Targeted Inhibition Of Id1 Mediated Autophagy Through The AKT/mTOR Signaling Pathway Influences The Malignant Biological Behavior And Osteogenic Differentiation Of Osteosarcoma MG63 Cells

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EFFECTS OF TARGETING REGULATION OF AKT/MTOR SIGNALING PATHWAY ON PROLIFERATION,APOPTOSIS,AUTOPHAGY AND OSTEOGENIC DIFFERENTIATION OF OSTEOSARCOMA CELL LINE MG63Objective:To investigate the effects of targeted regulation of AKT/mTOR signaling pathway on the proliferation,apoptosis,autophagy and osteogenic differentiation of human osteosarcoma cell line(MG63),and to explore its mechanism.Methods:(1)RT-PCR was used to detect the gene expression of AKT/mTOR in osteosarcoma cells of different malignant degrees.(2)Taking osteosarcoma cell line MG63 as the research object,MG63 cells were treated with mTOR inhibitor(rapamycin)and activator 3-benzyl-5-(2-nitrophenoxymethyl-3-butylactone).Western blot was used to verify the regulation of the two drugs on the AKT/mTOR signaling pathway.(3)Western blot method was used to detect autophagy related protein expression after targeted regulation of AKT/mTOR signaling pathway.(4)CCK-8 assay was used to detect the activity of osteosarcoma MG63 cells after targeting the AKT/mTOR signaling pathway.(5)DAPI staining and Annexin V-FITC /PI staining were used to detect apoptosis after targeted regulation of AKT/mTOR signaling pathway.(6)Alkaline phosphatase(ALP)staining was used to detect early osteogenic differentiation,and alizarin red staining was used to detect late osteogenic differentiation.(7)Expression of differentiation inhibitor(Id1)was detected by Western blot.Results:(1)The RT-PCR results showed that the expression levels of AKT and mTOR genes were 143 b,MG63 and TE85 from high to low.(2)Rapamycin could inhibit mTOR signaling pathway,and 3-BDO could activate mTOR signaling pathway.(3)Targeted inhibition of AKT/mTOR signaling pathway could up-regulate the expression of autophagy related proteins LC3II/I and Beclin1(P<0.05);targeting activation of AKT/mTOR signaling pathway could down-regulate the expressions of autophagy related proteins LC3II/I and Beclin1(P<0.05).(4)Targeted inhibition of the AKT/mTOR signaling pathway could inhibit the proliferation of MG63 cells in osteosarcoma(P<0.05);targeted activation of the AKT/mTOR signaling pathway had no significant effect on the proliferation of osteosarcoma MG63 cells.(5)Targeted inhibition of the AKT/mTOR signaling pathway could promote the apoptosis of osteosarcoma MG63 cells(P<0.05);targeted activation of the AKT/mTOR signaling pathway had no significant effect on the apoptosis of osteosarcoma MG63 cells.(6)Targeted inhibition of AKT/mTOR signaling pathway could inhibit early and late osteogenic differentiation of osteosarcoma cells.Targeted activation of AKT/mTOR signaling pathway could promote early and late osteogenic differentiation.(7)Targeted inhibition of AKT/mTOR signaling pathway,the expression level of differentiation inhibitor(Id1)was up-regulated(P<0.05);after targeted activation of AKT/mTOR signaling pathway,the expression level of differentiation inhibitor(Id1)was down-regulated(P<0.05).Conclusion:(1)The expression level of AKT/mTOR signaling pathway is positively correlated with the malignant biology of osteosarcoma cells;(2)In osteosarcoma cell line MG63,targeted inhibition of AKT/mTOR signaling pathway could up-regulate the level of autophagy,inhibit the proliferation of osteosarcoma cells,and promote apoptosis.Targeting activation of AKT/mTOR signaling pathway could down-regulate autophagy level and have no obvious effect on cell proliferation and apoptosis.(3)Targeted inhibition of AKT/mTOR signaling pathway inhibits osteoblastic differentiation,which might be related to up-regulation of differentiation inhibitor(Id1),further clarifying the pathogenesis of osteosarcoma and providing a theoretical basis for the treatment of induced differentiation.PART ? EFFECTS OF TARGETED INHIBITION OF ID1 COMBINED WITH MTOR INHIBITOR MEDIATED AUTOPHAGY ON MALIGNANT BIOLOGICAL BEHAVIOR AND OSTEOGENIC DIFFERENTIATION OF OSTEOSARCOMA MG63 CELLSObjective: After targeted inhibition of AKT/mTOR signaling pathway,the effects of Id1-mediated autophagy on malignant biological behavior and osteogenic differentiation of human osteosarcoma MG63 cells were investigated.Methods:(1)The recombinant adenovirus AdsiId1 expressing siId1 was amplified.The fluorescence expression of cells was observed by fluorescence microscope,and the m RNA and protein expressions of Id1 were verified by RT-PCR and Western blot.