Molecular Mechanism Of TSC2 Gene In Skeletal Muscle Cell Proliferation In Goat

TSC2(Tuberous sclerosis complex2;also known as tuberin),as a tumor suppressor,is upstream of mTOR signaling pathway and has GTPase-activating protein(GAP)activity,which regulates intracellular DNA transcription and protein synthesis.Studies have shown that TSC2 can serve as a hub in a variety of signals in cells,and plays an important role in regulating physiological processes,such as cell proliferation,differentiation,apoptosis and embryonic development.However,the regulation mechanism of TSC2 on skeletal muscle growth and development is still unclear.The investigation of the molecular mechanism of TSC2 on regulating the proliferation of goat myoblast cells has important theoretical significance and research value to regulate the growth and development of animal skeletal muscle.The objective is to investigate the function of TSC2 gene in skeletal muscle myoblast of dairy goat and its regulatory mechanism.Isolated and purified skeletal muscle myoblasts of dairy goats was used in present study.The coding region of TSC2 gene of dairy goat was cloned.Overexpression and RNAi techniques were used to study the function of TSC2 on myoblast proliferation.RT-qPCR and Western Blot were performed to detect the effects of overexpression and knockdown of TSC2 on myoblast proliferation and the expression of genes involved in mTOR signaling.Following are the main results obtained in this study:1.Isolation and identification of myoblasts of dairy goatsSemitendinosus muscle of dairy goat kids was used to isolate myoblast,following digestion procedure using collagenase I and trypsin and purification procedure using differential adhesion method.RT-PCR and immunofluorescence staining were conducted to further identify purified cells.Cell morphological results showed that purified cells were in spindle shape and maintained a stable status of growth.Myogenic regulatory factors,MyoD and Myf5,and desmin were positively expressed in purified cells.Purified myoblasts were able to fuse into myotubes after induced differentiation.Myosin heavy chain(MYHC),a specific marker of differentiated myotubes,was positively expressed.It is suggested that the isolated myoblast can be used in next step.2.TSC2 gene cloning and bioinformatics analysisThe goat TSC2 gene CDS sequence was cloned with the designed primers based on the TSC2 gene sequence of goat(MH643731)collected in GenBank using RT-PCR following total RNA extraction from dairy goat myoblasts.The length was 5 250 bp,1 749 amino acids with a molecular weight of 193.69 KDa,mainly stored in the cytoplasm.The genetic relationship analysis of TSC2 gene CDS region between dairy goats and other species revealed that dairy goats has the most similar genetic relationship with Ovis aries.3.Overexpression of TSC2 on myoblast proliferationWe successfully constructed the pcDNA3.1(+)vector containing TSC2 gene,which was transformed into myoblast.The mRNA expression of TSC2 gene increased by 18-fold,which significantly changed the mRNA and protein expression of Cyclin E,p27 and p57,and increased the number of cells in G1 phase,reduced the number of cells in G1 phase.It is suggested that overexpression of TSC2 inhibits myoblast proliferation in dairy goat.Overexpression of TSC2 gene can significantly down-regulate the mRNA and protein expression of mTOR and p70S6 K.After treatment with mTOR agonist(MYH1485),it can attenuate the inhibitory effect of TSC2 overexpression on myoblast proliferation.4.Interference with TSC2 on myoblast proliferationBased on CDS sequence of TSC2 gene,specifically designed siRNA transformed into myoblast of dairy goat leaded to the significantly decreased mRNA expression of TSC2 gene by about 70%,and the significantly changed mRNA and protein expression of proliferation-related genes Cyclin E,p27 and p57 and the number of cells in the S phase.It is suggested that interference of TSC2 promote myoblast proliferation in dairy goats.The interference of TSC2 significantly upregulated mRNA and protein expression of mTOR and p70S6 K,which was weakened by rapamycin treatment.