Modulatory Effect Of TENT5A On Myogenin In The Formation Of Muscle Fibers For Adolescent Idiopathic Scoliosis

Background:As the most common spinal deformity in adolescents,the pathogenesis of adolescent idiopathic scoliosis(AIS)is multi-factors,including hereditary factor,endocrine disorder,and neuromuscular system imbalance.There is still no consensus regarding its etiology,and it is of great significance to explore the pathogenesis for the diagnosis,treatment,and prevention of AIS.Objective:The main purposes of this research are:(1)To clarify the asymmetry of AIS paravertebral muscle,and to explore the molecular differences of the paravertebral muscle;(2)To investigate the function of TENT5A on myoblasts,as well as the formation of muscle fibers in vivo and in vitro.(3)To explore the regulating mechanism of TENT5A on muscle fiber formation.Materials and Methods:(1)In this study,the imaging parameters,including paravertebral muscle,the height of vertebral body and disc,and the transverse diameter of vertebral pedicle were measured in 30 AIS patients.Then the fiber types were compared between the concave and convex sides to explore the microscopic pathological differences of paravertebral muscle.Further,the differentially expressed genes and possible molecular pathways of paravertebral muscles on concave and convex sides were investigated based on RNA-seq.The differential expression of TENT5A was identified with q PCR and Western Blot.(2)In this study,LV-U6-shRNA(TENT5A)-CMV-EGFP-SV40 poly A lentivirus was constructed to knock down the expression of TENT5A,and the knockdown result was verified with q PCR and Western Blot.The effects of TENT5A on proliferation,migration,apoptosis,and myoblast differentiation of C2C12 cells were explored.In addition,the satellite cells were isolated and cultured.After the expression of TENT5A in myoblast cells was knocked down by lentivirus transfection.The myoblast cells were transplanted into the tibialis anterior muscle of mice after acute muscle injury to investigate the effect of TENT5A on myoblast differentiation in vivo,(3)The expression of TENT5A gene and myogenetic regulatory factors including myogenin,Myo D,Myf5 and Myf6 during myoblast differentiation were investigated.Furthermore,the expression of TENT5A in C2C12 cells was knocked down by lentivirus,and the influence on myogenic regulatory factors during myoblast differentiation were analyzed.Results:(1)The cross-sectional area(4.32 cm~2 vs.5.17cm~2;P=0.009)and fat infiltration of paravertebral muscle(14.9%vs.10.7%;P<0.0001),the vertebral height(1.71 cm vs.2.15 cm;P<0.0001),the transverse diameter of the pedicle(0.48cm vs.0.54 cm;P=0.0109),the disc height(0.27 cm vs.0.54 cm;P<0.0001)were asymmetry in AIS patients.Compared to the convex side,significantly higher proportion of muscle fibers type I was found in the concave side(69.2%vs.55.8%;P<0.0001),and the proportion of muscle fibers type IIA was lower than that of the convex side(16.6%vs.29.6%;P=0.0014),these results support paravertebral muscle asymmetry in AIS.Furthermore,RNA sequencing results of paravertebral muscles showed that the expression of 10genes were up-regulated and 48 genes were down-regulated in the concavity side,and the results of gene enrichment analysis showed that the differential genes were mainly concentrated in Insulin signaling pathway,PPAR signaling pathway and c AMP signaling pathway.23 susceptibility genes for AIS were selected from recent literatures,the TENT5A was asymmetric expression,which was verified with q PCR(1.91 vs.1.13;P=0.0111)and Western Blot(0.89 vs.0.70;P=0.0288).(2)After the lentiviral transfection of C2C12 cells,the knockdown effect of TENT5A was verified by q PCR and Western Blot(P<0.05).C2C12 cell proliferation(95.2%vs.103.6%;P=0.012)and mobility(73.3%vs.89.5%;P=0.008)were significantly decreased,while the apoptotic function did not change significantly(26.2%vs.30.4%;P=0.064).The results of myoblast differentiation showed that TENT5A knockdown significantly decreased the percentage of MYHC(+)nuclear(64.5%vs.29.7%;P<0.001),the percentage of My H7(+)nuclear(28.2%vs.20.7%;P=0.018),and the percentage of myogenin(+)nucleus(18.7%vs.29.3%;P=0.0122).Moreover,it mainly inhibited the expression of genes related to muscle fiber type I,including MYH7(P=0.024),TNNI1(P=0.011)and TNNC1(P=0.047).The results of in vivo transplantation showed that the TENT5A knockdown myoblasts was decreased to differentiate into muscle fiber type I(18.2%vs.29.4%;P=0.0238).In addition,the relative expression of MYH7(2.32 vs.0.69;P=0.042)and TNNC1(1.35 vs.0.75;P=0.043)was significantly decreased,while TNNC2(0.88 vs.1.12;P=0.045)was significantly increased.(3)During the induction of myoblast differentiation,the expression of myogenin was significantly up regulated on the first day of differentiation,and persistent overexpression of myogenin were found from the second to the fifth day.The expression of Myo D was downregulated on the first day of differentiation and continued to be low-level.My F5 and My F6 were slightly up regulated on the first day of differentiation,and slightly decreased on the second to fifth day.The expression of TENT5A was up-regulated gradually on the first to third day of differentiation and down-regulated gradually on the fourth to fifth day.The relative expression of myogenin in TENT5A knockdown C2C12 cells was significantly downregulated(0.82vs.1.21;P=0.010)on day 3 of differentiation.Conclusion:(1)There were significant differences in cross-sectional area,fat infiltration of paravertebral muscle,the proportion of type I,and type IIA muscle fibers between the concave-convex sides in the apical region of AIS,which supported the asymmetry of paravertebral muscle in AIS.Fifty-eight genes were differentially expressed in AIS paraspinal muscle,and the differentially expressed genes were mainly concentrated in Insulin signaling pathway,PPAR signaling pathway and c AMP signaling pathway.Correlation analysis with 23 AIS susceptibility genes,we found and verified the differential expression of TENT5A gene in the paravertebral muscle.(2)TENT5A gene plays an important role in maintaining the proliferation and migration ability of C2C12 cells,but it has no significant effect on the apoptotic function.TENT5A gene plays an important role in maintaining the myoblast differentiation ability of C2C12 cells,and mainly affects the expression of type I muscle fiber related genes,including MYH7,TNNI1 and TNNC1.(3)TENT5A gene affects the formation of muscle fiber in vivo,and mainly inhibits the maturation of type I muscle fiber,and the expression of MYH7 and TNNC1.TENT5A gene affects myoblast differentiation by regulating the expression of myogenin,a core member of the myogenic regulator family.AIS susceptibility gene TENT5A may be involved in the pathogenesis of AIS by regulating myogenin to affect muscle fiber formation.In conclusion,the paravertebral muscles on both sides of AIS shows asymmetry at the imaging level,histological level,and molecular level.The AIS susceptibility gene TENT5A is differentially expressed in the paravertebral muscle.TENT5A plays an important role in the proliferation,migration,and differentiation of myoblasts.In addition,it mainly affects the maturation of type I muscle fibers.TENT5A may affect myoblast differentiation by regulating myogenin.TENT5A,the susceptibility gene AIS,may be involved in the pathogenesis of AIS by regulating myogenin to affect muscle fiber formation.