MicroRNA-106a-5p Inhibited C2C12 Myogenesis via Targeting PIK3R1 and Modulating the PI3K/AKT Signaling.
ABSTRACT: The microRNA (miR)-17 family is widely expressed in mammalian tissues and play important roles in various physiological and pathological processes. Here, the functions of miR-106a-5p, a member of miR-17 family, were explored during myogenic differentiation in C2C12 cell line. First, miR-106a-5p was found to be relatively lower expressed in two-month skeletal muscle tissues and gradually decreased upon myogenic stimuli. Forced expression of miR-106a-5p significantly reduced the differentiation index, fusion index as well as the expression of myogenic markers (MyoD, MyoG, MyHC, Myomixer, Myomarker). Meanwhile, the levels of phosphorylated AKT were reduced by overexpression of miR-106a-5p, and administration of insulin-like growth factor 1 (IGF1), a booster of myogenic differentiation, could recover all the inhibitory effects above of miR-106a-5p. Furthermore, miR-106a-5p was elevated in aged muscles and dexamethasone (DEX)-treated myotubes, and up-regulation of miR-106a-5p significantly reduced the diameters of myotubes accompanied with increased levels of muscular atrophy genes and decreased PI3K/AKT activities. Finally, miR-106a-5p was demonstrated to directly bind to the 3'-UTR of PIK3R1, thus, repress the PI3K/AKT signaling.
Project description:Periodontal ligament stem cells (PDLSCs) are characterized by multiple differentiation potential and potent self-renewal ability, yet much remains to be elucidated that what determines these properties. Long noncoding RNAs (lncRNAs) have been suggested to involve in multiple biological process under physiological and pathological conditions, including osteogenic differentiation. In the present study, we performed comprehensive lncRNA profiling by lncRNA microarray analysis and identified prostate cancer-associated ncRNA transcript-1 (lncPCAT1) was gradually increased in PDLSCs during consecutive osteogenic induction, and it could further positively regulate the osteogenic differentiation both in vitro and in vivo, whereas lncPCAT1 inhibition led to suppressed osteogenic differentiation. Thereafter, we inferred a predicted interaction between lncPCAT1 and miR-106a-5p and then confirmed the direct binding sites of miR-106a-5p on lncPCAT1. Although miR-106a-5p upregulation led to decreased osteogenic differentiation, lncPCAT1 overexpression could reverse its suppression, indicating that lncPCAT1 act as a competing endogenous RNA for miR-106a-5p. Moreover, lncPCAT1 could sponge miR-106a-5p to upregulate miR-106a-5p-targeted gene BMP2, which was a crucial gene involved in osteogenic differentiation. Interestingly, we found that E2F5, another target of miR-106a-5p, could bind to the promoter of lncPCAT1 and then form a feed-forward regulatory network targeting BMP2. In conclusion, our study provided a novel lncRNA-miRNA feed-forward regulatory network and a promising target to modulate the osteogenic differentiation of PDLSCs.
Project description:Cryptotanshinone (CTS) has emerged as an anti-inflammatory agent in osteoarthritis (OA). However, the molecular mechanism underlying its potent therapeutic effect on OA remains largely unknown. MicroRNAs (miRNAs) act as crucial regulators in maintaining cartilage homeostasis. To investigate whether CTS protects against developing OA through regulation of miRNAs, we examined the potential CTS-mediated miRNA molecules using microarray analysis. We found that CTS significantly promoted miR-106a-5p expression in chondrocytes. Using the OA mouse model created by anterior cruciate ligament transection, we revealed that intra-articular injection of miR-106a-5p agomir attenuated OA. In addition, miR-106a-5p inhibited GLI-similar 3 (GLIS3) production by directly targeting the 3' untranslated region. CTS promoted miR-106a-5p expression through recruitment of a member of the paired box (PAX) family of transcription factors, PAX5, to the miR-106a-5p promoter. Inhibition of PAX5 mimicked the effect of miR-106a-5p and abolished the CTS ability to regulate miR-106a-5p expression. In OA patients, miR-106-5p is downregulated which is accompanied by downregulation of PAX5 and upregulation of GLIS3. Collectively, these data highlight that the PAX5/miR-106a-5p/GLIS3 axis acts as a novel pleiotropic regulator in CTS-mediated OA cartilage protection, suggesting that miR-106a-5p and PAX5 activation and GLIS3 inhibition might be useful and attractive for therapeutic strategies to treat OA patients.
