miR-103-3p Regulates the Proliferation and Differentiation of C2C12 Myoblasts by Targeting BTG2.

Skeletal muscle, a vital and intricate organ, plays a pivotal role in maintaining overall body metabolism, facilitating movement, and supporting normal daily activities. An accumulating body of evidence suggests that microRNA (miRNA) holds a crucial role in orchestrating skeletal muscle growth. Therefore, the primary aim of this study was to investigate the influence of miR-103-3p on myogenesis. In our study, the overexpression of miR-103-3p was found to stimulate proliferation while suppressing differentiation in C2C12 myoblasts. Conversely, the inhibition of miR-103-3p expression yielded contrasting effects. Through bioinformatics analysis, potential binding sites of miR-103-3p with the 3'UTR region of BTG anti-proliferative factor 2 (BTG2) were predicted. Subsequently, dual luciferase assays conclusively demonstrated BTG2 as the direct target gene of miR-103-3p. Further investigation into the role of BTG2 in C2C12 myoblasts unveiled that its overexpression impeded proliferation and encouraged differentiation in these cells. Notably, co-transfection experiments showcased that the overexpression of BTG2 could counteract the effects induced by miR-103-3p. In summary, our findings elucidate that miR-103-3p promotes proliferation while inhibiting differentiation in C2C12 myoblasts by targeting BTG2.

Saturated fatty acid-inducible miR-103-3p impairs the myogenic differentiation of progenitor cells by enhancing cell proliferation through Twinfilin-1/F-actin/YAP1 axis.

Actin dynamics play an essential role in myogenesis through multiple mechanisms, such as mechanotransduction, cell proliferation, and myogenic differentiation. Twinfilin-1 (TWF1), an actin-depolymerizing protein, is known to be required for the myogenic differentiation of progenitor cells. However, the mechanisms by which they epigenetically regulate TWF1 by microRNAs under muscle wasting conditions related to obesity are almost unknown. Here, we investigated the role of miR-103-3p in TWF1 expression, actin filament modulation, proliferation, and myogenic differentiation of progenitor cells. Palmitic acid, the most abundant saturated fatty acid (SFA) in the diet, reduced TWF1 expression and impeded myogenic differentiation of C2C12 myoblasts, while elevating miR-103-3p levels in myoblasts. Interestingly, miR-103-3p inhibited TWF1 expression by directly targeting its 3'UTR. Furthermore, ectopic expression of miR-103-3p reduced the expression of myogenic factors, i.e., MyoD and MyoG, and subsequently impaired myoblast differentiation. We demonstrated that miR-103-3p induction increased filamentous actin (F-actin) and facilitated the nuclear translocation of Yes-associated protein 1 (YAP1), thereby stimulating cell cycle progression and cell proliferation. Hence, this study suggests that epigenetic suppression of TWF1 by SFA-inducible miR-103-3p impairs myogenesis by enhancing the cell proliferation triggered by F-actin/YAP1.

miR-103-3p Regulates the Differentiation and Autophagy of Myoblasts by Targeting MAP4.

Skeletal muscle is the most abundant tissue in mammals, and myogenesis and differentiation require a series of regulatory factors such as microRNAs (miRNAs). In this study, we found that miR-103-3p was highly expressed in the skeletal muscle of mice, and the effects of miR-103-3p on skeletal muscle development were explored using myoblast C2C12 cells as a model. The results showed that miR-103-3p could significantly reduce myotube formation and restrain the differentiation of C2C12 cells. Additionally, miR-103-3p obviously prevented the production of autolysosomes and inhibited the autophagy of C2C12 cells. Moreover, bioinformatics prediction and dual-luciferase reporter assays confirmed that miR-103-3p could directly target the microtubule-associated protein 4 (MAP4) gene. The effects of MAP4 on the differentiation and autophagy of myoblasts were then elucidated. MAP4 promoted both the differentiation and autophagy of C2C12 cells, which was contrary to the role of miR-103-3p. Further research revealed that MAP4 colocalized with LC3 in C2C12 cell cytoplasm, and the immunoprecipitation assay showed that MAP4 interacted with autophagy marker LC3 to regulate the autophagy of C2C12 cells. Overall, these results indicated that miR-103-3p regulated the differentiation and autophagy of myoblasts by targeting MAP4. These findings enrich the understanding of the regulatory network of miRNAs involved in the myogenesis of skeletal muscle.

