Direct Target Of miR-124 Research Articles

Exercise increases MEF2A abundance in rat cardiac muscle by downregulating microRNA-223-5p

Exercise plays an important role in cardiac health and enhances the transport of glucose in cardiac muscle by increasing the glucose transporter-4 (GLUT4) content at the cell membrane. The GLUT4 gene is a target of myocyte enhancer transcription factor 2A (MEF2A). Several transcription factors are regulated by microRNAs (miRs), small non-coding RNAs that control gene expression at the posttranscriptional level. In this study we tested the hypothesis that exercise regulates the expression of miR-223 and that MEF2A is a direct target of miR-223. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot experiments showed that GLUT4 gene expression and protein abundance increased by 30 and 23%, respectively, in the microsomal fraction immediately after exercise, and had returned to control levels after 18 h. In contrast, the increase in GLUT4 in the membrane fraction was delayed. Exercise also increased the protein abundance of transcription factors involved in GLUT4 expression. Immediately after exercise, the protein abundance of MEF2A, nuclear respiratory factor 1 (NRF1), and forkhead box O1 (FOXO1) increased by 18, 30, and 40%, respectively. qRT-PCR experiments showed that miR-223-3p and miR-223-5p expression decreased immediately after exercise by 60 and 30%, respectively, and luciferase assays indicated that MEF2A is a target of the 5p strand of miR-223. Overexpression of miR-223-5p in H9c2 cells decreased the protein abundance of MEF2A. Our results suggest that the exercise-induced increase in GLUT4 content in cardiac muscle is partly due to the posttranscriptional increase in MEF2A protein abundance caused by the decrease in miR-223-5p expression. The exercise-induced decrease in miR-223-3p expression likely contributes to the increases in NRF1 and FOXO1 abundance and GLUT4 content.

MiR-145 as a Potential Biomarker and Therapeutic Target in Patients with Non-Small Cell Lung Cancer.

Our previous study found that miR-145 was downregulated in non-small cell lung cancer (NSCLC) tissues and that it could inhibit the cell proliferation in transfected NSCLC cells. In this study, we found that miR-145 was downregulated in NSCLC plasma samples compared to healthy controls. A receiver operating characteristic curve analysis indicated that plasma miR-145 expression was correlated with NSCLC in patient samples. We further revealed that the transfection of miR-145 inhibited the proliferation, migration, and invasion of NSCLC cells. Most importantly, miR-145 significantly delayed the tumor growth in a mouse model of NSCLC. We further identified GOLM1 and RTKN as the direct targets of miR-145. A cohort of paired tumors and adjacent non-malignant lung tissues from NSCLC patients was used to confirm the downregulated expression and diagnostic value of miR-145. The results were highly consistent between our plasma and tissue cohorts, confirming the clinical value of miR-145 in different sample groups. In addition, we also validated the expressions of miR-145, GOLM1, and RTKN using the TCGA database. Our findings suggested that miR-145 is a regulator of NSCLC and it plays an important role in NSCLC progression. This microRNA and its gene targets may serve as potential biomarkers and novel molecular therapeutic targets in NSCLC patients.

MiR-145 Modulates Fatty Acid Metabolism by Targeting FOXO1 to Affect SERBP1 Activity in Bovine Mammary Epithelial Cells

MicroRNA-mediated gene regulation is important for the regulation of fatty acid metabolism and synthesis. Our previous study uncovered that the miR-145 expression is higher in the lactating mammary gland of dairy cows than in the dry-period, but the underlying molecular mechanism is incompletely understood. In this study, we have investigated the potential role of miR-145 in bovine mammary epithelial cells (BMECs). We found that the expression of miR-145 gradually increased during lactation. CRISPR/Cas9-mediated knockout (KO) of miR-145 in BMECs results in the downregulated expression of fatty acid metabolism-associated genes. Further results revealed that miR-145 KO reduced total triacylglycerol (TAG) and cholesterol (TC) accumulation and altered the composition of intracellular fatty acids (C16:0, C18:0, and C18:1). Conversely, miR-145 overexpression had the opposite effect. Bioinformatics online program predicted that miR-145 targets the 3'-UTR of the Forkhead Box O1 (FOXO1) gene. Subsequently, FOXO1 was identified as a direct target of miR-145 by qRT-PCR, Western blot analysis, and luciferase reporter assay. Furthermore, siRNA-mediated silencing of FOXO1 promoted fatty acid metabolism and TAG synthesis in BMECs. Additionally, we observed the involvement of FOXO1 in the transcriptional activity of the sterol regulatory element-binding protein 1 (SREBP1) gene promoter. Overall, our findings indicated that miR-145 relieves the inhibitory effect of FOXO1 on SREBP1 expression by targeting FOXO1 and subsequently regulating fatty acid metabolism. Thus, our results provide valuable information on the molecular mechanisms for improving milk yield and quality from the perspective of miRNA-mRNA networks.

