Target Of miR-34a Research Articles

The miR-27a/FOXJ3 Axis Dysregulates Mitochondrial Homeostasis in Colorectal Cancer Cells.

Simple SummaryCellular and mitochondrial metabolism can be dysregulated during tumorigenesis. miR-27a plays a central role in redirecting cell metabolism in colorectal cancer. In this study, we searched for new miR-27a targets that could influence mitochondria and identified FOXJ3 a master regulator of mitochondrial biogenesis. We validated FOXJ3 as an miR-27a target in an in vitro cell model system that was genetically modified for miR-27a expression and showed that the miR-27a/FOXJ3 axis down-modulates mitochondrial biogenesis and regulates other members of the pathway. The miR-27a/FOXJ3 axis also influences mitochondrial dynamics, superoxide production, respiration capacity, and membrane potential. A mouse xenograft model confirmed that miR-27a downregulates FOXJ3 in vivo and a survey of the TCGA-COADREAD dataset supported the inverse relationship of FOXJ3 with miR-27a and the impact on mitochondrial biogenesis. The miR-27a/FOXJ3 axis is a major actor in regulating mitochondrial homeostasis, and its discovery may contribute to therapeutic strategies aimed at restraining tumor growth by targeting mitochondrial activities.miR-27a plays a driver role in rewiring tumor cell metabolism. We searched for new miR-27a targets that could affect mitochondria and identified FOXJ3, an apical factor of mitochondrial biogenesis. We analyzed FOXJ3 levels in an in vitro cell model system that was genetically modified for miR-27a expression and validated it as an miR-27a target. We showed that the miR-27a/FOXJ3 axis down-modulates mitochondrial biogenesis and other key members of the pathway, implying multiple levels of control. As assessed by specific markers, the miR-27a/FOXJ3 axis also dysregulates mitochondrial dynamics, resulting in fewer, short, and punctate organelles. Consistently, in high miR-27a-/low FOXJ3-expressing cells, mitochondria are functionally characterized by lower superoxide production, respiration capacity, and membrane potential, as evaluated by OCR assays and confocal microscopy. The analysis of a mouse xenograft model confirmed FOXJ3 as a target and suggested that the miR-27a/FOXJ3 axis affects mitochondrial abundance in vivo. A survey of the TCGA-COADREAD dataset supported the inverse relationship of FOXJ3 with miR-27a and reinforced cellular component organization or biogenesis as the most affected pathway. The miR-27a/FOXJ3 axis acts as a central hub in regulating mitochondrial homeostasis. Its discovery paves the way for new therapeutic strategies aimed at restraining tumor growth by targeting mitochondrial activities.

Analysis of Expression Profiles of CircRNA and MiRNA in Oviduct during the Follicular and Luteal Phases of Sheep with Two Fecundity (FecB Gene) Genotypes.

Simple SummaryThe oviduct is an important part of the female reproductive organs, but few people pay attention to its role in the reproductive process. In this study, we perform RNA-Seq to analyze the expression of circRNA and miRNA in the oviduct of sheep (FecBBB and FecB++) during follicular and luteal phases. Enrichment analysis showed that the host genes of differentially expressed (DE) circRNAs were mainly enriched in the Rap1 signaling pathway, PI3K–Akt signaling pathway and neuroactive ligand–receptor interactions. Enrichment analysis showed that the target genes of DE miRNAs were mainly enriched in insulin secretion, the cAMP signaling pathway, the cGMP–PKG signaling pathway, the Rap1 signaling pathway, the TGF-β signaling pathway and other pathways related to reproduction. Our study, although not establishing direct causal relationships of the circRNA and miRNA changes, provides data for further exploring the mechanism of sheep fecundity.CircRNA and miRNA, as classes of non-coding RNA, have been found to play pivotal roles in sheep reproduction. There are many reports of circRNA and miRNA in the ovary and uterus, but few in the oviduct. In this study, RNA-Seq was performed to analyze the expression profile of circRNA and miRNA in the oviduct during the follicular phase and luteal phase of sheep with FecBBB and FecB++ genotypes. The results showed that a total of 3223 circRNAs and 148 miRNAs were identified. A total of 15 DE circRNAs and 40 DE miRNAs were found in the comparison between the follicular phase and luteal phase, and 1 DE circRNA and 18 DE miRNAs were found in the comparison between the FecBBB genotype and FecB++ genotype. GO and KEGG analyses showed that the host genes of DE circRNAs were mainly enriched in the Rap1 signaling pathway, PI3K–Akt signaling pathway and neuroactive ligand–receptor interactions. Novel_circ_0004065, novel_circ_0005109, novel_circ_0012086, novel_circ_0014274 and novel_circ_0001794 were found to be possibly involved in the oviductal reproduction process. GO and KEGG analyses showed that the target genes of DE miRNAs were mainly enriched in insulin secretion, the cAMP signaling pathway, the cGMP–PKG signaling pathway, the Rap1 signaling pathway and the TGF-β signaling pathway, and the target genes LPAR1, LPAR2, FGF18, TACR3, BMP6, SMAD4, INHBB, SKP1 and TGFBR2 were found to be associated with the reproductive process. Miranda software was used to identify 27 miRNAs that may bind to 13 DE circRNAs, including miR-22-3p (target to novel_circ_0004065), miR-127, miR-136 (target to novel_circ_0000417), miR-27a (target to novel_circ_0014274) and oar-miR-181a (target to novel_circ_ 0017815). The results of this study will help to elucidate the regulatory mechanisms of circRNAs and miRNAs in sheep reproduction. Our study, although not establishing direct causal relationships of the circRNA and miRNA changes, enriches the sheep circRNA and miRNA database and provides a basis for further studies on sheep reproduction.

