| Objective: Our aim is:(1)to validate the promoting effects of Lnc RNA MALAT1 on osteosarcoma progression;(2)to elucidate the underlying regulation mechanism of MALAT1 through HIF-1a and EZH2/β-catenin pathway,and to investigate the possibility of inhibiting the progression of osteosarcoma by down-regulating MALAT1.Methods: In vitro:(1)We used RT-PCR to detect the MALAT1 expression level of MNNG/HOS cell line.(2)Through siMALAT1,mTOR inhibitor Rapamycin,mTOR activator MHY1485 and HIF-1a inhibitor BAY87-2243,as well as simulated hypoxic environment,we used RT-PCR,western-blot and ELISA to detect the expression level of MALAT1,p-mTOR,HIF-1a,VEGF-A and FGF2.Then we treated HMEC-1 cells with conditioned culture medium of osteosarcoma cells,and evaluated the ability of proliferation,migration and tubule formation of HMEC-1 cells through CCK8 assay,Transwell assay,and tubule formation assay.(3)By using siMALAT1 and GSK343 to inhibit MALAT1 and EZH2,we observed their inhibitory effects on osteosarcoma proliferation,migration and antiapoptosis activity through CCK8 assay,Transwell assay,and flow cytometry;Western blot was used to observe the effect of siMALAT1 and GSK343 on the expression and spatial distribution of E-Cadherin and β-catenin in osteosarcoma cells.In vivo: Heterotopic osteosarcoma model was established by BALB/c nude mice,divided to siMALAT1 group and control group.(1)The ability of siMALAT1 inhibiting angiogenesis was analysed,by messuring the volume of tumors,detecting the expression of Ki67,cleaved caspase-3,VEGFA and CD31 through immunofluorescence staining,and evaluating the expression of MALAT1 by PCR,and HIF-1a by western blot.(2)Through immunofluorescence staining to detect the expression of β-catenin,the ability of siMALAT1 to inhibit Wnt pathway was observed.Results: In vitro:(1)Lnc RNA MALAT1 was high expressed in osteosarcoma cells;(2)Inhibition of MALAT1 inhibited mTOR activation and expression of HIF-1a,VEGF-A and FGF2.Inhibition of mTOR or HIF-1a partially inhibited MALAT1 expression.Inhibition of Lnc RNA MALAT1/mTOR/HIF-1a loop of osteosarcoma cells inhibited the ability of proliferation,migration and tubule formation of HMEC-1 cells;(3)The similarity of inhibiting effects of siMALAT1 and GSK343 on osteosarcoma proliferation,migration and anti-apoptosis activity indicated the exist of Lnc RNA MALAT1/EZH2 axis.Inhibiting Lnc RNA MALAT1/EZH2 promoted E-Cadherin expression and inhibited expression and nucleus distribution of β-catenin.In vivo: chemically-modified siMALAT1 demonstrated inhibitory effect on angiogensis and Wnt pathway of osteosarcoma.Conclusion: Lnc RNA MALAT1 is closely related to osteosarcoma progression.Lnc RNA MALAT1/mTOR/HIF-1a loop and Lnc RNA MALAT1/EZH2/β-catenin pathway are major pathways of MALAT1 for regulating osteosarcoma progression.MALAT1 is a potential therapeutic target in osteosarcoma. |