Abstract 3756: miR-145 and its gene targets as therapeutic target and prognostic biomarkers for non-small cell lung cancer

Abstract Introduction: Our previous study found that miR-145 was downregulated in non-small cell lung cancer (NSCLC) tissues and it could inhibit cell proliferation in transfected NSCLC cells. This study aimed to investigate the potential anticancer effect and tumor suppressive function of miR-145, to identify its gene targets, as well as to assess the role of its gene targets as prognostic biomarkers for NSCLC patients. Materials and Methods: Cell proliferation, transwell migration, and invasion assays were performed on three miR-145 mimics transfected NSCLC cells. The effect of miR-145 in the cell cycle in vitro was assessed by flow cytometry analysis. In vivo transfection of miR-145 was performed to examine the cancer growth in eight BALB/c nude mice. Luciferase reporter assay was used to identify the gene targets. To reveal the role of the identified gene targets in patient survival, the clinical data of 500 lung adenocarcinoma patients from The Cancer Genome Atlas (TCGA) database was used for Kaplan-Meier survival analysis. Results: Upregulation of miR-145 significantly inhibited proliferation, migration, and invasion in NSCLC cells in vitro. Flow cytometry analysis revealed that the transfection of miR-145 into NSCLC cells did not show any effect in the cell cycle. After 37 days, transfection of miR-145 showed a reduction in tumor size (49% smaller) and tumor weight (57% lighter) than mimic negative in vivo. MicroRNA target prediction database miRTarBase and luciferase reporter assays identified GOLM1 and RTKN as the direct targets of miR-145. TCGA survival analysis found that the high expressions of GOLM1 (p < 0.0001) and RTKN (p < 0.0045) were significantly correlated with poor overall survival in lung adenocarcinoma patients. Conclusion: Our results revealed that miR-145 inhibited proliferation, migration, and invasion, but did not affect cell cycle regulation in NSCLC. It also caused a reduction in tumor growth and weight. Most importantly, we identified two direct targets (GOLM1 and RTKN) of miR-145, in which RTKN was first identified as a miR-145 target in NSCLC. Moreover, both GOLM1 and RTKN play an imperative role in patients’ overall survival. Taken together, our findings suggest that miR-145 may serve as a molecular therapeutic target for NSCLC, and its gene targets GOLM1 and RTKN may serve as prognostic biomarkers. Their potential clinical applications warrant further investigation. Citation Format: William C. Cho, Chi F. Wong, Leo K. Li, Alvin H. Fong. miR-145 and its gene targets as therapeutic target and prognostic biomarkers for non-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 3756.

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.

MicroRNA-1 attenuates the growth and metastasis of small cell lung cancer through CXCR4/FOXM1/RRM2 axis

BackgroundSmall cell lung cancer (SCLC) is an aggressive lung cancer subtype that is associated with high recurrence and poor prognosis. Due to lack of potential drug targets, SCLC patients have few therapeutic options. MicroRNAs (miRNAs) provide an interesting repertoire of therapeutic molecules; however, the identification of miRNAs regulating SCLC growth and metastasis and their precise regulatory mechanisms are not well understood.MethodsTo identify novel miRNAs regulating SCLC, we performed miRNA-sequencing from donor/patient serum samples and analyzed the bulk RNA-sequencing data from the tumors of SCLC patients. Further, we developed a nanotechnology-based, highly sensitive method to detect microRNA-1 (miR-1, identified miRNA) in patient serum samples and SCLC cell lines. To assess the therapeutic potential of miR-1, we developed various in vitro models, including miR-1 sponge (miR-1Zip) and DOX-On-miR-1 (Tet-ON) inducible stable overexpression systems. Mouse models derived from intracardiac injection of SCLC cells (miR-1Zip and DOX-On-miR-1) were established to delineate the role of miR-1 in SCLC metastasis. In situ hybridization and immunohistochemistry were used to analyze the expression of miR-1 and target proteins (mouse and human tumor specimens), respectively. Dual-luciferase assay was used to validate the target of miR-1, and chromatin immunoprecipitation assay was used to investigate the protein-gene interactions.ResultsA consistent downregulation of miR-1 was observed in tumor tissues and serum samples of SCLC patients compared to their matched normal controls, and these results were recapitulated in SCLC cell lines. Gain of function studies of miR-1 in SCLC cell lines showed decreased cell growth and oncogenic signaling, whereas loss of function studies of miR-1 rescued this effect. Intracardiac injection of gain of function of miR-1 SCLC cell lines in the mouse models showed a decrease in distant organ metastasis, whereas loss of function of miR-1 potentiated growth and metastasis. Mechanistic studies revealed that CXCR4 is a direct target of miR-1 in SCLC. Using unbiased transcriptomic analysis, we identified CXCR4/FOXM1/RRM2 as a unique axis that regulates SCLC growth and metastasis. Our results further showed that FOXM1 directly binds to the RRM2 promoter and regulates its activity in SCLC.ConclusionsOur findings revealed that miR-1 is a critical regulator for decreasing SCLC growth and metastasis. It targets the CXCR4/FOXM1/RRM2 axis and has a high potential for the development of novel SCLC therapies.Graphical MicroRNA-1 (miR-1) downregulation in the tumor tissues and serum samples of SCLC patients is an important hallmark of tumor growth and metastasis. The introduction of miR-1 in SCLC cell lines decreases cell growth and metastasis. Mechanistically, miR-1 directly targets CXCR4, which further prevents FOXM1 binding to the RRM2 promoter and decreases SCLC growth and metastasis.

