scholarly journals Murine miR-483-3p promotes proliferation and suppresses differentiation of C2C12 cells by targeting SRF

2017 ◽  
Author(s):  
Jing Huang ◽  
Ying Liu ◽  
Liangliang Fu ◽  
Hegang Li ◽  
Bingkun Xie ◽  
...  
Keyword(s):  
Muscle Development ◽  
Myogenic Differentiation ◽  
C2c12 Cells ◽  
Luciferase Reporter ◽  
Back Muscle ◽  
Mouse Development ◽  
Protein Levels ◽  
Complicated Process ◽  
Non Coding Rnas ◽  
Dual Luciferase Reporter Assay

Myogenesis is a complicated process, which is regulated by numerous regulators. MicroRNAs (miRNAs) are conserved non-coding RNAs of ~22 nucleotides, which regulate post-transcriptional gene expression in many physiological and pathophysiological processes. Recent studies have indicated that microRNAs are critical regulators of muscle development. Here, we report miR-483-3p as a new essential regulator of muscle development, mediating myoblast proliferation and myogenic differentiation. miR-483-3p is strongly and almost exclusively expressed in muscle-related tissues such as leg muscle, back muscle, and heart. Its expression is downregulated during mouse development. Overexpression of miR-483-3p in C2C12 cells promotes proliferation and suppresses myogenic differentiation. A dual-luciferase reporter assay demonstrated miR-483-3p direct targets to the 3′-UTR of the SRF gene. Overexpression of miR-483-3p reduced SRF protein levels in C2C12 myoblasts. These results reveal a novel function of miR-483-3p as a positive regulator of C2C12 proliferation and inhibitor of myogenic differentiation via SRF downregulation.

scholarly journals Murine miR-483-3p promotes proliferation and suppresses differentiation of C2C12 cells by targeting SRF

2017 ◽  
Author(s):  
Jing Huang ◽  
Ying Liu ◽  
Liangliang Fu ◽  
Hegang Li ◽  
Bingkun Xie ◽  
...  
Keyword(s):  
Muscle Development ◽  
Myogenic Differentiation ◽  
C2c12 Cells ◽  
Luciferase Reporter ◽  
Back Muscle ◽  
Mouse Development ◽  
Protein Levels ◽  
Complicated Process ◽  
Non Coding Rnas ◽  
Dual Luciferase Reporter Assay

Myogenesis is a complicated process, which is regulated by numerous regulators. MicroRNAs (miRNAs) are conserved non-coding RNAs of ~22 nucleotides, which regulate post-transcriptional gene expression in many physiological and pathophysiological processes. Recent studies have indicated that microRNAs are critical regulators of muscle development. Here, we report miR-483-3p as a new essential regulator of muscle development, mediating myoblast proliferation and myogenic differentiation. miR-483-3p is strongly and almost exclusively expressed in muscle-related tissues such as leg muscle, back muscle, and heart. Its expression is downregulated during mouse development. Overexpression of miR-483-3p in C2C12 cells promotes proliferation and suppresses myogenic differentiation. A dual-luciferase reporter assay demonstrated miR-483-3p direct targets to the 3′-UTR of the SRF gene. Overexpression of miR-483-3p reduced SRF protein levels in C2C12 myoblasts. These results reveal a novel function of miR-483-3p as a positive regulator of C2C12 proliferation and inhibitor of myogenic differentiation via SRF downregulation.

scholarly journals GATA1-induced upregulation of LINC01503 promotes carboplatin resistance in ovarian carcinoma by upregulating PD-L1 via sponging miR-766-5p

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Yao Li ◽  
Yan Zhai ◽  
Yuxuan Chen
Keyword(s):  
Ovarian Carcinoma ◽  
Inhibitory Concentration ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Invasive Ability ◽  
Protein Levels ◽  
Reverse Transcription Quantitative Pcr ◽  
Non Coding Rnas ◽  
Dual Luciferase Reporter Assay