Tumor-derived exosomes encapsulating miR-34a promote apoptosis and inhibit migration and tumor progression of colorectal cancer cells under in vitro condition.

MicroRNA (miR)-34a, as a master tumor suppressor in colorectal cancer (CRC), could regulate multiple genes participating in tumor proliferation, invasion, immune evasion, and inflammation-induced progression. Exosomes, as novel nano-carriers, were found to be capable of shuttling crucial mediators to various cells. Since the conventional CRC therapeutics currently are a matter of debate, implication of microRNAs in malignancy remedies have been addressed illustrating promising outlooks. In this study, we aimed to investigate the delivery of miR-34a to CRC cell line CT-26 by encapsulating into tumor-derived exosomes (TEXs), in order to evaluate the anti-proliferative and progressive effects of the novel nano-carrier complex under in vitro condition. Exosomes were purified from the starved CT-26 cells and then enriched by miR-34a using the calcium chloride (Cacl2) modified solution. Following the detection of miR-34a expression in the enriched TEXs, the viability of CT-26 cells treated by multiplicity concentrations of either TEXs or TEX-miR-34a was examined. Moreover, the apoptosis rate of the cells was evaluated, and the migration of CT-26 cells subjected to both TEX-miR-34a and TEX was also measured. Thereafter, the expressions of miR-34a target genes, as IL-6R, STAT3, PD-L1, and VEGF-A, which play roles in tumor progression, were determined in the treated CT-26 cells. The viability of CT-26 cells was harnessed following the treatment with TEX-miR-34a and the apoptosis levels of the cells were also observed to be enhanced dose-dependently. TEX-miR-34a was able to diminish the migration rate of the TEX-miR-34a treated cells and the expressions of IL-6R, STAT3, PD-L1, and VEGF-A were significantly restricted. Moreover, TEXs alone increased the apoptosis rate of tumor cells and repressed the proliferation and migration of these cells which were boosted by enrichment of TEXs with miR-34a. Exosomes isolated from the starved CT-26 cells were found to have a potential to deliver miR-34a into tumor cells properly with high functionality maintenance for miR-34a in case of regulating genes related to tumor progression and TEXs which showed no positive effect favoring cancer cells, presumably act as a favorable adjuvant in the CRC therapy.

USF2 reduces BMP3 expression via transcriptional activation of miR-34a, thus promoting osteogenic differentiation of BMSCs.

Osteoporosis is the most susceptible disease for people over 60. The main cause of osteoporosis is the decreased osteogenic differentiation of mesenchymal stem cells (MSCs). Here we showed that upstream stimulatory factor 2 (USF2)/microRNA-34a (miR-34a)/bone morphogenetic protein 3 (BMP3) axis regulated osteogenic differentiation of BMSCs. USF2 and miR-34a expression were examined using qPCR. Protein levels of BMP3 and osteogenic markers expression were evaluated using both western blot and qPCR. Activity of ALP was determined by ALP assay kit. Mineralization capacity of hBMSCs was assessed using ARS. Besides, CHIP assay was employed to verify whether USF2 could bind to miR-34a promoter. Finally, RIP assay and dual-luciferase reporter assay were employed to verify whether miR-34a directly bound to BMP3. Our results suggested that miR-34a was upregulated during osteogenic differentiation of BMSCs, and miR-34a overexpression could enhance osteogenic differentiation of BMSCs. USF2 could positively regulate miR-34a expression by interacting with its promoter. USF2 overexpression enhanced osteogenic differentiation of BMSCs, while miR-34a inhibition reversed the effect. Besides, BMP3 was the target of miR-34a. MiR-34a overexpression enhanced osteogenic differentiation of BMSCs, which was abolished by BMP3 overexpression. Taken together, USF2 enhanced osteogenic differentiation of BMSCs via downregulating BMP3 by interacting with miR-34a promoter.