The miR-106b-25 cluster mediates drug resistance in myeloid leukaemias by inactivating multiple apoptotic genes.

Drug resistance is a major obstacle to the successful treatment of cancer. The role of the miR-106b-25 cluster in drug resistance of haematologic malignancies has not yet been elucidated. Here, we show that the miR-106b-25 cluster mediates resistance to therapeutic agents with structural and mechanistic dissimilarity in vitro and in vivo. RNA sequencing data revealed that overexpression of the miR-106b-25 cluster or its individual miRNAs resulted in downregulation of multiple key regulators of apoptotic pathways. Luciferase reporter assay identified TP73 as a direct target of miR-93 and miR-106b, BAK1 as a direct target of miR-25 and CASP7 as a direct target of all three miRNAs. We also showed that inhibitors of the miR-106b-25 cluster and BCL-2 exert synergistic effects on apoptosis induction in primary myeloid leukaemic cells. Thus, the members of the miR-106b-25 cluster may jointly contribute to myeloid leukaemia drug resistance by inactivating multiple apoptotic genes. Targeting this cluster could be a promising combination strategy in patients resistant to therapeutic agents that induce apoptosis.

MiR-223 Exclusively Impairs In Vitro Tumor Growth through IGF1R Modulation in Rhabdomyosarcoma of Adolescents and Young Adults.

Adolescents and young adults (AYA) with rhabdomyosarcoma (RMS) form a subgroup of patients whose optimal clinical management and best possible access to care remain a challenge and whose survival rates lag behind that of children diagnosed with histologically similar tumors. A better understanding of tumor biology that differentiates children (PEDS-) from AYA-RMS could provide critical information and drive new initiatives to improve their final outcome. We investigated the functional role of miRNAs implicated in AYA-RMS development, as they have the potential to lead to discovery of new targets pathways for a more tailored treatment in these age groups of young RMS patients. MiR-223 and miR-486 were observed de-regulated in nine RMS tissues compared to their normal counterparts, yet only miR-223 replacement impaired proliferation and aggressiveness of AYA-RMS cell lines, while inducing apoptosis and determining cell cycle arrest. Interestingly, IGF1R resulted in the direct target of miR-223 in AYA-RMS cells, as demonstrated by IGF1R silencing. Our results highlight an exclusive functional role of miR-223 in AYA-RMS development and aggressiveness.

MicroRNA-223 and microRNA-126 are clinical indicators for predicting the plaque stability in carotid atherosclerosis patients.

Studies have demonstrated the essential functions of microRNAs (miRNAs) in cardiovascular disease. Herein, we explored the roles of miR-126 and miR-223 in the prediction of plaque stability in carotid atherosclerosis (CA).Patients with CA (N = 52) and healthy volunteers (N = 25) were recruited as the study subjects and controls. First, a miRNA microarray was performed to analyze the differentially expressed miRNAs in the serum of normal controls and patients with CA. Next, the correlations of miR-223 and miR-126 expression with plaque stability-related factors were analyzed. Then, the predictive efficacy of miR-223 and miR-126 on plaque stability was analyzed by the ROC curve, and the targeting relationships of miR-223 and miR-126 with COX2 were verified. Finally, the relationship between COX2 expression and CA plaque stability was analyzed. miR-223 and miR-126 were decreased in the serum of CA patients and had good diagnostic efficacy for CA. miR-223 and miR-126 in the serum of CA patients with unstable plaques were lower than that in patients with stable plaques. miR-223 and miR-126 were negatively correlated with plaque instability-related indicators, while COX2, a direct target of miR-223 and miR-126, was positively related to plaque instability-related indicators. Lowly expressed miR-223 and miR-126 in the serum of CA patients can be used as indicators for plaque stability.

