Kanchan, Naveenkumar Perumal, Mahek Fatima, Md Arafat Khan, Asad Ur Rehman, Imayavaramban Lakshmanan, Sidharth Mahapatra, Prakash Kulkarni, Apar Kishor Ganti, Maneesh Jain, Ravi Salgia, Surinder Kumar Batra, Mohd Wasim Nasser. MicroRNA-1 run down the growth and metastasis of small cell lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3760.

Abstract 5843: miR-224 activates cancer-associated fibroblasts to promote cancer cell invasion in lung tumor microenvironment

Abstract Cancer-associated fibroblasts (CAFs) promote EMT (epithelial-mesenchymal transition), invasion, and metastasis of cancer cells in the tumor microenvironment. To identify microRNAs (miRNAs) associated with the reprogramming of normal lung fibroblasts (LFs) into CAFs, global miRNA expression was profiled in LFs and CAFs through Nanostring nCounter. miR-224 was one of the increased miRNAs in CAFs, and its expression was higher in tumors than in normal tissues in dbDEMC and TCGA_lung adenocarcinoma data. miR-224 levels were then checked by qRT-PCR in paraffine-embedded tumor samples from patients with lung adenocarcinoma. Kaplan-Meier survival analysis showed that high miR-224 expression was related to poor survival. These data show that lung cancer progression is promoted by miR-224. To discover the roles of increased miR-224 in CAFs, miR-224 was overexpressed by lentiviral infection in LFs. LF_miR-224 overexpression enhanced the expression of CAF activation markers including Fap, S100a4, and Vim. In spheroid invasion assay using co-culture of LFs and cancer cells, as an experimental model of the tumor microenvironment, LF_miR-224 promoted invasion of lung cancer cells compared with control LFs. In transwell migration assay, LF_miR-224 stimulated migration of lung cancer cells more than control LFs. Collectively, these results indicate that CAFs reprogrammed by miR-224 promote the migration and invasion of lung cancer cells. Citation Format: Seonyeong Oh, Inyoung Cheon, Young-Ho Ahn. miR-224 activates cancer-associated fibroblasts to promote cancer cell invasion in lung tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5843.

Role of miR-128/216a Regulating Isl1 Expression during Differentiation of Human Umbilical Cord Mesenchymal Stem Cells into Insulin-Producing Cells

Islet-1 (Isl1), a LIM homeodomain protein, is expressed in the embryonic pancreatic epithelium. As a key transcription factor, Isl1 can not only regulate insulin gene expression in normal glucose condition but also maintain β-cell function and impact pancreatic β-cell target genes. Some experiments have suggested that MicroRNA (miRNA) can play a critical role during the induction of insulin-producing cells (IPCs). However, it is unclear whether miRNA may regulate Isl1 expression during differentiation of human umbilical cord mesenchymal stem cells (HUMSCs) into IPCs. In this investigation, we induced HUMSCs into IPCs with a modified two-step protocol, activin A, retinoic acid (step1) and conophylline, nicotinamide (step2). To find the miRNA regulating Isl1 expression, we respectively used TargetScan, miRDB and RNAhybrid to predict and got the result, miR-128 and miR-216a. The miRNAs can inhibit Isl1 expression by dual luciferase assay. The results of real-time Polymerase Chain Reaction (PCR) showed that Isl1 expression level was almost reciprocal to that of miR-128 and miR-216a during differentiation of HUMSCs into IPCs. Furthermore, over-expression of miR-128 or miR-216a down-regulated expression levels of Isl1 and MafA. Therefore, miR-128 or miR-216a may regulate expression of islet-specific transcription factors to control differentiation of HUMSCs into IPCs.

MiR-155 regulates macrophage polarization and alleviates myocardial fibrosis through paracrine secretion of transforming growth factor-beta 1 in ischemic heart failure

This study investigated the mechanism underlying miR-155 in ischemic heart failure. The left ventricular ejection fraction (LVEF) in rats was detected by color Doppler echocardiography. After the myocardial tissues were stained with Masson and Sirius red respectively, TGF-β expression was detected by immunohistochemical staining. Additionally, rat cardiomyocytes were transfected with miR-155 inhibitor, miR-155 mimic, TGF-β agonist or TGF-β inhibitor followed by analysis of levels of miR-155 and TGF-β by real time quantitative PCR. M1 polarization index of lipopolysaccharides (LPS) and M2 type polarization indexes of IL-4, IL-10, and TGF-β were examined by western blot. Among the three groups of rats, the LVEF of rats in model group was the lowest (p < 0.05) but it was elevated in miR-155 group, whilst the accumulated collagen fibers and hyperplasia in the myocardium were reduced upon miR-155 treatment. Of note, the myocardial tissue of modeled rats had the highest TGF-β level, but miR-155 overexpression or inhibitor dramatically decreased TGF-β level as well as protein expressions of LPS, IL-4, IL-10, and TGF-β, with the lowest level in miR-155 group. Interestingly, TGF-β agonist treatment resulted in a decrease in LPS while IL-4, IL-10, and TGF-β inhibitor caused opposite outcome. miR-155 can regulate the level of macrophage polarization and paracrine TGF-β and overexpressed miR-155 abrogates the effect of TGF-β on cardiac fibers, alleviating the myocardial ischemic in heart failure. TGF-β can also affect the biological function of LPS. miR-155 might become a target for the treatment of ischemic heart failure and myocardial fibrosis.

