MicroRNA-106b Induces Mitochondrial Dysfunction And Insulin Resistance In C2C12Myotubes By Targeting Mitofusin-2

Obesity and type2diabetes mellitus (T2DM) related to obesity are major health issues that have reached epidemic status worldwide. Resistance to the pleiotropic effects of insulin play a crucial role in pathogenesis of T2DM, and represents a key process in the development of it. Muscle tissue is the primary site of glucose uptake postprandially, with skeletal muscle accounting for approx.75%of insulin-dependant glucose removal from the plasma; The main feature of insulin-resistant states, including T2DM, is a reduction of nonoxidative glucose storage as glycogen in skeletal muscle during hyperinsulinemia. Therefor, skeletal muscle insulin resistance plays a major role in the development the whole body systematic insulin resistance. However, the underlying molecular mechanism(s) of skeletal muscle insulin resistance are only partially understood. The discovery of post-transcriptional gene silencing as an additional regulatory principle to control protein expression raises the possibility that MicroRNAs (miRNAs) are also involved in the development of obesity-induced insulin resistance and T2DM. MiRNAs are small22-25-nt-long non-coding RNA molecules that negatively regulate translation of target mRNAs. miRNAs normally bind to the3’untranslated region (UTR),5’UTR, coding sequence (CDS) of their target mRNA through imperfect base pairing, leading to translation inhibition and/or mRNA degradation. The present review examines the various miRNAs that have been identified as being potentially involved in T2DM, focusing on the insulin-sensitive organs:white adipose tissue, liver, skeletal muscle and the insulin-producing pancreatic β-cell. Interestingly, whole-genome association studies for T2DM susceptibility genes revealed that most of the associated variants were located in non-coding regions, further supporting the possibility that regulatory, non-coding RNAs such asmiRNAs may also contribute to the development of insulin resistance and T2DM.MiR-106b was reported to be upregulated in the skeletal muscle of db/db mice by our study group. miR-106b belonged to miR-106b-25cluster, was located in the thirteenth introns of Mcm7. It was reported that miR-106b is highly expressed in the skeletal muscle of diabetes patients and of mice with obesity and insulin resistance induced by a12-week high-fat diet. All of these indicated that miR-106b upreguation was highly correlated to skeletal muscle insulin resistance. Many compelling evidences indicated that mitochondrial dysfunction contributed to the pathogenesis of insulin resistance induced by obesity. However, the roles of miR-106b in regulating skeletal muscle insulin sensitivity and modulating mitochondrial function are still largely unknown.In order to clarify the role of miR-106b on skeletal muscle insulin resistance, we further showed that the3’UTR of Mfn2harbors two potential targeting site of miR-106b, one of which is highly conserved evolutionarily from Frog to Humans by bioinformatic analysis. Mfn2protein is a dynamin-related protein with GTPase activity anchored in the external mitochondrial membrane. In addition to mediating mitochondrial fusion, Mfn2localizes on the endoplasmic reticulum (ER) membrane, serving as a bridge to tether the ER to mitochondria. ERR-α transcription factor is a key regulator of Mfn2transcription and recruits PGC-1β and PGC-1α, which are potent, positive regulators of Mfn2expression in muscle cells. In coordination with PGCl-α, Mfn2maintain mitochondrial membrane potential and promoted oxidation phosphorylation. Document retrieval demonstrated that Mfn2was indispensable to regulate mitochondrial function and insulin sensitivity, and PGC-la/Mfn2signaling participated in regulation of skeletal muscle insulin sensitivity.In the first part, the effect of miR-106b on C2C12myoblast differentiation was observed, and the expression level of miR-106b was detected in the treatment of interleukin-6(IL-6), tumor necrosis factor-a (TNF-a) and palmitic acid (PA). In the second part, the role of miR-106b on the C2C12myotubes insulin sensitivity was studied by miR-106b gain of function and lose of function under condition of TNF-αtreatment, and the mechanism was explored by mitochondrial morphology and function, and ERR-α/PGC-lα/Mm2expression. Mfn2targeted by miR-106b was explored by luciferase activity assay combined with mutational analysis and immunoblotting.Part1. The biological function of miR-106b on C2C12myoblastObjective:①The expression level of miR-106b was detected in the period of C2C12differentiation.