Mechanism Of Long Noncoding RNA MALAT1 Regulating Biological Behavior Of Osteosarcoma Via MiR-124-3p/SphK1 Axis

Objectives This study aimed to explore the expression of long noncoding RNA(lncRNA)-MALAT1 in osteosarcoma(OS)and its effect on the biological behavior of osteosarcoma cells,such as proliferation and metastasis,and to analyze the mechanism of micro RNA-124-3p(miR-124-3p),Sphingosine Kinase 1(SphK1)and other molecules,so as to find new molecular markers of osteosarcoma as well as provide a theoretical basis for targeted therapy.Methods(1)The gene expression of MALAT1 in osteosarcoma was analyzed by bioinformatics,and the survival of MALAT1 was analyzed.The expression of LncRNA-MALAT1 in osteosarcoma tissues and surrounding non-tumor tissues were compared by RT-qPCR.(2)Two osteosarcoma cell lines with high expression of lncRNA-MALAT1 were screened and divided into OS group(osteosarcoma cells),Control group(osteoblasts),NC group(osteosarcoma cells transfected with nonsense sequence)and siRNA group(osteosarcoma cells transfected with si-MALAT1).The proliferation ability of cells was detected by cell counting kit-8(CCK-8),the migration ability was measured by cell scratch test,and the apoptosis and cycle of cells were identified by flow cytometry.The expressions of lncRNA-MALAT1,miR-124-3p,SphK1,caspase 3 mRNA and mammalian rapamycin target protein(mTOR)mRNA were detected by RT-qPCR.The expressions of SphK1,caspase 3,mTOR and p-mTOR proteins were measured by western blot.(3)Double luciferase assay was used to verify whether miR-124-3p targeted MALAT1 and SphK1 respectively.Results(1)(1)Bioinformatics analysis showed that a total of 536 genes were differentially expressed in osteosarcoma.The expression of 299 genes was up-regulated,237 genes were down regulated,and the expression of MALAT1 was significantly increased(P<0.001).The survival analysis of MALAT1 showed that the high expression of MALAT1 was closely related to the poor survival prognosis of patients(P < 0.05).(2)The results of RT-qPCR confirmed that the expression of lncRNA-MALAT1 in research group was obvious higher than that in peripheral non-tumor tissues(P < 0.01).(2)(1)The expression of MALAT1 increased most significantly in MG63 and U2OS osteosarcoma cell lines,while siRNA6108 had the most obvious inhibitory effect on MALAT1 expression,therefore they were selected for follow-up experiments.(2)The results of CCK-8 experiment showed that silencing MALAT1 significantly reduced the proliferation rate of MG63 and U2 OS cells(P < 0.01);(3)Cell scratch test showed that silencing MALAT1 significantly decreased the migration distance(P < 0.01),that was,the migration ability of MG63 and U2 OS cells decreased;(4)After silencing MALAT1,the apoptosis rate of MG63 and U2 OS cells increased significantly(P < 0.001),and the percentage of G0 / G1 phase(relatively inactive quiescent phase)increased significantly(P < 0.001).(5)In MG63 and U2 OS cells,silencing MALAT1 significantly increased the expression of Caspase3 mRNA and Caspase3 protein(P < 0.001),while mTOR mRNA,p-mTOR protein and SphK1 protein decreased significantly(P < 0.001).(6)After silencing MALAT1,the expression of MALAT1 in MG63 and U2 OS cells decreased,while the expression of miR-124-3p increased,and the expression of SphK1 decreased.There was a negative correlation between MALAT1 and miR-124-3p,and between miR-124-3p and SphK1.(3)(1)Double luciferase reporter experiment was used to explore the targeted binding relationship between miR-124-3p and lncRNA-MALAT1,and the result showed that only the co-transfection of MALAT1 wild-type double luciferase reporter vector + miR-124-3p mimic group significantly reduced the luciferase activity(P<0.001).Therefore,there was a targeted binding relationship between MALAT1 and miR-124-3p.(2)the targeted binding relationship between miR-124-3p and SphK1 was explored by double luciferase reporter experiment.The results showed that only the co-transfection of SphK1 wild-type double luciferase reporter vector + miR-124-3p mimic group significantly reduced the luciferase activity(P<0.001),Therefore,there is a targeted binding relationship between mir-124-3p and SphK1.Conclusions(1)LncRNA-MALAT1 is highly expressed in osteosarcoma,which is related to the poor survival of osteosarcoma.(2)LncRNA-MALAT1 promotes the proliferation and migration of osteosarcoma MG63 and U2 OS cells,and reduces the percentage of G0 / G1 phase and inhibits apoptosis.Meanwhile,MALAT1 inhibits the expression of Caspase3 but up-regulates the expression of mTOR.(3)LncRNA-MALAT1 promotes the expression of SphK1 by inhibiting the expression of miR-124-3p,so as to promote the malignant process of osteosarcoma.