Project description:Long noncoding RNAs (lncRNAs) have emerged as important regulators of human cancers. LncRNA GAS5 (GAS5) is identified as a tumor suppressor involved in several cancers. However, the roles of GAS5 and the mechanisms responsible for its functions in gastric cancer (GC) have not been well documented. Herein, the decreased GAS5 and increased miRNA-106a-5p levels were observed in GC and cell lines. GAS5 level was significantly inversely correlated with miRNA-106a-5p level in GC tissues. Moreover, dual-luciferase reporter and qRT-PCR assays showed that GAS5 bound to miRNA-106a-5p and negatively regulated its expression in GC cells. Functional experiments showed that GAS5 overexpression suppressed GC cell proliferation, migration and invasion capabilities, and promoted apoptosis, while miRNA-106a-5p overexpression inverted the functional effects induced by GAS5 overexpression. In vivo, GAS5 overexpression inhibited tumor growth by negatively regulating miRNA-106a-5p expression. Mechanistic investigations revealed that GAS5 overexpression inactivated the Akt/mTOR pathway by suppressing miRNA-106a-5p expression in vitro and in vivo Taken together, our findings conclude the GAS5 overexpression suppresses tumorigenesis and development of gastric cancer by sponging miR-106a-5p through the Akt/mTOR pathway.
Project description:MicroRNA-106a-5p (MiR-106a-5p), a small non-coding RNA, has been reported to be downregulated in astrocytoma, osteosarcoma and colorectal cancer. However, the expression levels and biological function in renal cell carcinoma (RCC) have not been studied yet. In this study, we found that the miR-106a-5p was significantly downregulated in RCC tissues and cell lines, and that overexpression of miR-106a-5p led to decreased cell metastasis ability in a xenograft model. Inhibition of miR-106a-5p in RCC cell lines altered the cell migration, invasion and wound healing abilities. Mechanistic studies demonstrated that miR-106a-5p directly bound to the 3'-UTR of the PAK5 mRNA and mediated a decrease in the protein expression of PAK5. We further proved that PAK5 protein levels were negatively correlated with the miR-106a-5p expression in both patient samples and xenograft model. In epigenetics, methylation specific PCR experiments indicated that the upstream gene promoter of miR-106a-5p was hypermethylated in RCC, which might be responsible for its downregulation. Our findings suggested that miR-106a-5p might be a potential gene therapy target for the treatment of RCC metastasis.
Project description:Astrocytomas are common malignant intracranial tumors that comprise the majority of adult primary central nervous system tumors. MicroRNAs (miRNAs) are small, non-coding RNAs (20-24 nucleotides) that post-transcriptionally modulate gene expression by negatively regulating the stability or translational efficiency of their target mRNAs. In our previous studies, we found that the downregulation of miR-106a-5p in astrocytomas is associated with poor prognosis. However, its specific gene target(s) and underlying functional mechanism(s) in astrocytomas remain unclear. In this study, we used mRNA microarray experiments to measure global mRNA expression in the presence of increased or decreased miR-106a-5p levels. We then performed bioinformatics analysis based on multiple target prediction algorithms to obtain candidate target genes that were further validated by computational predictions, western blot analysis, quantitative real-time PCR, and the luciferase reporter assay. Fas-activated serine/threonine kinase (FASTK) was identified as a direct target of miR-106a-5p. In human astrocytomas, miR-106a-5p is downregulated and negatively associated with clinical staging, whereas FASTK is upregulated and positively associated with advanced clinical stages, at both the protein and mRNA levels. Furthermore, Kaplan-Meier analysis revealed that the reduced expression of miR-106a-5p or the increased expression of FASTK is significantly associated with poor survival outcome. These results further supported the finding that FASTK is a direct target gene of miR-106a-5p. Next, we explored the function of miR-106a-5p and FASTK during astrocytoma progression. Through gain-of-function and loss-of-function studies, we demonstrated that miR-106a-5p can significantly inhibit cell proliferation and migration and can promote cell apoptosis in vitro. The knockdown of FASTK induced similar effects on astrocytoma cells as those induced by the overexpression of miR-106a-5p. These observations suggest that miR-106a-5p functions as a tumor suppressor during the development of astrocytomas by targeting FASTK.
