The Role And Mechanism Of MORC2 In Inducing C2C12 Muscle Cell Differentiation

Objective: MORC2 is a member of the Microrchidia family,CW type zinc finger protein(MORC)family.The MORC family is highly conservative in evolution,and has attracted widespread attention for its important functions in inhibiting transcription,epigenetic regulation,and DNA damage repair.MORC2 protein mainly includes four parts: ATPase domain,zinc finger CW domain,nuclear localization signal and coiled coil domain.It plays an important role in chromatin remodeling,DNA damage repair,adipogenesis,tumorigenesis and development.MORC2 mainly localize in the nucleus,but there is also a small amount in the cytoplasm.Studies have shown that the localization of MORC2 in the cytoplasm is a key player in lipid metabolism and homeostasis,and it can regulate the differentiation of adipocytes and adipogenesis in the cytoplasm.Studies have shown that changes in the nuclear and cytoplasmic distribution of proteins are often accompanied with structural and biological function changes.C2C12 cells are immortalized from C3 H mouse skeletal muscle satellite cells and belong to mouse skeletal muscle stem cells.This cell has the same biological behavior as skeletal muscle progenitor cells,and its cell characteristics and morphology are uniform,which can be infinitely subcultured.C2C12 cells have the ability to differentiate into muscle cells under 2% horse serum culture conditions,and can express a variety of marker proteins of mature skeletal muscle such as MyoD,myogenin,and myosin.Therefore,in the studys of muscle cell development and differentiation,it is often choosed as a classical experimental model.Myoblast regulatory factors(MRFs)play a vital role during the differentiation of myocytes.It mainly includes four members,MyoD,Myf5,myogenin,and MRF4.Among them,MyoD,myogenin,and myosin are often up-regulated in mature muscle cells,therefore they are often used as markers of muscle cell differentiation.They play a leading role in different stages of differentiation.MyoD and myosin,which are highly expressed at the early stage of differentiation,are often used as early differentiation markers,while myogenin,which is highly expressed in the middle and late stages of differentiation,is used as a metaphase differentiation marker.Inhibitor of differentiation(Id3)was first found in myoblasts.Like MyoD,a marker of muscle cell differentiation,they are members of the basic helix-loop-helix(bHLH)transcription factor family.Id family members lack the basic region necessary for binding to DNA in structure,so they can block the cell’s differentiation process and induce further cell proliferation.Generally,the higher degree of differentiation,the less the Id express.Previous studies have shown that overexpression of MyoD can promote the upregulation of Id3.Id3 can interfere with MyoD then affect its activity and inhibit differentiation.Studies have shown that Id3 expression is down-regulated during C2C12 differentiation.Our previous study indicated that over-expression of MORC2 can inhibit the differentiation of C2C12 cells,but the mechanism is unclear.In addition,we found that MORC2 can up-regulate the expression of Id3 from gene chip data,but it is not clear whether it can affect cell differentiation.Therefore,based on the previous work of our research group,this study aims at the effects of MORC2 on the induction and differentiation of C2C12 cells and its mechanism.Methods: Construct an over-expressing MORC2 lentiviral vector.After successful sequencing,the recombinant plasmid was co-transfected with the packaging plasmid into 293 T cells to obtain a lentiviral supernatant.C2C12 mouse myoblasts were cultured in vitro,C2C12 cells were infected with lentivirus,and cell lines stably expressing MORC2 protein were selected and identified.In order to study the effect of overexpressed MORC2 during the process of differentiation in C2C12 cells,we used low serum conditions(DMEM with 2% horse serums)to induce the differentiation.At the same time we detect the dynamic changes in MORC2 expression.To test and verify the effect on cell proliferation during the differentiation days,flow cytometry was used to detect the dynamic change of the proportion in G0/G1,S,G2/M;during the differentiation cells were collected on day 0,3,5 and the expression of intracellular differentiation markers MyoD and Id3 were analyzed by Real-time PCR and Western Blot.The localization of MORC2 in the cells was analyzed by fluorescence microscopy;changes in the expression of MORC2 and Id3 proteins in the nucleus and cytoplasm during differentiation were detected by a nuclear separation kit.Results:(1)The target gene fragment of MORC2 obtained by double digestion was ligated with the vector to obtain a recombinant plasmid.(2)The recombinant lentiviral vector was identified by double digestion and gene sequencing.(3)C2C12 cells were infected with the lentiviral vector carrying the MORC2 gene,and the C2C12 cells strain stably expression MORC2 was selected.(4)The results of Real-time PCR and western blot showed that MORC2 was highly expressed in C2C12 cells.(5)Statistical analysis of cell morphology showed that the degree of cell differentiation in the over-expressed MORC2 group was significantly lower than that in the negative control group.(6)The results of flow cytometry showed that the ratio of G0 / G1 phase of control group cells increased significantly and the ratio of S phase decreased with the time of induced differentiation,while the cell cycle of overexpressed MORC2 group did not change significantly.(7)MyoD,myogenin,and myosin were significantly up-regulated in the over-expressed MORC2 cells during the prolonged differentiation time(p <0.05).(8)Overexpression of MORC2 under the high serum conditions(DMEM with 10%FBS)can up-regulate the expression of differentiation inhibitor Id3 in C2C12 cells.Under conditions(DMEM with 2% horse serums),compared with the control group,overexpression of MORC2 can significantly promote the expression level of Id3.(9)The distribution of MORC2 fluorescence changed from nucleus to cytoplasm in cells on the third day of the differentiation process.(10)The results of Western Blot showed that the expression of MORC2 protein in the nucleus decreased during the differentiation process,and MORC2 in the cytoplasm increased.Conclusion:(1)A lentiviral vector carrying the MORC2 gene was successfully constructed.(2)Overexpression of MORC2 can inhibit cell differentiation and promote the maintenance of C2C12 cells’ proliferation status.(3)Under the low serum conditions(DMEM with 2% horse serums)the levels of the differentiation markers like MyoD,myogenin and myosin were up regulated,however at the same time we see overexpression of MORC2 can reduced their levels compared to the control group.(4)Overexpression MORC2 can up-regulate Id3 expression.(5)During the process of differentiation,the localization of MORC2 changed from nucleus to plasma.(6)During the differentiation process,the change of MORC2 from the nucleus to the plasma can up-regulate the expression of Id3 in the cytoplasm,and this process may play a role in inhibiting the cell differentiation.