Hypoxia Modulates Melanoma Cells Proliferation and Apoptosis via miRNA-210/ISCU/ROS Signaling.

In this study, luciferase reporter assay was used to establish the relationship between miR-210 and ISCU. This research was performed on both cell lines (A2058, G361, and 293T) and tissue samples. We found that miR-210 was upregulated in hypoxia and was elevated in melanoma in comparison with adjacent normal tissues. Expression of ISCU protein was decreased in melanoma tissues, and ISCU gene is a direct target of miR-210. ISCU knockdown with miR-210 enhanced ROS production. The results of our study showed that miR-210/ISCU/ROS axis can serve as a novel therapeutic target in melanoma.

MicroRNA-497 prevents pancreatic cancer stem cell gemcitabine resistance, migration, and invasion by directly targeting nuclear factor kappa B 1.

Objectives: Cancer stem cells (CSCs) comprise a small population of cells in cancerous tumors and play a critical role in tumor resistance to chemotherapy. miRNAs have been reported to enhance the sensitivity of pancreatic cancer to chemotherapy. However, the underlying molecular mechanism requires better understanding.Methods: Cell viability and proliferation were examined with CCK8 assays. Quantitative real-time polymerase chain reaction was executed to assess mRNA expression. StarBase database was used to select the target genes of miRNA, which were further affirmed by dual luciferase assay. Transwell assay was used to analyze cell invasion and migration.Results: We proved that miR-497 could be obviously downregulated in pancreatic cancer tissues and CSCs from Aspc-1 and Bxpc-3 cells. In addition, inhibition of miR-497 evidently accelerated pancreatic CSC gemcitabine resistance, migration and invasion. Moreover, we revealed that nuclear factor kappa B 1 (NFκB1) was prominently upregulated in pancreatic cancer tissues and pancreatic CSCs, and NFκB1 was also identified as a direct target of miR-497. Furthermore, we demonstrated that overexpression of NFκB1 could also notably promote the viability, migration, and invasion of gemcitabine-treated pancreatic CSCs, but this effect could be partially abolished by miR-497 overexpression.Conclusions: Those findings suggest that miR-497 overexpression could suppress gemcitabine resistance and the metastasis of pancreatic CSCs and non-CSCs by directly targeting NFκB1.

MicroRNA-155 expression is associated with pulpitis progression by targeting SHIP1.

Pulpitis is a commonly seen oral inflammation condition in clinical practice, it can cause much pain for the patient and may induce infections in other systems. Much is still unknown for the pathogenic mechanism of pulpitis. In this work, we discovered that the expression of miR-155 was associated with dental pulpal inflammation both in vivo and in vitro. Our experiments of LPS stimulated odontoblast cell line MDPC-23 showed miR-155 could act as a positive regulator by increasing the production of pro-inflammatory cytokines IL-1β and IL-6 during inflammatory responses, whereas knockdown of miR-155 can reverse the effects. Bioinformatics analysis demonstrated that SHIP1 is a direct target of miR-155 in odontoblasts, this result was further verified at both mRNA and protein level. Inhibition of miR-155 resulted in the downregulation of inflammation factors, while co-transfection of si-SHIP1 and miR-155 inhibitor promoted the inflammatory responses. Treatment with miR-155 mimic or si-SHIP1 up-regulated the protein level of p-PI3K and p-AKT. By contrast, miR-155 inhibitor exerted the opposite effects. miR-155 mimics could upregulate the gene expression of IL-1β and IL-6. Co-transfection of LY294002 and miR-155 mimic attenuated the inflammatory responses. Consistent with in vitro results, miR-155-/- mice could alleviate inflammatory response, as well as decrease the activation of p-PI3K and p-AKT, whereas increase the activation of SHIP1. Our data revealed a novel role for miR-155 in regulation of dental pulpal inflammatory response by targeting SHIP1 through PI3K/AKT signaling pathway.