Project description:Novel noninvasive biomarkers with high sensitivity and specificity for the diagnosis of breast cancer (BC) are urgently needed in clinics. The aim of this study was to explore whether miRNAs from the miR-106a-363 cluster can be detected in the circulation of BC patients and whether these miRNAs can serve as potential diagnostic biomarkers.The expression of 12 miRNAs from the miR-106a-363 cluster was evaluated using qRT-PCR in 400 plasma samples (from 200 BC patients and 200 healthy controls (HCs)) and 406 serum samples (from 204 BC patients and 202 HCs) via a three-phase study. The identified miRNAs were further examined in tissues (32 paired breast tissues), plasma exosomes (from 32 BC patients and 32 HCs), and serum exosomes (from 32 BC patients and 32 HCs).Upregulated levels of four plasma miRNAs (miR-106a-3p, miR-106a-5p, miR-20b-5p, and miR-92a-2-5p) and four serum miRNAs (miR-106a-5p, miR-19b-3p, miR-20b-5p, and miR-92a-3p) were identified and validated in BC. A plasma 4-miRNA panel and a serum 4-miRNA panel were constructed to discriminate BC patients from HCs. The areas under the receiver-operating characteristic curves of the plasma panel were 0.880, 0.902, and 0.858, and those of the serum panel were 0.910, 0.974, and 0.949 for the training, testing, and external validation phases, respectively. Two overlapping miRNAs (miR-106a-5p and miR-20b-5p) were consistently upregulated in BC tissues. Except for the expression of the plasma-derived exosomal miR-20b-5p, the expression patterns of exosomal miRNAs were concordant between plasma and serum, indicating the potential use of exosomal miRNAs as biomarkers.We identified four plasma miRNAs and four serum miRNAs from the miR-106a-363 cluster as promising novel biomarkers for the diagnosis of BC.
Project description:Novel long non-coding RNA Fer-1-like protein 4 (FER1L4) has been confirmed to play crucial regulatory roles in tumor progression. It exerts an impact on tumor suppression and functions as a competing endogenous RNA (ceRNA) by sponging miR-106a-5p in gastric cancer. However, its clinical significance in colon cancer is completely unknown. The aim of the present study was to annotate the role of FER1L4 and its clinical value in colon cancer. The results showed the aberrant expression of FER1L4 and miR-106a-5p in colon cancer tissues. In addition, significant negative correlation between FER1L4 and miR-106a-5p expression levels was observed. Among the colon cancer cell lines, FER1L4 levels were relatively lower, with concurrent high levels of miR-106a-5p. Restoration of FER1L4 decreased the expression of miR-106a-5p, and had a significant influence on colon cancer cell proliferation, migration and invasion. The FER1L4 expression was correlated with depth of tumor invasion, lymph node metastasis, vascular invasion and clinical stage. Moreover, striking differences in overall survival and disease-free survival were observed for the cases with both low FER1L4 expression and high miR-106a-5p expression compared with cases with high FER1L4 expression and low miR-106a-5p expression. Circulating FER1L4 and miR-106a-5p levels were decreased and increased, respectively, in colon cancer patients after surgery. Our findings indicated that FER1L4 could exert a tumor suppressive impact on colon cancer, which at least, in part, through suppressing miR-106a-5p expression, and depletion of FER1L4, alone or combined with overexpression of miR-106a-5p, is predictive of poor prognosis in colon cancer and may play a crucial role in cancer prevention and treatment.