②To confirm the establishment of cell lines in which miR-106b gain of function or loss of function.③The affection of miR-106b gain of function and loss of.function on C2C12myoblasts differentiation was explored.④The expression level of miR-106b was detected and in the treatment of TNF-α、IL-6、 PA.Methods:①The expression level of miR-106b was detected by real-time PCR in the period of C2C12differentiation, and in the treatment of TNF-α、IL-6、PA for24h,48h.②The stable cell lines overexpression and silence of miR-106b were obtained by lentivirus transfection, and were confirmed by real-time PCR.③During C2C12differentiation by2%horse, real-time PCR was used to detect the level of MyoD and myogene mRNA, and the morphology of C2C12myotubes was observed by giemsa staining. Result:①The level of miR-106b was not affected during C2C12myoblasts differentiation.②The stable cell lines miR-106b gain of function and loss of function was obtained.③MyoD expression was gradually increased during C2C12myoblasts differentiation, attached to the maximum at the sixth day after differentiation. Myogene was increased at the third day, and then was reduced at the sixth day after differentiation. Overexpression and silence of miR-106b did not chang the expressional pattern of myogenic regulatory factors (MyoD and myogenin mRNA). C2C12myoblasts appeared star shape, usiformate, and mononuclear. After differentiation, elongated and multinucleated myotubes formed by cells fusing with each other were observed by Giemsa staining. Overexpression and silence of miR-106b did not affect myotubes morphology compared with the control.④TNF-a could rapidly induce miR-106b expression in a time-and dose-dependent manner, and this effect was more prominent at the concentration of20ng/ml after48h. PA could upregulate the expression of miR-106b in a dose-dependent manner, and this effect was not affected by the treatment time. IL-6treatment did not change the level of miR-106b.Conclusions:The expression of miR-106b was not changed significantly during C2C12myoblasts differentiation, which was not affected by miR-106b gain of function and loss of function. TNF-a and PA could rapidly induce miR-106b expression.Part2. The role of miR-106b gain of function and lose of function on the C2C12myotubes insulin sensitivity and mitochondrial mechanism analysisObjective:(1) The role of miR-106b on the C2C12myotubes insulin sensitivity was studied by miR-106b gain of function and the mechanisms were explored by mitochondrial morphology and function, and ERR-a/PGC-la/Mfn2expression.(2) MiR-106b was validated to target the3’untranslated region (3’UTR) of mitofusin-2(Mfn2) mRNA through miR-106b binding sites and to downregulate Mfn2protein abundance at the post-transcriptional level.(3) The role of miR-106b on the C2C12myotubes insulin sensitivity was studied by miR-106b lose of function under condition of TNF-a treatment, and the mechanisms were explored by mitochondrial morphology and function, and ERR-a/PGC-la/Mfn2expression.Methods:(1) miR-106b gain of function by lentivirus:Insulin sentivity was determined by insulin-stimulated2-Deoxy-D-[3H] glucose uptake in C2C12myotubes. Glucose transporter-4(GLUT4) transportation to the plasma membrane and phosphorylation of insulin recepter substrate-1(IRS-1) at Ser1101were examined by Western Blot. Mitochondria ultramicrostructure was displayed by electromicrograph morphometry, and the mitochondrial DNA copynumber was detected by real-time PCR. The ATP content of the C2C12myotubes was measured with ATP lite-glo, a luciferase-based luminescence assay kit. Mitochondrial membrane potential (△Ψ) and intracellular ROS was detemined by fluorescent molecular probes and FACS. PGC-la/ERR-a mRNA and protein level were detected by real-time PCR and Western Blot.