MiR-203 is an anti-obese microRNA by targeting apical sodium-dependent bile acid transporter

SummaryObesity is characterized by excessive fat deposition within the body. Bile acids (BA) are important regulators for controlling the absorption of lipid. Here we show that miR-203 exerts weight-loss and lipid-lowering effects by increasing total BA excretion in obese rodents. miR-203 overexpression transgenic mice are resistant to high-fat diet (HFD)-induced obesity and dyslipidemia. Moreover, the knockdown of miR-203 deteriorates metabolic disorders. ASBT plays important role in regulating BA homeostasis and is a direct target of miR-203. In human intestinal epithelial cells, overexpression of miR-203 decreases the cellular uptake of BA by inhibiting ASBT. Furthermore, TCF7L2 is downregulated in obese mice and acts as a transcription factor of miR-203. The ASBT mRNA level was positively correlated with the body mass index (BMI) of population, while the miR-203 level was negatively associated with BMI. Taken together, these data suggest miR-203 could be a new therapeutic BA regulator for obesity and dyslipidemia.

Abstract 1553: miR-1 targets Cdk6 and controls cell cycle progression and apoptosis in colitis-associated dysplasia

Abstract Detection of colorectal dysplasia during surveillance colonoscopy is currently the best method of determining risk of colitis-associated colorectal cancer (CAC). An understanding of the early molecular changes associated with the development of these lesions will inform the identification of new biomarkers for earlier detection. miRNAs (miRs), highly conserved noncoding RNAs, show great promise as stable, tissue-specific biomarkers of neoplasia. We previously identified 12 miRs that are differentially-expressed in colitis-associated dysplasias (flat and polypoid) vs. inflamed colonic mucosa from mice treated with AOM/DSS. miR-1, a putative tumor suppressor, was downregulated in colitis-associated dysplasias. Analysis of the mRNA expression profile of AOM/DSS-induced dysplasias and prediction of the interactions between the miRs and their targets led to the selection of the Cdk6 as the target of miR-1 to be further investigated, based on its: 1) upregulation in AOM/DSS-induced dysplasia; and 2) association with cell cycle progression and inflammatory signaling. The goal of the present study was to validate the predicted interaction between miR-1 and Cdk6 and assess the biological function of miR-1 in vitro. The ability of miR-1 to interact with the 3’UTR of Cdk6 mRNA was assessed using a dual luciferase assay. Co-transfection of HCT116 or RKO colon carcinoma cells with Cdk6-WT and miR-1 mimics led to a significant reduction in relative luciferase activity in both cell lines (30%, p=0.0117 and 47%, p=0.0269; respectively). Transfection with the Cdk6-Mut did not alter relative luciferase activity, confirming the Cdk6 binding site was specific for miR-1. The biological function of miR-1 was assessed in HCT116 and RKO cells reverse-transfected with miR-1 and cel-miR-67 (negative control) for 48 hrs. Apoptosis (AnnexinV+ cells) and cell cycle progression (% of cells in G0/G1, G2/M and S phase) were evaluated by flow cytometry, and proliferation by cell count (Trypan Blue). HCT116 and RKO transfected with the miR-1 mimic exhibited a higher proportion of apoptotic cells than the negative control (30% and 20% increase, p=0.0022 and 0.0013, respectively). Cell cycle analyses revealed the miR-1 mimic induced cell cycle arrest (G0/G1) in both cell lines (p&amp;lt;0.05). In HCT116 cells, this was accompanied by a reduction in the percentage of cells in G2/M (p=0.002) and S (p&amp;lt;0.001) phase. No effect of miR-1 on total cell number was observed. These results demonstrate that Cdk6 is a direct target of miR-1, and suggest that downregulation of this miR in dysplastic lesions contributes to CAC by inducing cell cycle progression and inhibiting apoptosis. These data provide novel insight into the early molecular changes that accompany the development of colitis-associated dysplasia and may serve as biomarkers for early detection of neoplasia. Supported by the Timothy P. and Aurora M. Hughes Fund for Colon Cancer Research. Citation Format: Mariana F. Fragoso, Geysson J. Fernandez, Lisa Vanderveer, Harry S. Cooper, Michael Slifker, Margie L. Clapper. miR-1 targets Cdk6 and controls cell cycle progression and apoptosis in colitis-associated dysplasia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1553.

[Effect of miR-203/CREB1 Signaling Regulation Mediated by DNA Methylation on the Proliferation and Apoptosis of Multiple Myeloma Cells].