Abstract Background Ovarian Carcinoma (OCa) is a high-mortality malignancy derived from female reproductive system. Increasing evidence has identified long non-coding RNAs (lncRNAs) as important regulators in OCa chemoresistance. In this study, we intended to explore the role of LINC01503 in OCa resistance to carboplatin (CBP). Methods Gene expression was measured by reverse transcription-quantitative PCR (RT-qPCR) in OCa cells. Western blot was adopted to detect protein levels of GATA1, PD-L1, E-cadherin, N-cadherin, Vimentin, Bcl-2, Bax, cleaved caspase-3. To assess the effects of LINC01503 on the resistance of OCa cells to CBP, Cell Counting Kit-8 (CCK-8), colony formation, Transwell, and flow cytometry experiments were performed to evaluate half-maximal inhibitory concentration (IC50), cell viability, migrative and invasive ability, as well as cell apoptosis. Dual-luciferase reporter assay was employed to assess the associations between the genes. Results LINC01503 was upregulated in CBP-resistant OCa cells. LINC01503 knockdown reduced CBP resistance in OCa cells. Besides, GATA-binding protein 1 (GATA1) activated LINC01503 transcription in CBP-resistant OCa cells. MiR-766-5p was lowly expressed in CBP-resistant cells and confirmed as a target for LINC01503. In addition, miR-766-5p overexpression increased CBP sensitivity in OCa cells. PD-L1 was verified as the target of miR-766-5p. Besides, LINC01503 upregulated PD-L1 level by regulating miR-766-5p. Furthermore, rescue experiments showed that PD-L1 overexpression abrogated the inhibited impacts of blocking LINC01503 on CBP resistance in OCa cells. Conclusion GATA1-induced LINC01503 expedited CBP resistance in OCa cells via the miR-766-5p/PD-L1 axis, providing a new target for improving the efficacy of OCa chemotherapy. Graphical Abstract

scholarly journals MiR-501-3p Forms a Feedback Loop with FOS, MDFI, and MyoD to Regulate C2C12 Myogenesis

Cells ◽  
2019 ◽  
Vol 8 (6) ◽  
pp. 573 ◽  
Author(s):  
Lianjie Hou ◽  
Linhui Zhu ◽  
Huaqin Li ◽  
Fangyi Jiang ◽  
Lingbo Cao ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Feedback Loop ◽  
Muscle Development ◽  
Myogenic Differentiation ◽  
C2c12 Cells ◽  
Luciferase Reporter ◽  
Myoblast Differentiation ◽  
Regulation Mechanism ◽  
Reporter System ◽  
Regulatory Relationship

Skeletal muscle plays an essential role in maintaining body energy homeostasis and body flexibility. Loss of muscle mass leads to slower wound healing and recovery from illness, physical disability, poor quality of life, and higher health care costs. So, it is critical for us to understand the mechanism of skeletal muscle myogenic differentiation for maintaining optimal health throughout life. miR-501-3p is a novel muscle-specific miRNA, and its regulation mechanism on myoblast myogenic differentiation is still not clear. We demonstrated that FOS was a direct target gene of miR-501-3p, and MyoD regulated miR-501-3p host gene Clcn5 through bioinformatics prediction. Our previous laboratory experiment found that MDFI overexpression promoted C2C12 myogenic differentiation and MyoD expression. The database also showed there is an FOS binding site in the MDFI promoter region. Therefore, we hypothesize that miR-501-3p formed a feedback loop with FOS, MDFI, and MyoD to regulate myoblast differentiation. To validate our hypothesis, we demonstrated miR-501-3p function in the proliferation and differentiation period of C2C12 cells by transfecting cells with miR-501-3p mimic and inhibitor. Then, we confirmed there is a direct regulatory relationship between miR-501-3p and FOS, MyoD and miR-501-3p, FOS and MDFI through QPCR, dual-luciferase reporter system, and ChIP experiments. Our results not only expand our understanding of the muscle myogenic development mechanism in which miRNA and genes participate in controlling skeletal muscle development, but also provide treatment strategies for skeletal muscle or metabolic-related diseases in the future.

scholarly journals Downregulation of lncRNA ANRIL Inhibits Osteogenic Differentiation of Periodontal Ligament Cells via Sponging miR-7 through NF-κB Pathway

2021 ◽  
Author(s):  
Xinwei Liu ◽  
Yue Zhou
Keyword(s):  
Western Blot ◽  
Osteogenic Differentiation ◽  
Periodontal Ligament ◽  
Molecular Mechanisms ◽  
Luciferase Reporter ◽  
Periodontal Ligament Cells ◽  
Protein Levels ◽  
Alp Activity ◽  
Non Coding Rnas ◽  
Dual Luciferase Reporter Assay