Aberrant Expression of FGFRL1 in Esophageal Cancer and Its Regulation by miR-107.

Fibroblast growth factor receptors are growth factor receptor tyrosine kinases, exerting their roles in embryogenesis, tissue homeostasis, and development of cancer. However, little is known about the expression and function of FGFRL1 in esophageal cancer (EC). We systematically evaluated the expression of FGFRL1 in TCGA and GETex datasets followed by expression analysis in EC cell lines and clinical specimens using immunofluorescence (IF) and immunohistochemistry (IHC) respectively. GEPIA analysis on TCGA and GETex datasets identified significant upregulation of FGFRL1 in EC patients (n=182) compared to normal controls (n=286, p<0.05). IHC analysis showed significantly higher FGFRL1 expression in EC tissues as compared to the distant matched non-malignant tissues (p<0.001). Immunoflourescence in EC cells suggested increased expression of FGFRL1 from WDSCC (KYSE30) to MDSCC (KYSE140) and finally to PDSCC (KYSE410). In-silico tools predicted miR-107 as most significant miRNA regulating FGFRL1 expression. qRT-PCR revealed miR-107 expression to be significantly and inversely correlated with FGFRL1 expression in 73% (22/30) EC tissues (p=0.015) and over-expression of miR-107 resulted in significantly decreased expression of FGFRL1 at mRNA (fold change=0.11, p=0.0016) as well as protein level in miR-107 versus NC treated cells. Luciferase reporter assay using FGFRL1-3'UTR further confirmed it to be a direct target of miR-107. Our results herein document clinical as well as functional relevance of FGFRL1 in EC and its regulation by miR-107.

Bone Marrow Mesenchymal Stem Cell-Derived Exosomal miR-126 Inhibits Colon Cancer by Targeting PLEXIN-B2

Recent studies demonstrated that bone mesenchymal stem cells (BMSCs) can be recruited to the tumor microenvironment, and exosomes secreted by BMSCs have new function in the intercellular communication of human cancer. To explore the effects of human BMSCs-derived exosomal miR-126 on the proliferation, migration and invasion of colon cancer. BMSCs were transfected with mimic and inhibitor of miR-126, respectively. Then after BMSCs treated with mimic or inhibitor, we isolated exosomes from BMSCs. The viability, migration and invasion ability of Colon cells were detected via methyl thiazolyl tetrazolium (MTT) assay and Transwell assay, respectively. The targeting relation between miR-126 and plexin-B2 (PLXNB2) was verified by using bioinformatics analysis and dual luciferase reporter assay. The expressions of PLXNB2 and related proteins in Colon cells were determined by Western blot. miR-126 expressed higher in exosomes from BMSCs, compared with control group. Moreover, overexpression of miR-126 inhibited cell viability, migration and invasion. In addition, Exosomal miR-126 lead to targeted inhibition of PLXNB2 in Colon cells. What’s more, according to the analysis of exosome content, miR-126 could mediate the inhibitory effect of exosomes on HCT116 and SW620 cells via negative regulating of PLXNB2. The results of our study showed that BMSCs-derived exosomal miR-126 could inhibit cell viability, cell migration and cell invasion.

Transplantation of endothelial progenitor cells overexpressing mir-126-3p improves vascular repair in a diabetic rat model.

Transplantation of endothelial progenitor cells overexpressing mir-126-3p improves vascular repair in a diabetic rat model.

MiR-194 Up-Regulates Cholesterol 7 Alpha-Hydroxylase Expression via β-Catenin Signaling and Aggravates Cholestatic Liver Diseases