To investigate the effect of miR-203/CREB1 signaling regulation mediated by DNA methylation on the proliferation, invasion and apoptosis of multiple myeloma (MM) cells. The methylation level of miR-203 in the RPMI 8226 cells was detected by bisulfite sequcucing polymerase chain reaction (BSP). The mRNA expression of miR-203 was measured by quantitative real-time polymerase chain reaction. RPMI 8226 cells were treated with DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR). The miR-203 mimic in MM cell line RPMI 8226 was transfected to establish overexpressed miR-203 cell. The proliferation, invasion ability and apoptosis of RPMI 8226 cell was detected by CCK-8 assay, Transwell, and flow cytometry, respectively. The targeting relationship between miR-203 and CREB1 was verified by double luciferase report assay. Western blot was used to detect the expression of CREB1 protein. Hypermethylation of miR-203 promoter region and low expression level of miR-203 mRNA were detected in the RPMI 8226 cells, which showed that demethylation could induce the expression of miR-203. The proliferation and invasion ability of RPMI 8226 cells after treated by 5-Aza-CdR were inhibited, and showed statistical significance as compared with blank control group (both P<0.05)，while the apoptosis rate was promoted (P<0.05). The proliferation, invasion ability and apoptosis of overexpressed miR-203 were the same as the demethylation group. Double luciferase report assay confirmed that CREB1 was the direct target of miR-203. The protein level of CREB1 was inhibited by demethylation and showed statistical significance as compared with control group (P<0.05). MiR-203 targeting CREB1 mediated by DNA methylation leads to maintain the malignant biological behaviors of MM cells.

MiR-203, fine-tunning neuroinflammation by juggling different components of NF\u2010\u03baB signaling

BackgroundmiR-203 was first indicated in maintaining skin homeostasis and innate immunity. Aberrant expression of miR-203 was found associated with pathological progressions of immune disorders, cancers, as well as neurodegenerations. Recently, increasing data on miR-203 in regulating neuroinflammation and neuronal apoptosis has raised extensive concern about the biological function of this microRNA.MethodsMouse model with ectopic miR-203 expression in the hippocampus was constructed by stereotactic injection of lentiviral expression vector of pre-miR-203. Association of miR-203 and mRNA of Akirin2, as well as the competition for miR-203 targeting between Akirin2 3ʹUTR and another recently characterized miR-203 target, 14-3-3θ, was verified using Dual-Luciferase Reporter Gene Assay and western blot. Microglia activation and pro-inflammatory cytokines expression in the hippocampus of mice overexpressing miR-203 was evaluated using immunohistochemistry analysis and western blot. Neuronal cell death was monitored using anti-caspase 8 in immunohistochemistry as well as TUNEL assay. Cognition of mice was assessed with a behavior test battery consisting of nesting behavior test, Barnes maze and fear conditioning test.ResultsAkirin2, an activator of NF‐κB signaling, was identified as a direct target of miR-203. By also targeting 14-3-3θ, a negative regulator of NF‐κB signaling, miR-203 displayed an overall pro-inflammatory role both in vitro and in vivo. Promoted nuclear translocation of NF‐κB and increased expression of proinflammatory cytokines were observed in cultured BV2 cells transfected with miR-203 mimics. Microglia activation and upregulation of NF‐κB, IL-1β and IL-6 were observed in mouse hippocampus with overexpression of miR-203. In addition, promoted neuronal cell death in the hippocampus and impaired neuronal activities resulted in cognitive dysfunction of mice with ectopic miR-203 expression in the hippocampus.ConclusionA pro-inflammatory and neurodisruptive role of miR-203 was addressed based on our data in this study. Given the identification of Akirin2 as a direct target of miR-203 and the competition with 14-3-3θ for miR-203 targeting, together with the findings of other signaling molecules in NF‐κB pathway as targets of miR-203, we proposed that miR-203 was a master modulator, fine-tunning neuroinflammation by juggling different components of NF‐κB signaling.

MiR-124 Regulates IQGAP1 and Participates in the Relationship Between Morphine Dependence Susceptibility and Cognition.

BackgroundLong-term excessive use of morphine leads to addictive diseases and affects cognitive function. Cognitive performance is associated with genetic characteristics.MiR-124 plays a critical regulatory role in neurogenesis, synaptic development, brain plasticity, and the use of addictive substances. As a scaffold protein, IQGAP1 affects learning and memory dose-dependent. However, the role of miR-124 and its target protein as potential addiction biomarkers and the impact on cognitive function have not been fully explored.MethodA total of 40 patients with morphine dependence and 40 cases of healthy people were recruited. We collected basic and clinical information about the two groups. The Generalized Anxiety Disorder Scale (GAD-7), Patient Health Questionnaire-9(PHQ-9), Montreal Cognition Assessment Scale (MoCA), Pittsburgh Sleep Quality Index (PSQI) were used to assess the severity of depression, anxiety, depressive symptoms, cognitive dysfunction, and sleep quality.ResultsCompared to the control group, the morphine-dependent group had higher GAD-7, PHQ-9, PSQI scores, and more elevated miR-124 levels but lower MOCA scores and IQGAP1 levels. MiR-124, IQGAP1, the average intake last year were related to OASI scores.MiR-124, IQGAP1, PHQ-9 were associated with MOCA scores. In the multiple regression model, the levels of miR-124 and IQGAP1 were independent factors influencing the severity of morphine dependence. The level of miR-124 was an independent factor influencing the severity of cognitive impairment in patients with morphine dependence. In addition, the luciferase report confirmed that IQGAP1 mRNA is the direct target of miR-124.ConclusionMiR-124 and its target protein IQGAP1 are involved in the regulation of addiction and cognitive function in patients with morphine dependence.