Abstract Background Long non-coding RNAs (lncRNAs) are dysregulation in periodontitis development and involved in osteogenesis. The current study aimed was to investigate the function of lncRNA ANRIL in periodontal ligament cells (PDLCs) and potential molecular mechanisms.Methods Firstly, the level of ANRIL was tested by qPCR. Then PDLCs were treated with a mineralizing solution to induce the osteogenic differentiation. ALP activity was measured and protein levels of BMP2, Osterix, and OCN were measured by western blot. A target of ANRIL was verified using dual-luciferase reporter assay. MiR-7 level was measured by qPCR and the signalings of NF-κB pathway were tested by western blot.Results ANRIL expression was downregulated in PDL tissues. Next, ALP activity and protein levels of BMP2, Osterix, and OCN were reduced to show PDLCs were differentiated. ANRIL level was increased in differential PDLCs, and which knockdown inhibited osteogenic differentiation. Then, miR-7 was found as a target of ANRIL. The miR-7 level was upregulated in PDL tissues and reduced in differential PDLCs. Inhibition of miR-7 suppressed ALP activity and BMP2, Osterix, and OCN expression. Moreover, inhibition of miR-7 reversed the effects on the osteogenic differentiation induced by knockdown of ANRIL. Besides, the levels of p-P65 and p-IκBα were elevated by ANRIL downregulation and were rescued by suppressing miR-7.Conclusions Knockdown of ANRIL inhibited osteogenic differentiation via sponging miR-7 through the NF-κB pathway, suggesting that ANRIL might be a therapeutic target for periodontitis.

scholarly journals MiR-34b-5p Mediates the Proliferation and Differentiation of Myoblasts by Targeting IGFBP2

Cells ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 360 ◽  
Author(s):  
Wang ◽  
Zhang ◽  
Li ◽  
Abdalla ◽  
Chen ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Cell Cycle Progression ◽  
Muscle Development ◽  
Muscle Growth ◽  
Flow Cytometric Analysis ◽  
Luciferase Reporter ◽  
Cycle Progression ◽  
Flow Cytometric ◽  
Proliferation And Differentiation ◽  
Dual Luciferase Reporter Assay

As key post-transcriptional regulators, microRNAs (miRNAs) play an indispensable role in skeletal muscle development. Our previous study suggested that miR-34b-5p and IGFBP2 could have a potential role in skeletal muscle growth. Our goal in this study is to explore the function and regulatory mechanism of miR-34b-5p and IGFBP2 in myogenesis. In this study, the dual-luciferase reporter assay and Western blot analysis showed that IGFBP2 is a direct target of miR-34b-5p. Flow cytometric analysis and EdU assay showed that miR-34b-5p could repress the cell cycle progression of myoblasts, and miR-34b-5p could promote the formation of myotubes by promoting the expression of MyHC. On the contrary, the overexpression of IGFBP2 significantly facilitated the proliferation of myoblasts and hampered the formation of myotubes. Together, our results indicate that miR-34b-5p could mediate the proliferation and differentiation of myoblasts by targeting IGFBP2.

MiR-155 aggravates impaired autophagy of pancreatic acinar cells through targeting Rictor

2020 ◽  
Vol 52 (2) ◽  
pp. 192-199 ◽  
Author(s):  
Xueming Zhang ◽  
Jiangtao Chu ◽  
Haijun Sun ◽  
Dali Zhao ◽  
Biao Ma ◽  
...  
Keyword(s):  
Acinar Cells ◽  
Immunofluorescence Assay ◽  
Pancreatic Acinar Cell ◽  
Pancreatic Acinar Cells ◽  
Luciferase Reporter ◽  
Cellular Model ◽  
Protein Levels ◽  
Ar42j Cells ◽  
Direct Target ◽  
Dual Luciferase Reporter Assay

Abstract The aim of this study was to investigate the role and mechanism of miR-155 in regulating autophagy in a caerulein-induced acute pancreatitis (AP) cellular model. GFP-LC3 immunofluorescence assay was performed to detect autophagy vesicle formation in pancreatic acinar cell line AR42J. AR42J cells were transfected with miR-155 mimic, inhibitor, and corresponding controls to explore the effect of miR-155 on autophagy. The protein levels of LC3-I, LC3-II, Beclin-1, and p62 were analyzed by western blot analysis. Dual-luciferase reporter assay was performed to verify the interaction between miR-155 and Rictor (RPTOR independent companion of MTOR complex 2). The results showed that caerulein treatment induced impaired autophagy as evidenced by an increase in the accumulation of p62 together with LC3-II in AR42J cells, accompanied by miR-155 upregulation. Furthermore, miR-155 overexpression aggravated, whereas miR-155 silencing reduced the caerulein-induced impairment of autophagy. Mechanistically, Rictor was confirmed to be a direct target of miR-155, which could rescue the miR-155 overexpression-mediated aggravation of impaired autophagy. Collectively, these findings indicate that miR-155 aggravates impaired autophagy in caerulein-treated pancreatic acinar cells by targeting Rictor.