miR-194 is abundantly expressed in hepatocytes, and its depletion increases hepatic resistance to acetaminophen-induced acute injuries. In this study, the biological role of miR-194 in cholestatic liver injury was investigated by using miR-194/miR-192 cluster liver-specific knockout (LKO) mice, in which no liver injuries or metabolic disorders were predisposed. Bile duct ligation (BDL) and 1-naphthyl isothiocyanate (ANIT) were applied to LKO and matched control wild-type (WT) mice to induce hepatic cholestasis. Periportal liver damage, mortality rate, and liver injury biomarkers in LKO mice were significantly less than in WT mice after BDL and ANIT injection. Intrahepatic bile acid level was significantly lower in the LKO liver within 48 hours of BDL- and ANIT-induced cholestasis compared with WT. Western blot analysis showed that β-catenin (CTNNB1) signaling and genes involved in cellular proliferation were activated in BDL- and ANIT-treated mice. The expression levels of cholesterol 7 alpha-hydroxylase (CYP7A1), pivotal in bile synthesis, and its upstream regulator hepatocyte nuclear factor 4α were reduced in primary LKO hepatocytes and liver tissues compared with WT. The knockdown of miR-194 using miRNA inhibitors reduced CYP7A1 expression in WT hepatocytes. In contrast, the knockdown of CTNNB1 and overexpression of miR-194, but not miR-192, in LKO hepatocytes and AML12 cells increased CYP7A1 expression. In conclusion, the results suggest that the loss of miR-194 ameliorates cholestatic liver injury and may suppress CYP7A1 expression via activation of CTNNB1 signaling.

Abstract PD2-10: MiR-150 expression in breast cancer cells attract infiltration of lymphocytes to tumor microenvironment and is associated with better survival of the patients

Abstract MicroRNA (miRNA) are known as a key player in tumor biology and is shown to epigenetically regulate a large number of protein-coding genes, including tumor-related genes. MiR-150, a hematopoietic cell-specific miRNA, has been suggested to have various effects on cell proliferation, differentiation, apoptosis, migration, and invasion. However, there has been no study that investigated the role of miR-150 in the tumor microenvironment (TME) of breast cancer patients. We hypothesized that miR-150 expressed in breast cancer cells attract infiltration of lymphocytes to tumor microenvironment and is associated with better survival of the patients. In silico analyses of 1961 breast cancer patients were performed using multiple independent large cohorts, and in vitro studies by overexpressing miR-150 using its mimic in triple negative breast cancer (TNBC) cell lines were conducted. We found that miR-150 expression in patient breast tumors were strongly correlated with immune-related gene set scores, Allograft rejection, IL6/JAK/STAT3 signaling, Interferon-γ response, Inflammatory response, IL2/STAT5 signaling, and complement, in Hallmark collection of gene set variation analyses consistently in both METABRIC and TCGA cohorts (all spearman’s rank correlation coefficient (r) &amp;gt; 0.50, all p &amp;lt; 0.01). MiR-150 expression was also strongly correlated with cytolytic activity (CYT) score in both cohorts (r = 0.824 and 0.786, respectively, both p &amp;lt; 0.01). Furthermore, miR-150 expression was significantly correlated with infiltrating fraction of CD8+ T cells (r = 0.799 and 0.525, respectively), CD4+ memory T cells (r = 0.759 and 0.656, respectively), dendritic cells (r = 0.735 and 0.696, respectively), and B cells (r = 0.759 and 0.576, respectively), as well as mRNA expression of major immune checkpoint molecules, including PD-1, CTLA4, IDO1, TIGIT, BTLA, and LAG3, in both cohorts (all r &amp;gt; 0.50, and all p &amp;lt; 0.01). MiR-150 expression in triple negative breast cancer (TNBC) was the highest among the subtypes (both p &amp;lt; 0.001). MiR-150 expression level was significantly correlated with Nottingham histologic grade (both p &amp;lt; 0.001). MiR-150 high tumor enriched not only immune-related gene sets but also apoptosis, KRAS signaling up, MTORC1, and p53 pathway by gene set enrichment analysis in both cohorts. High miR-150 expression patients were significantly associated with better overall survival (OS) in both cohorts (p &amp;lt; 0.001 and p = 0.030, respectively). Subgroup analysis revealed that a high miR-150 was associated with better OS in ER-positive/HER2-negative breast cancer in both cohorts (p = 0.002 and 0.044, respectively), and in TNBC in the METABRIC cohort (p = 0.006). Unexpectedly, overexpression of miR-150 by its mimic shown no significant effect on cell proliferation, migration, nor invasion consistently in two TNBC cell lines. On the other hand, overexpression of miR-150 by its mimic significantly attracted Jurkart immortalized lymphocytes consistently in two cell lines, which effect was abolished by addition of miR-150 inhibitor. Finally, we found that mimic overexpression of miR-150 significantly expressed multiple cytokines and multiple inflammatory signaling pathways which at least partially explained the increased attraction of T cells in patient samples and in vitro condition. In conclusion, miR-150 expression in breast cancer cells may be associated with better patient survival outcomes by attracting and activating immune cells in breast cancer TME. Citation Format: Masanori Oshi, Aparna Maiti, Raj.G Vaghjiani, Rongrong Wu, Li Yan, Akimitsu Yamada, Nitai Hait, Takashi Ishikawa, Itaru Endo, Kazuaki Takabe, Yoshihisa Tokumaru. MiR-150 expression in breast cancer cells attract infiltration of lymphocytes to tumor microenvironment and is associated with better survival of the patients [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD2-10.