MiR-106b-25 Promotes Chemoresistance in Acute Myeloid Leukemia Via Abolishing Multiple Apoptotic Pathways

Introduction: Chemoresistance and disease relapse remain the main obstacles responsible for treatment failure in leukemia. MicroRNAs (miRNAs) play essential roles in various physiological and pathological processes, including cell proliferation, differentiation, metabolism, and cancer development. The miR-106b-25 cluster consists of three miRNAs: miR-106b, miR-93 and miR-25. We have previously reported that miR-106b-25 was associated with chemoresistance by negatively regulated EP300 in breast cancer, but its role in hematological malignancies has not yet been elucidated. Here, we aim to clarify the biological role and underlying mechanisms of miR-106b-25 on drug resistance in leukemia.Methods: To see whether the miR-106b-25 was associated with the poor prognosis of AML patients, enriched LSCs (CD34 + cells) were isolated from the bone marrow of 18 newly diagnosed AML patients, the expression of miR-106b, miR-93, and miR-25 were examined, respectively. The expression levels of miR-106b, miR-93 and miR-25 were further determined in the doxorubicin-resistant leukemia cell line K562/A02 and HL60/ADR, compared with their parental cell lines. In addition, K562 cells were transduced with lentiviral vectors carrying miR-106b-25, and cell proliferation, drug resistance, colony-forming assay, apoptosis assays were performed to explore the function of miR-106b-25 overexpression in leukemia cells in vitro. To investigate the role of miR-106b-25 on tumor growth and overall survival after drug treatment, we performed xenotransplantation in nude mice using miR-106b-25 overexpressed K562 cells. To further clarify the function of each microRNA function in this cluster, K562 cells were also transduced with lentiviral vectors carrying individual miR-25, miR-93, or miR-106b separately. Cell proliferation, colony forming assay and cell apoptosis assay were also carried out subsequently. Simultaneously, RNA-sequencing was performed to reveal the underlying mechanisms of miR-106b-25 in the chemoresistance of myeloid leukemia. To experimentally confirm the direct target of the miR-106b-25 cluster in AMLs, we further performed a dual-luciferase reporter assay.Results: Upregulated miR-106b, miR-93 and miR-25 expression in enriched LSCs were significantly associated with shortened overall survival of AML patients. We also found miR-106b, miR-93 and miR-25 were significantly upregulated in drug-resistant leukemia cell lines compared with its parental cell lines. Overexpression of miR-106b-25 cluster promoted cell proliferation, led to resistance of K562 cells to doxorubicin, imatinib and ABT-737 (BCL-2 inhibitor) in liquid culture and drug-resistant colony-forming assays. Overexpression of miR-93 or miR-106b accelerated cell growth, and all the three miRNAs can promote drug-resistant colony-forming and inhibit cell apoptosis. RNA-sequencing (RNA-Seq) data revealed that multiple critical genes related to apoptotic pathways were downregulated after overexpressing miR-25, miR-93, miR-106b as well as the whole cluster, such as TP73, BAX, BAK1, Caspase-7, CDKN1A and BTG2. RT-qPCR confirmed that these genes are reduced with or without ABT-737 treatment. Luciferase assay further identified TP73 was a direct target of miR-93 and miR106b, BAK1 was a direct target of miR-25, and CASPASE-7 was a direct target of all these three miRNAs.Conclusions: In summary, we made the novel observation that miR-106-25 is associated with AML drug-resisitance and disease prognosis and identified TP73, BAK1 caspase-7 as a novel direct target of this cluster. Further studies revealed that the biological effects of miR-106b-25 cluster on leukemic cell proliferation, chemoresistance and apoptosis were mediated through regulation of apoptotic pathway. These findings indicate a promising diagnostic biomarker and a potential target therapeutic strategy for AML patients. DisclosuresNo relevant conflicts of interest to declare.

