The Effect Of NRSN2 On Proliferation And Differentiation Of Osteosarcoma Cells And Its Molecular Mechanism

Objective:(1)To investigate the relationship between NRSN2 and osteosarcoma by detecting the expression of NRSN2 in osteosarcoma tissue and osteosarcoma cells.(2)To determine the effect of NRSN2 on the proliferation and differentiation of osteosarcoma cells by in vitro and vivo experiments by silencing/overexpressing NRSN2.(3)To determine whether NRSN2 regulates PI3K/AKT/mTOR and Wnt/?-catenin signaling pathways,to study the molecular mechanism of NRSN2 in the proliferation and differentiation of osteosarcoma cells.Methods:(1)To investigate the expression differences of NRSN2 in tumor tissue and matched adjacent tissue,the expression of NRSN2 mRNA and protein level was detected by real-time quantitative PCR and IHC,of 18 cases of osteosarcoma.(2)To study the expression differences of NRSN2 in osteosarcoma cell lines,the expression of NRSN2 m RNA and protein level in U20 S,Saos2 and MG63 were detected by real-time quantitative PCR and Western blot.(3)Construction of silencing NRSN2 U20 S,Saos2 by RNA interference technology(NRSN2 relatively high expression in U20 S,Saos2),to verify the silence effect of NRSN2 by Western blot.In vitro and vivo,to investigate the effect of silenced NRSN2 on the proliferation and differentiation of U2 OS,Saos2 were screened by CCK8,colony formation assays,in vivo tumor formation assays.(4)Construction of overexpressing NRSN2 MG63 by RNA interference technology(NRSN2 relatively low expression in MG63),verify the overexpression of NRSN2 by Western blot.In vitro and vivo,to investigate the effect of overexpressing NRSN2 on the proliferation and differentiation of MG63 were screened by CCK8,colony formation assays,in vivo tumor formation assays.(5)To investigate the regulation of silencing NRSN2 on PI3K/AKT/m TOR and Wnt/?-catenin signaling pathways,the expression of Akt,p-Akt,m TOR,p-mTOR,GSK3?,p-Akt,m TOR,p-m TOR,GSK3?,p-GSK3?,nu-?-catenin protein level were detected by Western blot,which are PI3K/AKT/mTOR and Wnt/?-catenin signaling pathway related proteins.The expression of CCND1 and c-myc mRNA were detected by real-time quantitative PCR respectively,which are Wnt/?-catenin signaling pathway target protein.(6)To investigate the regulation of overexpressing NRSN2 on PI3K/AKT/mTOR and Wnt/?-catenin signaling pathways,the expression level of Akt,p-Akt,mTOR,p-mTOR,GSK3?,p-GSK3? and nu-?-catenin were detected by Wesrtern blot,the expression of Wnt/?-catenin signaling pathway target protein CCND1 and c-myc mRNA were detected by real-time quantitative PCR.The proliferation and differentiation of osteosarcoma cells were detected by CCK8 method after adding ?-catenin inhibitor IWR-1-endo.Results:(1)The mRNA and protein expression of NRSN2 in 18 cases of osteosarcoma tumor tissue were significantly higher than matched adjacent tissue(P<0.01),suggesting that NRSN2 was associated with osteosarcoma.(2)The levels of NRSN2 mRNA and protein in U20 S,Saos2 and MG63 osteosarcoma cells were higher than control group.NRSN2 was correlated with the proliferation and differentiation of osteosarcoma cells(P<0.01).U20 S and Saos2 were relatively higher than MG63(P<0.05).There was no significant difference between U20 S and Saos2(P>0.05).(3)The proliferation and differentiation of U2 OS and Saos2 were significantly inhibited by silencing NRSN2(P<0.01).The proliferation and differentiation of U2 OS and Saos2 were increased again by re-introduced NRSN2.The number of cloned cells in the NRSN2 group was less than control group(P<0.01).The weight of the tumor in vitro was significantly lighter than control group(P<0.01).(4)The proliferation and differentiation of MG63 were significantly increased by overexpressing NRSN2(P<0.01).The proliferation and differentiation of MG63 were declined again by re-silenced NRSN2.The number of cloned cells in the NRSN2 group was more than control group(P<0.01).The weight of the tumor in vitro in overexpression NRSN2 group was significantly heavier than control group(P<0.01).(5)The silencing NRSN2 of U2 OS protein expression levels of p-Akt,p-mTOR,p-GSK3?,nu-?-catenin and the mRNA expression levels of CCND1 and c-myc were lower than control group(P<0.01).(6)The overexpressing NRSN2 of MG63 protein expression levels of p-Akt,p-mTOR,p-GSK3?,nu-?-catenin and the mRNA expression levels of CCND1 and c-myc were higher than control group(P<0.01).The proliferation and differentiation of MG63 osteosarcoma cell lines were inhibited by the addition of ?-catenin signaling pathway inhibitor IWR-1-endo(P < 0.01).Conclusion:(1)There was correlation between NRSN2 and osteosarcoma by detecting the mRNA and protein expression of NRSN2 in osteosarcoma tissue and U20 S,Saos2 and MG63 osteosarcoma cell lines.(2)The effect of NRSN2 promotes the proliferation and differentiation of osteosarcoma cells demonstrated by in vitro and in vivo studies of silencing/overexpressing NRSN2.(3)The study of molecular mechanism NRSN2 suggested that NRSN2 regulates PI3k/AKT/m TOR and Wnt/?-catenin signaling pathway to promote osteosarcoma cell proliferation and differentiation.