MiR-103-3p promotes hepatic steatosis to aggravate nonalcoholic fatty liver disease by targeting of ACOX1.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a major risk factor for hepatocellular carcinoma, and alterations in miRNA expression are related to the development of NAFLD. However, the role of miRNAs in regulating the development of NAFLD is still poorly understood. METHODS: We used qRT-PCR to detect the level of miR-103-3p in both cell and mouse models of NAFLD. Biochemical assays, DCF-DA assays, Oil red O staining and HE staining were used to detect the role of miR-103-3p in NAFLD development. Target genes of miR-103-3p were predicted using the TargetScan database and verified by qRT-PCR, western blot and dual-luciferase assays. RESULTS: The expression of miR-103-3p increased in both NAFLD model cells and liver tissues from the NAFLD mouse model. Inhibition of miR-103-3p significantly alleviated the accumulation of lipid droplets in free fatty acid-treated L02 cells and liver tissues from mice with NAFLD. Inhibition of miR-103-3p reduced the contents of H2O2, TG, ALT, and AST and ROS production while increasing the ATP content. Moreover, the miR-103-3p antagomir alleviated liver tissue lesions in mice with NAFLD. Further studies identified ACOX1, a key enzyme for the oxidation and decomposition of fatty acids, as a direct target of miR-103-3p. CONCLUSIONS: These findings identified a negative regulatory mechanism between ACOX1 and miR-103-3p that promotes the pathogenesis of NAFLD and suggested that inhibition of miR-103-3p may be a potential treatment strategy for NAFLD.

Overexpression of cytosolic long noncoding RNA cytb protects against pressure-overload-induced heart failure via sponging microRNA-103-3p.

Long noncoding RNAs (lncRNAs) play crucial roles in cardiovascular diseases. To date, only limited studies have reported the role of mitochondria-derived lncRNAs in heart failure (HF). In the current study, recombinant adeno-associated virus 9 was used to manipulate lncRNA cytb (lnccytb) expression in vivo. Fluorescence in situ hybridization (FISH) assay was used to determine the location of lnccytb, while microRNA (miRNA) sequencing and bioinformatics analyses were applied to identify the downstream targets. The competitive endogenous RNA (ceRNA) function of lnccytb was evaluated by biotin-coupled miRNA pull-down assays and luciferase reporter assays. Results showed that lnccytb expression was decreased in the heart of mice with transverse aortic constriction (TAC), as well as in the heart and plasma of patients with HF. FISH assay and absolute RNA quantification via real-time reverse transcription PCR suggested that the reduction of the lnccytb transcripts mainly occurred in the cytosol. Upregulation of cytosolic lnccytb attenuated cardiac dysfunction in TAC mice. Moreover, overexpression of cytosolic lnccytb in cardiomyocytes alleviated isoprenaline-induced reactive oxidative species (ROS) production and hypertrophy. Mechanistically, lnccytb acted as a ceRNA via sponging miR-103-3p, ultimately mitigating the suppression of PTEN by miR-103-3p. In summary, we demonstrated that the overexpression of cytosolic lnccytb could ameliorate HF.

LncRNA H19 sponges miR-103-3p to promote the high phosphorus-induced osteoblast phenotypic transition of vascular smooth muscle cells by upregulating Runx2.

Elucidating the mechanism of the osteogenic phenotypic transdifferentiation of vascular smooth muscle cells (VSMCs) is the key to determining the diagnosis and treatment of arterial medial calcification (AMC). Long noncoding RNAs (lncRNAs) have been reported to participate in the regulation of vascular physiology and pathology. Here, we investigated the effect and mechanism of the lncRNA H19 on the osteoblastic differentiation of VSMCs induced by high phosphorus. H19 was expressed at high levels in high phosphorus-induced primary rat VSMCs. Further experiments indicated that H19 played a positive role in the osteoblast phenotypic transition by suppressing miR-103-3p expression and subsequently promoting osteoblast-specific marker expression, including bone morphogenetic protein 2 (BMP-2) and osteopontin (OPN). Mechanistically, we recognized RUNX family transcription factor 2 (Runx2) as a direct target of miR-103-3p. Moreover, H19 directly interacted with miR-103-3p, and overexpression of miR-103-3p reversed the upregulation of Runx2 induced by H19. Therefore, H19 positively regulated Runx2 expression by sponging miR-103-3p and promoted the osteoblast phenotypic transition in VSMC calcification. Collectively, the lncRNA H19 promoted osteogenic differentiation by modulating the miR-103-3p/Runx2 axis in the process of VSMC calcification induced by a high phosphorus concentration. The current study provided new insights into an important role for the lncRNA H19 as a miRNA sponge in VSMCs and supplied novel insights into lncRNA-directed diagnostics and therapeutics for vascular calcification.

miR-103-3p targets Ndel1 to regulate neural stem cell proliferation and differentiation.