Targeting of Marcks By MiR-34a Overcomes Drug Resistance in Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy characterized by abnormal proliferation of clonal plasma cells in the bone marrow. Current therapies, such as the proteasome inhibitor bortezomib (BTZ), have improved the outcome of patients. Nevertheless MM remains an incurable disease with a high rate of relapse and development of drug resistance. So far, the pathogenic mechanisms underlying drug resistance of MM are not fully elucidated. Our previous study showed the overexpression of MARCKS (Myristoylated alanine-rich C-kinase substrate) protein in MM cells played an important role in drug resistance. However; the mechanism (s) underlying MARCKS overexpression and its association with drug resistance in MM have not been elucidated.In the current study, to investigate whether miRNAs play a role in aberrant upregulation of MARCKS in drug resistance, several miRNA-target prediction algorithms were exploited to generate a selective miRNA library for subsequent screening. Among 52 miRNA candidates that target MARCKS, the expression level of several miRNAs with higher score was assessed in drug resistant cell lines (8226-R5 and MM1.R) in comparison to the parental cells (8226 and MM1.S). We observed that miR-34a expression was most significantly downregulated in 8226-R5 and MM1.R cell lines relative to their parental lines, 8226 and MM1S. We also identified a negative correlation between MARCKS and miR-34a expression levels in 6 primary patient samples and two healthy donor samples. Next, to confirm direct targeting of MARCKS by miR-34a, two MM resistant cell lines (8226-R5 and MM1R) were transduced with lentiviral vectors harboring miR-34a precursor gene or scramble control and mRNA and protein level of MARCKS were assessed in miR-34a overexpressing cell lines in comparison to scramble control. Overexpression of miR-34a reduced MARCKS expression level in both mRNA and protein levels. In addition, the luciferase reporter assay was performed to validate MARCKS as a direct target of miR-34a. To this end, the 3' UTR sequence of human MARCKS was cloned into the luciferase-expressing vector pEZX-MT01 to the downstream of the firefly luciferase gene. Co-transfection of MARCKS-UTR or negative control luciferase reporter vectors together with miR-34a expressing or control vector showed that miR-34a overexpression significantly reduced the luciferase activity, indicating that miR-34a can directly target the MARCKS 3′ UTR.To examine the functional effect of miR-34a on cell proliferation and apoptosis in MM cells, the miR-34a overexpressing or scramble control cell lines were treated with BTZ. Overexpression of miR-34a in drug resistant cell lines re-sensitized them to BTZ. Furthermore, FACS analysis revealed that overexpression of miR-34a could induce apoptosis in drug resistant MM cell lines as indicated by an increase in the percentage of annexin-V positive cells compared to the control, suggesting that downregulation of miR-34a is associated with acquired BTZ-resistance in MM.To further elucidate the interaction of miR-34a and MARCKS, we knocked down MARCKS expression by transfecting shMARCKS lentiviral vector into 8226-R5 and MM1.R resistant cell lines and examined the miR-34a level in MARCKS-knockdown cells. We found that miR-34a is also regulated by MARCKS as silencing of MARCKS could restore the miR-34a expression level in MM resistant cell lines. In compatible with miR-34a overexpression experiments, following the knockdown of MARCKS, both cell lines had significantly lower viability after treatment with BTZ, in comparison to negative controls. We also showed that knockdown of MARCKS by shMARCKS silencing or suppression of MARCKS by miR-34a overexpression increased the mRNA and protein levels of some pro-apoptotic genes including PUMA which may be involved in induction of apoptosis in drug resistant MM cells.In conclusion, our data indicate that miR-34a is downregulated in drug resistant MM cell lines and primary MM cells, which contributes to the drug resistance of MM through upregulation of MARCKS. Moreover, regulatory interaction between miR-34a and MARCKS provides the rationale that restoration of miR-34a may serve as a promising therapeutic approach by targeting MARCKS in patients with refractory/relapsed MM. DisclosuresNo relevant conflicts of interest to declare.

Oar-miR-16b and oar-miR-27a: negatively correlated with milk yield and milk protein in sheep

The characterization of miRNAs from sheep milk and their effect on milk yield and composition in sheep are remain unclear. Therefore, the aim of this study was to determine the expression pattern of several important miRNAs, which are associated with lactation in the sheep milk between high and low lactating-yield ewe groups. In addition to experimentally obtained miRNA expression results, the miRNA target genes were determined by bioinformatics analysis to identify biological pathways involved. miRNAs found to differ significantly in the expression level between the groups were oar-miR-181a, oar-miR-23a, oar-miR-27a, oar-miR-16b and oar-miR-374. Also, oar-miR-27a was shown negative correlation with milk protein and lactose contents while oar-miR-16b was shown negative correlation with milk yield in the high milk yield group. The highest connected hub genes for miR-27a target genes were determined as MAPK14 and PPARG. Also, six genes (HSPA4L, DNAJA2, ATP6V1B2, PPP2R1A, PPP2R1B, and PRKAR2A) were detected as hub genes for miR-16b. In this study, the relationship between expression profiles of several important miRNAs in sheep milk and milk yield and milk composition were investigated for the first time in high and low lactating yield groups.

Histone deficiency and accelerated replication stress in T cell aging.

With increasing age, individuals are more vulnerable to viral infections such as with influenza or the SARS-CoV-2 virus. One age-associated defect in human T cells is the reduced expression of miR-181a. miR-181ab1 deficiency in peripheral murine T cells causes delayed viral clearance after infection, resembling human immune aging. Here we show that naive T cells from older individuals as well as miR-181ab1-deficient murine T cells develop excessive replication stress after activation, due to reduced histone expression and delayed S-phase cell cycle progression. Reduced histone expression was caused by the miR-181a target SIRT1 that directly repressed transcription of histone genes by binding to their promoters and reducing histone acetylation. Inhibition of SIRT1 activity or SIRT1 silencing increased histone expression, restored cell cycle progression, diminished the replication-stress response, and reduced the production of inflammatory mediators in replicating T cells from old individuals. Correspondingly, treatment with SIRT1 inhibitors improved viral clearance in mice with miR-181a-deficient T cells after LCMV infection. In conclusion, SIRT1 inhibition may be beneficial to treat systemic viral infection in older individuals by targeting antigen-specific T cells that develop replication stress due to miR-181a deficiency.