Project description:Osteosarcoma (OS) is a common primary malignant bone tumor among adolescences, and the emergence of multidrug resistance poses a huge challenge for clinical treatment of OS. LncRNA HOTAIR (HOX antisense intergenic RNA) has been reported to be associated with many malignancies, including OS. However, the underlying mechanisms of HOTAIR involved in drug resistance in OS are obscure. Our study showed that HOTAIR was upregulated in cisplatin (DDP)-resistant OS tissues and cells. HOTAIR knockdown decreased the DDP resistance, drug resistance-related gene expression, cell proliferation, and invasion and promoted apoptosis of Saos2/DDP, MG-63/DDP, and U2OS/DDP cells. Mechanism researches displayed that miR-106a-5p was downregulated in DDP-resistant OS tissues and cells. MiR-106a-5p directly bound with HOTAIR and was regulated by HOTAIR. Moreover, STAT3 was inhibited by miR-106a-5p at a post-transcriptional level, and the transfection of miR-106a-5p reversed the upregulation of STAT3 caused by HOTAIR overexpression. The increase or decrease of miR-106a-5p suppressed the effect of HOTAIR upregulation or downregulation on DDP resistance, cell proliferation, invasion, and apoptosis of Saos2/DDP, MG-63/DDP, and U2OS/DDP cells. What's more, the transfection of STAT3 siRNA reversed the decrease of DDP resistance, cell proliferation, and invasion and rescued the increase of apoptosis induced by miR-106a-5p inhibition. These data suggested that HOTAIR enhanced DDP resistance of Saos2/DDP, MG-63/DDP, and U2OS/DDP cells by affecting cell proliferation, invasion, and apoptosis via miR-106a-5p/STAT3 axis.
Project description:MicroRNAs are evolutionarily conserved, small non-coding RNAs that play critical post-transcriptional regulatory roles in skeletal muscle development. We previously found that miR-9-5p is abundantly expressed in chicken skeletal muscle. Here, we demonstrate a new role for miR-9-5p as a myogenic microRNA that regulates skeletal muscle development. The overexpression of miR-9-5p significantly inhibited the proliferation and differentiation of skeletal muscle satellite cells (SMSCs), whereas miR-9-5p inhibition had the opposite effect. We show that insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) is a target gene of miR-9-5p, using dual-luciferase assays, RT-qPCR, and Western Blotting, and that it promotes proliferation and differentiation of SMSCs. In addition, we found that IGF2BP3 regulates IGF-2 expression, using overexpression and knockdown studies. We show that Akt is activated by IGF2BP3 and is essential for IGF2BP3-induced cell development. Together, our results indicate that miR-9-5p could regulate the proliferation and differentiation of myoblasts by targeting IGF2BP3 through IGF-2 and that this activity results in the activation of the PI3K/Akt signaling pathway in skeletal muscle cells.
Project description:In patients with Duchenne muscular dystrophy (DMD), the absence of a functional dystrophin protein results in sarcolemmal instability, abnormal calcium signaling, cardiomyopathy, and skeletal muscle degeneration. Using the dystrophin-deficient sapje zebrafish model, we have identified microRNAs (miRNAs) that, in comparison to our previous findings in human DMD muscle biopsies, are uniquely dysregulated in dystrophic muscle across vertebrate species. MiR-199a-5p is dysregulated in dystrophin-deficient zebrafish, mdx(5cv) mice, and human muscle biopsies. MiR-199a-5p mature miRNA sequences are transcribed from stem loop precursor miRNAs that are found within the introns of the dynamin-2 and dynamin-3 loci. The miR-199a-2 stem loop precursor transcript that gives rise to the miR-199a-5p mature transcript was found to be elevated in human dystrophic muscle. The levels of expression of miR-199a-5p are regulated in a serum response factor (SRF)-dependent manner along with myocardin-related transcription factors. Inhibition of SRF-signaling reduces miR-199a-5p transcript levels during myogenic differentiation. Manipulation of miR-199a-5p expression in human primary myoblasts and myotubes resulted in dramatic changes in cellular size, proliferation, and differentiation. MiR-199a-5p targets several myogenic cell proliferation and differentiation regulatory factors within the WNT signaling pathway, including FZD4, JAG1, and WNT2. Overexpression of miR-199a-5p in the muscles of transgenic zebrafish resulted in abnormal myofiber disruption and sarcolemmal membrane detachment, pericardial edema, and lethality. Together, these studies identify miR-199a-5p as a potential regulator of myogenesis through suppression of WNT-signaling factors that act to balance myogenic cell proliferation and differentiation.