Inhibition of Drp1-dependent mitochondrial division impairs myogenic differentiation

2013 ◽  
Vol 305 (8) ◽  
pp. R927-R938 ◽  
Author(s):  
Boa Kim ◽  
Ji-Seok Kim ◽  
Yisang Yoon ◽  
Mayra C. Santiago ◽  
Michael D. Brown ◽  
...  
Keyword(s):  
Muscle Development ◽  
Myogenic Differentiation ◽  
Mitochondrial Dynamics ◽  
C2c12 Cells ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Dominant Negative Mutant ◽  
Serum Starvation ◽  
Dependent Manner ◽  
Mitochondrial Division

Mitochondria are dynamic organelles forming a tubular network that is continuously fusing and dividing to control their morphology and functions. Recent literature has shed new light on a potential link between the dynamic behavior of mitochondria and muscle development. In this study, we investigate the role of mitochondrial fission factor dynamin-related protein 1 (Drp1) in myogenic differentiation. We found that differentiation of C2C12 myoblasts induced by serum starvation was accompanied by a gradual increase in Drp1 protein expression (to ∼350% up to 3 days) and a fast reduction of Drp1 phosphorylation at Ser-637 (to ∼30%) resulting in translocation of Drp1 protein from the cytosol to mitochondria. During differentiation, treatment of myoblasts with mitochondrial division inhibitor ( mdivi-1), a specific inhibitor of Drp1 GTPase activity, caused extensive formation of elongated mitochondria, which coincided with increased apoptosis evidenced by both enhanced caspase-3 activity and increased number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells. Furthermore, the mdivi-1-treated myotubes ( day 3 in differentiation media) showed a reduction in mitochondrial DNA content, mitochondrial mass, and membrane potential in a dose-dependent manner indicating defects in mitochondrial biogenesis during myogenic differentiation. Most interestingly, mdivi-1 treatment significantly suppressed myotube formation in both C2C12 cells and primary myoblasts. Likewise, stable overexpression of a dominant negative mutant Drp1 (K38A) dramatically reduced myogenic differentiation. These data suggest that Drp-1-dependent mitochondrial division is a necessary step for successful myogenic differentiation, and perturbation of mitochondrial dynamics hinders normal mitochondrial adaptations during muscle development. Therefore, in the present study, we report a novel physiological role of mitochondrial dynamics in myogenic differentiation.

scholarly journals LncRNA SNHG14 regulates the DDP-resistance of non-small cell lung cancer cell through miR-133a/HOXB13 pathway

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Li Xu ◽  
Yan Xu ◽  
Min Yang ◽  
Jia Li ◽  
Fang Xu ◽  
...  
Keyword(s):  
Cell Line ◽  
A549 Cells ◽  
Parental Cell ◽  
Luciferase Reporter ◽  
Inhibitory Effects ◽  
Through Flow ◽  
Non Coding Rnas ◽  
Apoptosis Related Protein ◽  
Dual Luciferase Reporter Assay

Abstract Background Recently, long non-coding RNAs (lncRNAs) have been reported to be involved in regulating chemo-resistance of NSCLC, however, the role of lncRNA SNHG14 in the DDP-resistance of NSCLC remains unexplored. Methods Relative expression of SNHG14, HOXB13 and miR-133a in DDP-resistant A549 (A549/DDP) cell and its parental cell A549 were measured using qRT-PCR. Cell proliferation viability of indicated A549/DDP cell was estimated via CCK-8 and colony formation experiments. Cell cycle and apoptosis were analyzed through flow cytometry. Expression of apoptosis-related protein and HOXB13 were detected via western blot. The interaction among SNHG14, HOXB13 and miR-133a was predicted by bioinformatics and validated by dual-luciferase reporter assay. Results LncRNA SNHG14 and HOXB13 were upregulated while miR-133a was downregulated in A549/DDP cell line compared to A549 cell line. SNHG14 knockdown or miR-133a overexpression was demonstrated to increase the DDP-sensitivity of A549/DDP cells. SNHG14 was revealed to compete with HOXB13 for miR-133a binding in A549/DDP cells. Inhibition of miR-133a in A549 cells could reverse the promotive effects of SNHG14 knockdown on DDP-sensitivity, as well as the inhibitory effects on HOXB13 expression. HOXB13 overexpression was revealed to abolish the enhanced effects of miR-133a on the sensitivity of A549/DDP cell to DDP. Conclusion Our findings demonstrated that SNHG14 was involved in the development of DDP-resistance of A549/DDP cells through miR-133a/HOXB13 axis, which may present a path to novel therapeutic stratagems for DDP resistance of NSCLC.