CircMYLK promotes the growth, migration, invasion, and survival of bladder cancer cells by upregulating CCND3 level via competitively binding to miR‐34a

Bladder cancer is one of the most common types of urothelial carcinoma with a rising incidence rate worldwide. Circular RNAs (circRNAs) are involved in the development of numerous cancers, including bladder cancer. We aimed to uncover the role and associated mechanism of circMYLK in bladder cancer. The expression levels of circMYLK, miRNA-34a (miR-34a) and Cyclin D3 (CCND3) mRNA were investigated using real-time quantitative polymerase chain reaction. The protein level of CCND3 was investigated using western blot. In functional assays, flow cytometry assays were utilized for cell cycle analysis and cell apoptosis analysis. Transwell assays were used for cell migration and invasion analysis. Caspase-3 activity was examined to monitor cell apoptosis. The putative relationship between miR-34a and circMYLK or CCND3 was validated by dual-luciferase reporter assay and RNA immunoprecipitation assay. CircMYLK was highly expressed in bladder cancer tissues and cells. CircMYLK downregulation inhibited bladder cancer cell migration and invasion, and promoted cancer cell apoptosis and cell cycle arrest. MiR-34a, a target of circMYLK, was downregulated in bladder cancer tissues and cells. MiR-34a inhibition reversed the effects of circMYLK downregulation and then recovered bladder cell malignant behaviors. Further analysis showed that CCND3 was a downstream target of miR-34a, and CCND3 was upregulated in bladder cancer tissues and cells. MiR-34a overexpression blocked bladder cancer cell migration and invasion, and induced cell apoptosis and cycle arrest, while these effects were abolished by CCND3 overexpression. CircMYLK contributed to the malignant development of bladder cancer cells partly through the miR-34a/CCND3 regulatory network, showing the significance of circMYLK in bladder cancer pathogenesis.

Shikonin treatment ameliorates Lipopolysaccharides (LPS) induced acute liver failure in mice via regulating the miR-106/MCL1 and miR-34a/SIRT1/TP53 signaling

IntroductionThe treatment with shikonin (SKN) suppresses the expression of miR-106 and miR-34a. Furthermore, SIRT1 and MCL1 are targets of miR-34a and miR-106, respectively. In this study, we treated an animal model of ALF with high dose (1.0 mg/kg) and low dose (0.5 mg/kg) of SKN to investigate its effect on liver functions and signaling pathways of SKN/miR-106/MCL1 and SKN/miR-34a/SIRT1/TP53.Material and methodsALF animal model was established and the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed to evaluate the effects of different doses of SKN. TUNNEL was performed to assess hepatocyte apoptosis. Luciferase assay, RT-qPCR and Western blot analysis were performed to measure the relationship between miR-106, miR-34a, SIRT1 and MCL1.ResultsIn the ALF mice models, the administration of SKN decreased the levels of ALT and AST in a dose-dependent manner, along with a significantly decreased number of apoptotic hepatocytes. And SKN may protect liver during ALF via reducing the level of inflammation. Luciferase assay showed that the co-transfection of wild-type MCL1/SIRT1 and miR-106/miR-34a significantly decreased the luciferase activity of LO2 cells, thus indicating that MCL1 and SIRT1 are identified as targets of miR-106 and miR-34a, respectively, while SIRT1 could act as a regulator of TP53. Moreover, the expression of miR-106, miR-34a and TP53 was decreased over an increasing concentration of SKN, along with the increasing mRNA and protein levels of MCL1 and SIRT1.ConclusionsIn this study, we showed that SKN alleviated ALF in a dose-dependent manner via regulating the signaling pathways of SKN/miR-106/MCL1 and SKN/miR-34a/SIRT1/TP53.

MiR-34a suppression targets Nampt to ameliorate bone marrow mesenchymal stem cell senescence by regulating NAD+-Sirt1 pathway

BackgroundExpansion-mediated replicative senescence and age-related natural senescence have adverse effects on mesenchymal stem cell (MSC) regenerative capability and functionality, thus severely impairing the extensive applications of MSC-based therapies. Emerging evidences suggest that microRNA-34a (miR-34a) has been implicated in the process of MSC senescence; however, the molecular mechanisms with regard to how miR-34a influencing MSC senescence remain largely undetermined.MethodsMiR-34a expression in MSCs was evaluated utilizing RT-qPCR. The functional effects of miR-34a exerting on MSC senescence were investigated via gene manipulation. Relevant gene and protein expression levels were analyzed by RT-qPCR and western blot. Luciferase reporter assays were applied to confirm that Nampt is a direct target of miR-34a. The underlying regulatory mechanism of miR-34a targeting Nampt in MSC senescence was further explored by measuring intracellular NAD+ content, NAD+/NADH ratio and Sirt1 activity.ResultsIn contrast to Nampt expression, miR-34a expression incremented in senescent MSCs. MiR-34a overexpression in young MSCs resulted in senescence-associated characteristics as displayed by senescence-like morphology, prolonged cell proliferation, declined osteogenic differentiation potency, heightened senescence-associated-β-galactosidase activity, and upregulated expression levels of the senescence-associated factors. Conversely, miR-34a suppression in replicative senescent and natural senescent MSCs contributed to diminished senescence-related phenotypic features. We identified Nampt as a direct target gene of miR-34a. In addition, miR-34a repletion resulted in prominent reductions in Nampt expression levels, NAD+ content, NAD+/NADH ratio, and Sirt1 activity, whereas anti-miR-34a treatment exerted the opposite effects. Furthermore, miR-34a-mediated MSC senescence was evidently rescued following the co-treatment with Nampt overexpression.ConclusionThis study identifies a significant role of miR-34a playing in MSC replicative senescence and natural senescence via targeting Nampt and further mediating by NAD+-Sirt1 pathway, carrying great implications for optimal strategies for MSC therapeutic applications.