MiRNA-124 regulates the sensitivity of renal cancer cells to cisplatin-induced necroptosis by targeting the CAPN4-CNOT3 axis.

BackgroundCurrently, drug-resistance is a major challenge in the treatment of renal cancer. Although microRNAs (miRNAs) have been reported to contribute to the incidence of drug resistance in renal cancer, the bio-functional roles and underlying regulatory mechanisms of novel miRNAs in cisplatin resistance remain largely unclear.MethodsIn this study, miRNA microarray analysis was applied to evaluate miRNA changes induced by cisplatin on RCC (renal cell carcinoma) cell lines. Then, Caki-1 and 786-0 cells were transfected with miR (miRNA)-124 mimics to observe cisplatin resistance in RCC cell lines after up-regulation of miR-124. TargetScan was used to identify putative protein-coding gene targets of miR-124. Further, the interaction between calpain small subunit 1 (Capn4) and CCR4-NOT transcription complex subunit 3 (CNOT3) was detected by quantitative real-time PCR (qPCR) and western blotting, and confirmed by co-immunoprecipitation. The effect of Capn4 and/or CNOT3 on cell viability and half maximal inhibitory concentration (IC50) value of miR-124 overexpressed Caki-1 and 786-O cells to cisplatin was evaluated using the Cell Counting Kit-8 (CCK-8) assay. And the effect of Capn4 and/or CNOT3 on the level of necroptosis in miR-124 overexpressed Caki-1 and 786-O cells to cisplatin was evaluated by flow cytometric analysis. Then, four groups of 786-0 cells (miR-124, miR-124+ Capn4, miR-124+ CNOT3, miR-124+ Capn4+ CNOT3) were inoculated into nude mice to observe the effect of cisplatin on tumor formation.ResultsmiR-124 was found to be markedly elevated in renal cancer cells by cisplatin. Functionally, the overexpression of miR-124 reduced the sensitivity of renal cancer cells to cisplatin and CAPN4 was found to be a direct target of miR-124, which can negatively regulated CAPN4 expression. Moreover, ectopic expression of CAPN4 reversed the impairment of miR-124 on cisplatin-sensitivity and cisplatin-induced necroptosis. Mechanically, the present study revealed that CAPN4 could directly interact with CNOT3 and promote its degradation, and that the cisplatin-resistant phenotype was reversed by up-regulation of CNOT3.ConclusionsTherefore, miR-124 is an important inhibitor in cisplatin-induced necroptosis, and the miR-124-CAPN4-CNOT3 signaling axis plays a critical role in the emergence of cisplatin-resistance.

Overexpression of lncRNA-NEF regulates the miR-155/PTEN axis to inhibit adipogenesis and promote osteogenesis.

Osteoporosis is characterized by osteopenia and bone tissue microstructure degradation. Adipose-derived stem cells (ADSCs) are multipotent adult stem cells that have the ability to yield mesenchymal stem cells and have the potential to undergo osteogenesis and bone regeneration. Therefore, ADSCs have the potential to treat osteoporosis, but the molecular mechanism of these cells in the process of osteogenesis and osteoclasts is still not clear. In the present study, we collected serum samples from 10 clinical osteoporosis patients to detect long noncoding RNA-neighboring enhancer of FOXA2 (lncRNA-NEF) and miR-155 expression levels. Half of these patients were senile and half were postmenopausal women, and nine of them have used steroids for a long time, in which ADSCs were cultured and induced to adipogenic and osteogenic differentiations. Quantitative real-time polymerase chain reaction was used to detect the expression of genes in ADSCs. Overexpression of lncRNA-NEF in ADSCs were undertaken to verify its regulatory function on cell osteogenic and adipogenic differentiations. A luciferase activity experiment was performed to determine the relationship between miR-155 and phosphatase and tensin homologue deleted on chromosome 10 (PTEN). The level of lncRNA-NEF was downregulated, and miR-155 was upregulated, in serum samples from patients with clinical osteoporosis. LncRNA-NEF showed different expression levels in the induction of osteogenic or adipogenic differentiation, which increased during osteogenic induction and decreased during adipogenic induction. Overexpression of lncRNA-NEF or downregulation of miR-155 in ADSCs promoted osteogenic differentiation and inhibited adipogenesis progression. PTEN was the direct target of miR-155 and was involved in the regulation of osteogenic differentiation. Overexpression of lncRNA-NEF regulated the miR-155/PTEN axis to inhibit adipogenesis and promote osteogenesis in ADSCs.