The regulation of adult neural stem cells (NSCs) is critical for lifelong neurogenesis. MicroRNAs (miRNAs) are a type of small, endogenous RNAs that regulate gene expression post-transcriptionally and influence signaling networks responsible for several cellular processes. In this study, miR-103-3p was transfected into neural stem cells derived from embryonic hippocampal neural stem cells. The results showed that miR-103-3p suppressed neural stem cell proliferation and differentiation, and promoted apoptosis. In addition, miR-103-3p negatively regulated NudE neurodevelopment protein 1-like 1 (Ndel1) expression by binding to the 3' untranslated region of Ndel1. Transduction of neural stem cells with a lentiviral vector overexpressing Ndel1 significantly increased cell proliferation and differentiation, decreased neural stem cell apoptosis, and decreased protein expression levels of Wnt3a, ß-catenin, phosphor-GSK-3ß, LEF1, c-myc, c-Jun, and cyclin D1, all members of the Wnt/ß-catenin signaling pathway. These findings suggest that Ndel1 is a novel miR-103-3p target and that miR-103-3p acts by suppressing neural stem cell proliferation and promoting apoptosis and differentiation. This study was approved by the Animal Ethics Committee of Nantong University, China (approval No. 20200826-003) on August 26, 2020.

Inhibition of miR-103-3p Preserves Neurovascular Integrity Through Caveolin-1 in Experimental Subarachnoid Hemorrhage.

Caveolin-1 (Cav-1) is a constitutive structural protein of caveolae in the plasma membrane. It plays an important role in maintaining blood brain barrier (BBB) integrity. In this study, we identified that miR-103-3p, a hypoxia-responsive miRNA, could interact with Cav-1. In endothelial cells, miR-103-3p mimic diminished the expression of Cav-1 and tight junction proteins, which were rescued by the inhibition of miR-103-3p. We found a substantial increase of miR-103-3p and decease of Cav-1 in the rat subarachnoid hemorrhage (SAH) model. Pre-SAH intracerebroventricularly injection of miR-103-3p antagomir relieved Cav-1 loss, sequentially reduced BBB permeability and improved neurological function. Finally, we demonstrated that the salutary effects of miR-103-3p antagomir were abolished in Cav-1 knock-out mice, suggesting that Cav-1 was required for the miR-103-3p inhibition-induced neurovascular protection. Taken together, our findings suggest that the inhibition of miR-103-3p could exert neuroprotective effects through preservation of Cav-1 and BBB integrity, making miR-103-3p a novel therapeutic target for SAH.

MiR-103-3p targets the m6 A methyltransferase METTL14 to inhibit osteoblastic bone formation.

Impaired osteoblast function is involved in osteoporosis, and microRNA (miRNA) dysregulation may cause abnormal osteoblast osteogenic activity. However, the influence of miRNA on osteoblast activity and the underlying mechanisms remain elusive. In this study, miR-103-3p was found to be negatively correlated with bone formation in bone specimens from elderly women with fractures and ovariectomized (OVX) mice. Additionally, miR-103-3p directly targeted Mettl14 to inhibit osteoblast activity, and METTL14-dependent N6 -methyladenosine (m6 A) methylation inhibited miR-103-3p processing by the microprocessor protein DGCR8 and promoted osteoblast activity. Moreover, miR-103-3p inhibited bone formation in vivo, and therapeutic inhibition of miR-103-3p counteracted the decreased bone formation in OVX mice. Further, METTL14 was negatively correlated with miR-103-3p but positively correlated with bone formation in bone specimens from elderly women with fractures and OVX mice. Collectively, our results highlight the critical roles of the miR-103-3p/METTL14/m6 A signaling axis in osteoblast activity, identifying this axis as a potential target for ameliorating osteoporosis.

LncRNA BC083743 Promotes the Proliferation of Schwann Cells and Axon Regeneration Through miR-103-3p/BDNF After Sciatic Nerve Crush.