The comprehensive landscape of miR-34a in cancer research.

MicroRNA-34 (miR-34) plays central roles in human diseases, especially cancers. Inactivation of miR-34 is detected in cancer cell lines and tumor tissues versus normal controls, implying its potential tumor-suppressive effect. Clinically, miR-34 has been identified as promising prognostic indicators for various cancers. In fact, members of the miR-34 family, especially miR-34a, have been convincingly proved to affect almost the whole cancer progression process. Here, a total of 512 (miR-34a, 10/21), 85 (miR-34b, 10/16), and 114 (miR-34c, 10/14) putative targets of miR-34a/b/c are predicted by at least ten miRNA databases, respectively. These targets are further analyzed in gene ontology (GO), KEGG pathway, and the Reactome pathway dataset. The results suggest their involvement in the regulation of signal transduction, macromolecule metabolism, and protein modification. Also, the targets are implicated in critical signaling pathways, such as MAPK, Notch, Wnt, PI3K/AKT, p53, and Ras, as well as apoptosis, cell cycle, and EMT-related pathways. Moreover, the upstream regulators of miR-34a, mainly including transcription factors (TFs), lncRNAs, and DNA methylation, will be summarized. Meanwhile, the potential TF upstream of miR-34a/b/c will be predicted by PROMO, JASPAR, Animal TFDB 3.0, and GeneCard databases. Notably, miR-34a is an attractive target for certain cancers. In fact, miR-34a-based systemic delivery combined with chemotherapy or radiotherapy can more effectively control tumor progression. Collectively, this review will provide a panorama for miR-34a in cancer research.

Expression of miR-34a and miR-15b during the progression of cervical cancer in a murine model expressing the HPV16 E7 oncoprotein.

The high-risk human papillomavirus (HR-HPV) E7 oncoprotein appears to be a major determinant for cell immortalization and transformation altering critical processes such as cell proliferation, apoptosis, and immune response. This oncoprotein plays an essential role in cervical carcinogenesis, but other cofactors such as long-term use of hormonal contraceptives are necessary to modulate the risk of cervical cancer (CC). The role of HR-HPVs in the alteration of microRNA (miRNA) levels in persistent viral infections currently remains unclear. The aim of this study was to evaluate the miR-34a and miR-15b expression levels in the murine HPV16K14E7 (K14E7) transgenic model after chronic estrogen (E2) treatment and their involvement in CC. Interestingly, results showed that, although miR-34a expression is elevated by the HPVE7 oncogene, this expression was downregulated in the presence of both the E7 oncoprotein and chronic E2 in cervical carcinoma. On the other hand, miR-15b expression was upregulated along cervical carcinogenesis mainly by the effect of E2. These different changes in the expression levels of miR-34a and miR-15b along cervical carcinogenesis conduced to low apoptosis levels, high cell proliferation and finally, to cancerous cervical tissue development. In this work, we also determined the relative mRNA expression of Cyclin E2 (Ccne2), Cyclin A2 (Ccna2), and B cell lymphoma 2 (Bcl-2) (target genes of miR-34a and miR-15b); Sirtuin 1 (Sirt1), Cmyc, and Bax (miR-34a target genes); and p21/WAF1 (mir15b target gene) and the H-ras oncogene. Given the modifications in the expression levels of miR-34a and miR-15b during the development of cervical cancer, it will be useful to carry out further investigation to confirm them as molecular biomarkers of cancer.

FGF-2-Dependent Signaling Activated in Aged Human Skeletal Muscle Promotes Intramuscular Adipogenesis