RyR1-mediated moderate endoplasmic reticulum stress is required for myogenic differentiation of myoblasts

2021 ◽  
Author(s):  
Kai Qiu ◽  
Yubo Wang ◽  
Doudou Xu ◽  
Linjuan He ◽  
Xin Zhang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Cell Fate ◽  
Muscle Development ◽  
Myogenic Differentiation ◽  
C2c12 Cells ◽  
Stress Signaling ◽  
Congenital Myopathies ◽  
Myotube Formation

Abstract BackgroundCytosolic Ca2+ plays vital roles in myogenesis and muscle development. Key mutations of ryanodine receptor 1 (RyR1), a major Ca2+ release channel of endoplasmic reticulum (ER), are main causes of severe congenital myopathies. The role of RyR1 in myogenic differentiation has attracted intense research interest, however, it remains unclear. MethodsThis study employed RyR1-knockdown myoblasts and CRISPR/Cas9-based RyR1-knockout myoblasts cells to explore the role of RyR1 in myogenic differentiation, myotube formation as well as the potential mechanism of RyR1-related myopathies.ResultsCytoplasmic Ca2+ concentration was significantly elevated during myogenic differentiation of both primary myogenic cells and myoblasts C2C12 cells, accompanied with a dramatic increase in RyR1 expression and resultant ER stress. Inhibition of RyR1 by siRNA-mediated silence or chemical inhibitor, dantrolene, significantly reduced cytosolic Ca2+, alleviated ER stress, and blocked multinucleated myotube formation. Moderate activation of ER stress effectively relieved myogenic differentiation stagnation induced by RyR1 suppression and demonstrated that RyR1 modulates myogenic differentiation via activation of Ca2+ -induced ER stress signaling. RyR1 knockout-induced Ca2+ leakage led to severe ER stress and excessive unfolded protein response, and drove cell fate from differentiation into apoptosis. ConclusionsTherefore, we concluded that dramatic increase in RyR1 expression is required for myogenic differentiation, and RyR1-mediated Ca2+ release leading to the activation of ER stress signaling serves a double-edged sword role during myogenic differentiation. This study contributes to a novel understanding of the role of RyR1 in muscle development and related congenital myopathies, and provides a potential target for regulation of muscle regeneration and tissue engineering.

scholarly journals The long non-coding RNA TUG1-miR-9a-5p axis contributes to ischemic injuries by promoting cardiomyocyte apoptosis via targeting KLF5

2019 ◽  
Vol 10 (12) ◽  
Author(s):  
Di Yang ◽  
Jie Yu ◽  
Hui-Bin Liu ◽  
Xiu-Qing Yan ◽  
Juan Hu ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Cardiomyocyte Apoptosis ◽  
Luciferase Reporter ◽  
Apoptotic Pathway ◽  
Non Coding Rna ◽  
Non Coding Rnas ◽  
Long Non Coding Rna ◽  
Cultured Cardiomyocytes ◽  
Competitive Endogenous Rna ◽  
Dual Luciferase Reporter Assay

AbstractNon-coding RNAs participate in many cardiac pathophysiological processes, including myocardial infarction (MI). Here we showed the interplay between long non-coding RNA taurine-upregulated gene 1 (lncR-TUG1), miR-9a-5p (miR-9) and Krüppel-like factor 5 (KLF5). LncR-TUG1 was upregulated in ischemic heart and in cultured cardiomyocytes exposed to H2O2. Knockdown of lncR-TUG1 markedly ameliorated impaired cardiac function of MI mice. Further study showed that lncR-TUG1 acted as a competitive endogenous RNA of miR-9, and silencing of lncR-TUG1 inhibited cardiomyocyte apoptosis by upregulating miR-9 expression. Furthermore, the miR-9 overexpression obviously prevented ischemia injury and significantly inhibited H2O2-induced cardiomyocyte apoptosis via inhibition of mitochondrial apoptotic pathway. KLF5, as a target gene of miR-9 by dual-luciferase reporter assay, was involved in the process of miR-9 in regulating cardiomyocyte apoptosis. Our data identified the KLF5 was downregulated by miR-9 overexpression and knockdown of KLF5 inhibited cardiomyocyte apoptosis induced by H2O2. MiR-9 exerts anti-cardiomyocyte apoptotic affects by targeting KLF5. Collectively, our data identify a novel function of lncR-TUG1/miR-9/KLF5 axis in regulating cardiomyocyte apoptosis that affects myocardial infarction progression.

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