To investigate the underlying mechanism of lncRNA BC083743 in regulating the proliferation of Schwann cells (SCs) and axon regeneration after sciatic nerve crush (SNC), we used a rat model. Sciatic function index and the atrophy ratio of gastrocnemius muscle were evaluated. The relationship among BC083743, miR-103-3p, and brain-derived neurotrophic factor (BDNF) and their regulation mechanism in the repair of SNC were investigated using in vivo and in vitro experiments. The expression changes of BC083743 were positively associated with that of BDNF following SNC, but the expression changes of miR-103-3p were inversely associated with that of BDNF. The SC proliferation and BDNF expression could be promoted by overexpression of BC083743, while they were inhibited by a miR-103-3p mimic. In addition, BC083743 interacted with and regulated miR-103-3p, thereby promoting BDNF expression and SC proliferation. BC083743 overexpression also promoted axon regeneration through miR-103-3p. In vivo experiments also indicated that BC083743 overexpression promoted the repair of SNC. In conclusion, LncRNA BC083743 promotes SC proliferation and the axon regeneration through miR-103-3p/BDNF after SNC.

Satb2 expression in Foxc1-promoted osteogenic differentiation of MC3T3-E1 cells is negatively regulated by microRNA-103-3p.

The forkhead transcription factor C1 (Foxc1) is a cell-fate-determining factor that controls cranial bone development and osteogenic differentiation. Previously, it was demonstrated that various microRNAs (miRNAs) play important roles in osteogenesis and regulate the complex process of osteogenic differentiation. However, it remains unclear how miRNA expression changes during Foxc1-promoted osteogenic differentiation. In this study, we successfully overexpressed the Foxc1 gene in MC3T3-E1 cells and investigated the alterations in the miRNA expression profile on day 3 after osteogenic induction by using a miRNA microarray. Nine downregulated miRNAs and eight upregulated miRNAs were found to be differentially expressed. Among these miRNAs, miR-103-3p was consistently downregulated in the Foxc1-overexpressing MC3T3-E1 cells and was identified as a negative regulator of osteogenic differentiation by using a gain- and lose-of-function assay. The special AT-rich sequence-binding protein 2 (Satb2), a pivotal osteogenic transcription factor, was identified as the miR-103-3p targeting gene and was verified by real-time polymerase chain reaction, western blot analysis, and luciferase assay. Overexpression of miR-103-3p markedly inhibited the expression of Satb2 and attenuated Foxc1-promoted osteogenic differentiation. Taken together, our results elucidated the miRNA expression profiles of MC3T3-E1 cells in the early stage of Foxc1-promoted osteogenic differentiation and suggested that miR-103-3p acts as a negative regulator of the osteogenic differentiation of MC3T3-E1 cells by directly targeting Satb2.

Chicken gga-miR-103-3p Targets CCNE1 and TFDP2 and Inhibits MDCC-MSB1 Cell Migration.

Marek's disease (MD) is a highly contagious viral neoplastic disease caused by Marek's disease virus (MDV), which can lead to huge economic losses in the poultry industry. Recently, microRNAs (miRNAs) have been found in various cancers and tumors. In recent years, 994 mature miRNAs have been identified through deep sequencing in chickens, but only a few miRNAs have been investigated further in terms of their function. Previously, gga-miR-103-3p was found downregulated in MDV-infected samples by using Solexa deep sequencing. In this study, we further verified the expression of gga-miR-103-3p among MDV-infected spleen, MD lymphoma from liver, noninfected spleen, and noninfected liver, by qPCR. The results showed that the expression of gga-miR-103-3p was decreased in MDV-infected tissues, which was consistent with our previous study. Furthermore, two target genes of gga-miR-103-3p, cyclin E1 (CCNE1) and transcription factor Dp-2 (E2F dimerization partner 2) (TFDP2), were predicted and validated by luciferase reporter assay, qPCR, and western blot analysis. The results suggested that CCNE1 and TFDP2 are direct targets of gga-miR-103-3p in chickens. Subsequent cell proliferation and migration assay showed that gga-miR-103-3p suppressed MDCC-MSB1 migration, but did not obviously modulate MDCC-MSB1 cell proliferation. In conclusion, gga-miR-103-3p targets the CCNE1 and TFDP2 genes, and suppresses cell migration, which indicates that it might play an important role in MD tumor transformation.