Abstract Background: A unique feature of muscle during aging, obesity and type 2 diabetes is the appearance of adipose tissue between skeletal muscle fibers, the intramuscular adipose tissue (IMAT). IMAT is generally associated with systemic insulin resistance, decreased muscle strength and, in older adults, impaired mobility. A large body of work over the last years have addressed the origin of IMAT. Skeletal muscle contains preadipocyte progenitors termed fibro/adipogenic progenitors (FAPs) that normally do not form adipocytes, but proliferate during muscle injury to support the commitment of myogenic progenitor cells during muscle repair. However, under the conditions outlined above, FAPs differentiate to adipocytes and give rise to IMAT. The molecular cues that trigger this pathogenesis are unknown and strategies that can modulate the fate of FAPs and their propensity to differentiate to adipocytes in disease states are urgently needed. Methods: We have used molecular cloning and genome editing techniques together with a variety of biochemical, molecular, genomic, transcriptomic and proteomic analyses in pre-adipocyte and muscle cell lines, primary cells isolated from human and mouse skeletal muscle, human skeletal muscle biopsies, and animal models. Results: Using multiple screening assays upstream and downstream of a master regulator of myogenesis, microRNA (miR)-29a, we located the secreted protein and adipogenic inhibitor SPARC to an FGF-2 signaling pathway that is conserved between skeletal muscle cells from mice and humans and that is activated in skeletal muscle of aged mice and humans. We identified FGF-2 as an upstream regulator that increases miR-29a promoter activity and gene expression via MEK1/2/MAPK signaling and consequently decreases the conserved miR-29a target SPARC. FGF-2 induces the miR-29a/SPARC axis through transcriptional activation of FRA-1, which binds and activates an evolutionary conserved AP-1 site element proximal in the miR-29a promoter. CRISPR/Cas9-mediated genomic deletions of miR-29a recognition elements located in the Sparc 3′UTR of muscle cells and AAV-mediated overexpression of FGF-2 or SPARC in mouse skeletal muscle revealed that this axis regulates differentiation of FAPs in vitro and IMAT formation in vivo. Skeletal muscle from human donors aged > 75 years versus < 55 years showed activation of FGF-2-dependent signaling and increased IMAT. Conclusion: FGF-2 not only stimulates muscle growth, but also promotes intramuscular adipogenesis via targeting the fate of FAPs. Thus, our data highlights a disparate role of FGF-2 in adult skeletal muscle and reveals a novel pathway, which has the potential to provide innovative diagnostic and therapeutic approaches to combat fat accumulation in aged human skeletal muscle.

Inhibiting Glucose Metabolism By miR-34a and miR-125b Protects Against Hyperglycemia-Induced Cardiomyocyte Cell Death.

ResumoFundamento:É sabido que a resistência à insulina e a hiperglicemia são causas patológicas importantes no desenvolvimento de cardiomiopatia diabética (CMD). Entretanto, seus mecanismos moleculares precisos na patogênese da CMD ainda não estão claros.Objetivos:Estudos recentes revelam que os microRNAs (miRNAs) desempenham papéis essenciais na patogênese da CMD. Este projeto tem o objetivo de determinar os papéis de miR-34a e miR-125b na morte celular de cardiomiócitos causada por hiperglicemia.Métodos:Cardiomiócitos primários de ratos foram isolados e expostos a concentrações de glicose normais e altas. A viabilidade das células foi medida utilizando-se o ensaio MTT. As expressões de miR-34a e miR-125b foram detectadas por qRT-PCR. Alvos potenciais de miR-34a e miR-125b foram previstos pelo www.Targetscan.org, e validados a partir de tecidos cardíacos humanos. Um p<0,05 foi considerado significância estatística.Resultados:Demonstra-se neste estudo que o miR-34a e o miR-125b têm resposta celular reduzida no coração humano diabético. Além disso, os dados in vitro de cardiomiócitos primários de ratos demonstraram que o tratamento com glicose alta em curto prazo estimula a expressão de miR-34a e miR-125b. Demonstrou-se que, em condições de glicose alta, os cardiomiócitos de ratos apresentaram metabolismo de glicose intracelular, e a captação de glicose e a produção de lactato aumentaram significativamente. Foi identificado que as principais enzimas metabólicas da glicose, hexoquinase 2 (HK2) e lactato desidrogenase-A (LDHA) eram alvos diretos de miR-125b e miR-34a, respectivamente. A superexpressão de miR-125b e miR-34a poderia evitar a morte de celular de cardiomiócitos causada por hiperglicemia. Por fim, a recuperação de HK2 e LDHA em cardiomiócitos com superexpressão de miR-125b e miR-34a restaurou a sensibilidade de cardiomiócitos à hiperglicemia.Conclusões:Nossos resultados propõem um mecanismo molecular para proteção cardiovascular diabética mediada por microRNA e contribuirão para o desenvolvimento de estratégias de tratamento de disfunção cardiovascular associada a diabetes.

MiR-27a promotes osteogenic differentiation in glucocorticoid-treated human bone marrow mesenchymal stem cells by targeting PI3K.

MicroRNA-27a (miR-27a) modulates osteogenic differentiation (OD); however, the mechanism by which it influences osteoclastic activity in the glucocorticoid (GC)-elicited osteoporotic bone is still unclear. Bone marrow was obtained from the proximal femur of patients (n = 3) with a femoral neck fracture and those (n = 3) with steroid-related osteonecrosis of the femoral head (ONFH). GC was applied to an established ONFH cell model from human bone marrow mesenchymal stem cells (hBMSCs). The miR-27a expression profiles were found to be downregulated in ONFH samples and GC-induced hBMSCs using microarray analysis and real-time quantitative polymerase chain reaction, whereas the OD capacity of hBMSCs was significantly reduced in the GC group compared with the control group. Subsequent transfection of an miR-27a mimic in hBMSCs revealed that the OD capacity of cells was remarkably strengthened in the GC group compared with the miR-control group. Bioinformatics software (TargetScan) predicted that phosphoinositide 3-kinase (PI3K) might be a potential miR-27a target, which was indicated by dual-luciferase reporter assay. Compared with the control group, the GC group exhibited a significantly downregulated protein expression level of PI3K and its downstream protein kinase B (Akt) and mammalian target of rapamycin (mTOR) expression. Furthermore, administration of 10μM 740 Y-P, a cell-permeable phosphopeptide activator of PI3K, to hBMSCs increased the expression of Akt and mTOR. Treatment with 740 Y-P reversed the effect of miR-27a on OD in hBMSCs. In conclusion, miR-27a is thought to relieve ONFH and the OD repression in GC-induced hBMSCs by targeting the PI3K/Akt/mTOR pathway.