(2) Mfn2targeted by miR-106b was explored by Luciferase activity assay combined with mutational analysis and immunoblotting.(3) MiR-106b silence by lentivirus:Differentiated C2C12myotubes were treated with TNF-α at10ng/ml or20ng/ml for24h or48h. Insulin sentivity was determined by insulin-stimulated2-Deoxy-D-[3H] glucose uptake in C2C12myotubes. GLUT4level in the plasma membrane were examined by Western Blot. Mitochondria ultramicrostructure was displayed by electromicrograph morphometry, and the mitochondrial DNA copynumber was detected by real-time PCR. The ATP content of the C2C12myotubes was measured with ATP lite-glo, a luciferase-based luminescence assay kit. Mfn2/PGC-la/ERR-a mRNA and protein level were detected by real-time PCR and Western Blot.Results:(1) miR-106b overexpression:①MiR-106b overexpression significantly decreased insulin-stimulated glucose uptake and the translocation of GLUT4to the cell surface, and enhanced phosphorylation of IRS-1at Ser1101.②The transmission electron microscopy data revealed a large number of double and multimembrane autophagosome-like vacuolar structures in myotubes overexpression of miR-106b. The miR-106b overexpression resulted in obvious destruction of mitochondrial ultrastructure, the mitochondria swelling, hollow with reduced density and absent cristae. Mitochondria typically were larger in diameter and appeared round upon upregulation of miR-106b, compared with the elongated, tubular mitochondria detected in control conditions.③Intracellular△Ψ were greatly decreased and ATP production were slightly decreased in C2C12myotubes overexpressing miR-106b. Indeed, overexpression of miR-106b enhanced ROS production.④miR-106b overexpression upregulated PGC-1α and ERR-α both at the mRNA and protein level, which was paralleled by the downregulation of Mfn2protein.(2)①Overexpression of miR-106b decreased the reporter gene activity by37%compared with normal controls (NC). Suppression of reporter gene activity was most efficiently reversed by mutation of target site2, and mutation of both binding sites resulted in a significant20%increase in luciferase activity.②Overexpression of miR-106b markedly downregulated Mfn2protein levels, while Mfn2mRNA levels were increased slightly but not significantly. Inhibition of miR-106b significant upregulated Mfn2expression both at the mRNA and protein level.(3) miR-106b loss of function:①TNF-α treatment for48h at the concentration of20ng/ml significantly reduced C2C12myotubes2-deoxyglucose uptake. Silencing of miR-106b significantly increased GLUT4transportation without TNF-a treatment. TNF-a treatment at10ng/ml significantly decreased GLUT4transportation to the plasma membrane in control group but not in miR-106b silence group.②It was found that TNF-α decreased levels of Mfn2mRNA and protein in C2C12myotubes, which were significantly attenuated by the loss of miR-106b function.③TNF-α resulted in obvious destruction of the mitochondrial ultrastructure, along with alteration of mitochondrial vacuolar structures, the mitochondria were condensed (smaller and higher density) compared with those without TNF-α treatment. MiR-106b loss of function partly improved mitochondrial morphological lesions impaired by TNF-α.④TNF-α reduced production of intracellular ATP, with no significant change in mtDNA levels. Meanwhile, miR-106b loss of function increased ATP production impaired by TNF-α treatment and enhanced mtDNA levels without TNF-α treatment.⑤TNF-α at20ng/ml led to decrease PGC-1α mRNA expression, while it did not change PGC-la protein expression. TNF-α at20ng/ml downregulated ERR-α protein expression but upregulated ERR-α mRNA expression. MiR-106b silencing with the sponge inhibitor upregulated PGC-1α both at the mRNA and protein level under TNF-α treatment, and upregulated ERR-α mRNA and protein without TNF-α treatment.Conclusions:miR-106b overexpression probalbly contributed to mitochondrial dysfunction and insulin resistance in C2C12myotubes by targeting Mfn2. MiR-106b silence possibly improved C2C12myotubes mitochondrial function and insulin sensitivity impaired by TNF-α treatment though upregulating Mfn2.