(2)The MG63 cells were divided into control group,mTOR inhibitor group(Rapamycin group),siId1 group(AdsiId1 infection group),and siId1+Rapamycin group(AdsiId1 +Rapamycin group).Western blot was used to detect the expression of autophagy key proteins.(3)m GFP-RFP-LC3 double fluorescence plasmid transfection was used to detect the level of autophagy in each group.(4)The number of autophagosomes in each group was determined by transmission electron microscopy.(5)CCK-8 assay was used to detect the activity of MG63 cells.(6)Transwell assay was used to detect the migration and invasion of MG63 cells.(7)Flow cytometry was used to detect cell cycle and apoptosis of MG63 cells.(8)ALP staining was used to detect the early osteogenic differentiation of cells,and alizarin red staining was used to detect the late osteogenic differentiation of cells.(9)Western blot assay was used to detect the expression of AKT/mTOR signaling pathway.Results:(1)AdsiId1 could successfully infect MG63 cells,and the m RNA and protein expression of Id1 in MG63 cells are down-regulated after adenovirus siId1 infection(P<0.05).(2)Compared with the control group,Beclin1 and LC3II/I expressions of autophagy related proteins in the siId1 group were increased,and the protein expression of P62 was down-regulated(P<0.05).After the addition of the mTOR inhibitor rapamycin,Beclin1 and LC3II/I protein expression levels were higher than those in the siId1 group,and the protein expression level of P62 was lower than that in the siId1 group(P<0.05).(3)After autophagy double-labeled plasmid was transfected,the confocal results showed that the number of autophagy flow increased in the siId1 group compared with the control group(P<0.05),and the autophagy flow increased further after the addition of the mTOR inhibitor rapamycin(P<0.001).(4)The results of transmission electron microscopy showed that the number of autophagosomes in the siId1 group was higher than that in the control group,and the number of autophagosomes was further increased after the addition of the mTOR inhibitor rapamycin.(5)Compared with the control group,siId1 group could inhibit cell proliferation(P<0.05),and the addition of mTOR inhibitor rapamycin could further inhibit cell proliferation.(6)Compared with the control group,siId1 group could inhibit cell migration and invasion(P<0.05),and mTOR inhibitor rapamycin could further inhibit cell migration and invasion(P<0.001).(7)Compared with the control group,siId1 group could block cell cycle in G0/G1 phase(P<0.05),and the proportion of cells in G0/G1 phase increased significantly after the addition of mTOR inhibitor rapamycin(P<0.001).siId1 group could promote apoptosis(P<0.05),and the apoptosis rate was further increased after the addition of mTOR inhibitor rapamycin(P<0.001).(8)Compared with the control group,the siId1 group could promote the early osteogenic differentiation of osteosarcoma cells.siId1 group had no significant effect on late osteoblastic differentiation of osteosarcoma cells,and the ability of late osteoblastic differentiation was significantly enhanced after the addition of mTOR inhibitor rapamycin.(9)Compared with the control group,the expression levels of p-AKT and p-mTOR in the siId1 group were down-regulated(P<0.05),and the expression levels of p-AKT?p-mTOR were further down-regulated after the addition rapamycin,an mTOR inhibitor(P<0.001).Conclusion:(1)siId1 combined with mTOR inhibitor could significantly inhibit MG63 malignant biological behavior of osteosarcoma cells and promote osteoblastic differentiation of osteosarcoma cells;(2)Osteosarcoma is a differentiated disease.This study showed that Id1 promoted the osteoblast differentiation of osteosarcoma cell through AKT/mTOR signaling pathway mediated autophagy,providing a basis for revealing the pathogenesis of osteosarcoma.Meanwhile,siId1 combined with mTOR inhibitor may be a new therapeutic strategy for osteosarcoma.