Metformin attenuates ischemia/reperfusion-induced apoptosis of cardiac cells by downregulation of p53/microRNA-34a via activation of SIRT1.

Metformin has been demonstrated to be beneficial for the treatment of an impaired myocardium as a result of ischemia/reperfusion (I/R) injury, and miR-34a may be involved in this process. The aim of the present study was to determine the mechanisms by which metformin attenuated myocardial I/R injury-induced apoptosis. In the in vivo I/R model using Sprague-Dawley rats, metformin reduced the area of damaged myocardium and serum creatine MB isoform (CKMB) activity resulting in protection of the myocardium. Metformin also reduced apoptosis and the expression of apoptosis associated proteins, including caspase 3 and cleaved caspase, and decreased the expression of miR-34a, which is upregulated during I/R injury, which in turn resulted in corresponding changes in expression of Bcl-2, a direct target of miR-34a both in vitro and in vivo. To further examine the role of miR-34a in this process, H9C2 cells were transfected by a miR-34a mimic and inhibitor. Overexpression of miR-34a increased apoptosis in H9C2 cells induced by oxygen-glucose deprivation/recovery and knockdown of miR-34a expression-reduced apoptosis under the same conditions. Therefore, the effect of metformin on miR-34a in vitro were assessed. Metformin decreased the deacetylation activity of silent information regulator 1 resulting in reduced Ac-p53 levels, which reduced the levels of pri-miR-34a, and thus in turn reduced miR-34a levels. To confirm these results clinically, 90 patients with ST-segment elevation myocardial infarction following percutaneous coronary intervention were recruited. Patients who took metformin regularly before infarction had lower miR-34a levels and lower serum CKMB activity. Metformin also improved the sum ST-segment recovery following I/R injury. In conclusion, metformin may be helpful in the treatment of myocardial I/R.

Autophagy Regulates Cancer Stem Cell Properties in Triple Negative Breast Cancer Via miR-181a-Mediated Regulation of ATG5/ATG2B

Background: Cancer stemness, miRNA, and autophagy flux are known to contribute to the maintenance of cancers; however, determination of the precise interactions among autophagy, miRNAs, and cancer stemness requires further investigation. Methods: Western blot, immunocytochemistry, immunofluorescence staining, quantitative real-time PCR were performed to detect gene expression. Tumorsphere formation assay, Aldefluor assay, limiting dilution assay were carried out to measure cancer stemness. Acridine orange and Cyto-ID® staining were used to detect autophagy vacuoles. Findings: Autophagy flux was inhibited in triple-negative breast cancer (TNBC) cancer stem cells (CSCs). Moreover, miRNA-181a (miR-181a) is upregulated both in TNBC CSCs and patients. ATG5 and ATG2B participate in the early formation of autophagosomes and were revealed as targets of miR-181a. Inhibition of miR-181a expression led to attenuation of cancer stemness and an increase in autophagy flux. Furthermore, treatment with curcumin led to attenuation of cancer stemness in TNBC CSCs; the expression of ATG5 and ATG2B was enhanced and there was an increase of autophagy flux. Interpretation: It could be suggested that luminal and triple-negative breast cancer require distinct therapeutic approaches because of their different expressions of autophagy flux. These results indicated that ATG5 and ATG2B are involved in the suppression of cancer stemness in TNBC. Autophagy inhibits cancer stemness through the miR-181a-regulated mechanism in TNBC. Promoting tumor-suppressive autophagy using curcumin may be a potential method for the treatment of TNBC. Funding: This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MSIP) (2019R1A2B5B03069738, NRF- 2016R1A5A1011974). Declaration of Interest: All the authors declared no competing interests. Ethical Approval: This study was approved by the 235 Institutional Review Board of Seoul National University Hospital (IRB number: 1704-236 019-843). A breast cancer tissue array (BC081120f, US Biomax Inc, USA).

Hypoxia-reoxygenation induces macrophage polarization and causes the release of exosomal miR-29a to mediate cardiomyocyte pyroptosis.

To investigate the mechanism by which hypoxia-reoxygenation (HR) mediates macrophage polarization to the M1 phenotype and then mediates cardiomyocyte (CM) pyroptosis through exosome release. Mouse bone marrow macrophages and CMs were cultured in vitro under hypoxia for 12h and reoxygenation for 6h to establish an HR cell model. qPCR was used to detect the M1 or M2 macrophage markers IL-1β, TNF-α, MR, and Arg, and a macrophage and CM coculture system was then established. Macrophages were transfected with an exosome-CD63-red fluorescent protein (RFP) lentivirus, allowing secretion of exosomes expressing RFP, and GW4869 was used to inhibit exosome release by macrophages. qPCR detected miR-29 expression in macrophage-derived exosomes, and macrophages were transfected with miR-29a inhibitors to obtain exosomes with low miR-29a expression (siR-exos). Pyroptosis indicators were detected by Western blot and ELISA. Importantly, LPS induced bone marrow macrophage polarization to the M1 type as a positive control to further verify that these exosomes (LPS-exos) regulated CM pyroptosis by delivering miR29a. Dual luciferase reporter and Western blot assays were adopted to analyze the miR-29a and MCL-1 target relationship. In addition, MCL-1 overexpression was used as a rescue experiment to determine whether miR-29a regulates pyroptosis in CM by targeting MCL-1. Macrophages expressed the M1 macrophage markers IL-1β and TNF-α after HR exposure. After CM coculture, RFP expression was significantly higher in the HR group than in the normal (Nor) group but significantly reduced in the GW4869 group. Immunofluorescence showed that caspase-1 mRNA and protein expression in the HR group was significantly higher than that in the Nor group (P < 0.05). Caspase-1 expression was significantly decreased in the GW4869 group compared with the HR group (P< 0.05). Western blotting showed that the pyrolysis-related NLRP3 and ASC protein expression levels were significantly upregulated in the HR group compared with the control (Ctr) and Nor groups (P< 0.05). However, GW4869 effectively inhibited pyroptosis-related protein expression (P< 0.05). In addition, ELISA showed that the expression of the inflammation indicators IL-1β and IL-18 was significantly increased in the HR group compared to the Ctr group (P< 0.05) but decreased in the GW4869 group (P< 0.05). qPCR showed that miR-29a was upregulated in the HR group compared to the Nor group. Moreover, HR-induced exosomes (HR-exos) from macrophages exacerbated HR-induced CM pyroptosis, while inhibition of miR-29a in exosomes partially offset CM pyroptosis induction. LPS-exos promoted pyroptosis-related protein expression, as the IL-1β and IL-18 concentrations were increased in the LPS-exos group. However, pyroptosis-related proteins were observably decreased, and IL-1β and IL-18 were also significantly decreased after miR-29a inhibition when compared with that in the HR-exos and LPS-exos groups. Mcl-1 overexpression reversed miR-29a-mediated CM pyroptosis in an HR environment. HR treatment induced macrophage polarization towards the M1 phenotype, which mediated CM pyroptosis through exosomal miR-29a transfer by targeting MCL-1.

MiR-34a regulates phenotypic modulation of vascular smooth muscle cells in intracranial aneurysm by targeting CXCR3 and MMP-2.

MicroRNAs (miRNAs) dysregulation is tightly related to diseases including tumor, neuro disease and cardiovascular disease. In this study, we investigated the potential biological effects of miR-34a and its target CXCR3 in phenotypic modulation of vascular smooth muscle cells (VSMCs) of intracranial aneurysms (IAs). MiR-34a was found to be down-regulated in IAs patients tested by Real-time PCR and decreased in GEO data. Meanwhile, our study also showed miR-34a inhibited matrix metalloproteinases (MMPs) and migration of VSMCs. Besides, CXCR3 is a direct target of miR-34a identified via luciferase assay. CXCR3 showed inhibitory effect on SM-MHC, SM22 while promoted MMPs expression, cell proliferation and migration in VSMCs. MiR-34a reversed the effect of CXCR3 in VSMCs. In addition, MMP-2 is a competitive endogenous RNA (ceRNA) of CXCR3 sharing common miR-34a target. CXCR3 increased MMP-2 level through competitive endogenous RNA regulation by sponging endogenous miR-34a. In conclusion, miR-34a is down-regulated in IAs while CXCR3 is the direct target of miR-34a that regulates phenotypic modulation of VSMCs. CXCR3 increased MMP-2 level through competitive endogenous RNA regulation by sharing common miR-34a targets.

MiR-34a attenuates myocardial fibrosis in diabetic cardiomyopathy mice via targeting Pin-1.

Diabetic cardiomyopathy (DCM) is characterized by myocardial hypertrophy and fibrosis. This study aimed to investigate the effects of microRNA (miR)-34a on myocardial fibrosis in DCM and its potential mechanism of targeting Pin-1 signaling. Vimentin and Pin-1 proteins in mouse cardiac tissues were detected by immunohistochemical staining. Locked nucleic acid in situ hybridization was used to measure miR-34a expression in cardiac tissues. Primary mouse cardiac fibroblasts (CFs) were transfected with a mimics control/miR-34a mimics or Pin-1 plasmid and cultured in high-glucose (HG) Dulbecco's modified Eagle's medium. The miR-34a levels were measured by quantitative polymerase chain reaction. The apoptosis and viability of transfected cells were detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling and Cell Counting Kit-8 assays respectively. A cell migration experiment and dual-luciferase reporter assay were also performed. The body weight and fasting blood glucose of DCM mice were significantly higher than those in the control (CTL) group. In addition, DCM mice had decreased serum insulin levels and impaired cardiac function. The number of CFs in the DCM group was higher than in the CTL group and Pin-1 expression was upregulated. The expression level of miR-34a in the cardiac tissue of mice in the DCM group was obviously downregulated compared with the CTL group. The HG stimulation of CFs for 48 h significantly downregulated the expression level of miR-34a and was associated with increased Type I collagen expression, cell viability, and migration and decreased apoptosis. However, these effects could be reversed by overexpressing miR-34a in HG-induced CFs. Furthermore, we found that Pin-1 was a direct target of miR-34a. Our results suggest that miR-34a can attenuate myocardial fibrosis in DCM by reducing Type I collagen production, cell viability, and migration and increasing the apoptosis of CFs by targeting Pin-1 signaling.