The Study Of The Role And Molecular Mechanism Of LncRNA-ASBEL On Proliferation,Migration And Invasion In Osteosarcoma

Osteosarcoma(OS)is the most common malignant bone tumor in children and adolescents under 20 years old with high rate of incidence,high frequency of recurrence and high degree of metastasis.OS is the main cause of death in in children and adolescents.Because of poor prognosis and great burden bought to society,the study of OS becomes more and more imperative in bioscience area.Despite relevant studies are more,the mechanism of OS has not been explained well.Numerous researches in recent years have demonstrated that lnc RNAS are critical regulatory molecules which participates in the various cellular biological processes,including proliferation,differentiation,migration,invasion,embryogenesis,neurogenesis and tumorgenesis and so on.This study aimed to investigate the effects of long noncoding RNA antisense nc RNA in the Abundant in neuroepithelium area(ANA)/B-cell translocation gene 3(BTG3)locus(lnc RNA ASBEL)on the pathogenesis of osteosarcoma.Our studies will be helpful for further understand the tumor promoting effect of ASBEL and may provide a possible therapeutic target for osteosarcoma treatment,and have the important practical significance.[Objective](1)To illuminate expression profile of lnc RNA-ASBE in osteosarcoma and its effects on proliferation,migration,invasion of osteosarcoma cells.(2)To reveal the Molecular Mechanism of lnc RNA-ASBEL involving biological behavior of osteosarcoma cells by targeting mi R-21.[Method]The first part: expression profile of lnc RNA-ASBE in osteosarcoma and its effects on proliferation,migration,invasion of osteosarcoma cells.(1)Human osteoblast cell line h FOB1.19 and human osteosarcoma cell line MG63 were cultured.Extracting the total RNA and detecting the expression of lnc RNA-ASBEL by real-time RT-PCR.Results were statistically analyzed byone-way ANOVA analysis.(2)Osteosarcoma cell line MG63 was cultured and Cell transfection was performed using Lipofectamine 3000 reagent(Life Technologies Corporation,MD,USA)following with the manufacturer's protocol.Cells was divided into three groups:group Control(1ipofectamine only),group sh NC(lipofectamine+sh NC),group sh-ASBEL(lipofectamine+sh-ASBEL).Then after 1,2,3 and 4 days culture,quantitative real-time PCR(QPCR)was performed to examine expression of lnc RNA-ASBE in MG63 cells in three groups.Cell viability was detected using trypan blue exclusion assay.Cell migration was measured using cell transwell assay with a pore size of 8mm.The rate of cell invasion was measured similar with the rate of cell migration except that the transwell membrane was pre-incubated using Matrigel(BD Bioscience,NJ,USA).The second part:the molecular mechanism of lnc RNA-ASBE involving biological behavior of osteosarcoma cells by targeting mi R-21.(1)The effect of lnc RNA-ASBEL on expression of mi R-21 in osteosarcoma cell MG631)Osteosarcoma cell line MG63 was cultured and Cell transfection was performed using Lipofectamine 3000 reagent(Life Technologies Corporation,MD,USA).Cells was divided into three groups:group Control(1ipofectamine only),group sh NC(lipofectamine+sh NC),group sh-ASBEL(lipofectamine+sh-ASBEL).Then after 1,2,3 and 4 days culture,quantitative real-time PCR(QPCR)was performed to examine expression of mi R-21 in MG63 cells in three groups.2)Osteosarcoma cell line MG63 was cultured and Cell transfection was performed using Lipofectamine 3000 reagent(Life Technologies Corporation,MD,USA).Cells was divided into three groups: group NC(lipofectamine+NC),group mi R-21 mimic(lipofectamine+mi R-21 mimic),group mi R-21 inhibitor(lipofectamine+mi R-21inhibitor).Then after 1,2,3 and 4 days culture,quantitative real-time PCR(QPCR)was performed to evaluate the efficiency of transfection.3)Osteosarcoma cell line MG63 was cultured and Cell transfection was performed using Lipofectamine 3000 reagent.Cells was divided into four groups: group Control,group sh NC+NC,group sh-ASBEL+NC,group sh-ASBEL+mi R-21 mimic.Then after1,2,3 and 4 days culture,cell viability was detected using trypan blue exclusion assay.Cell migration was measured using cell transwell assay with a pore size of 8 mm.The rate of cell invasion was measured similar with the rate of cell migration except that the transwell membrane was pre-incubated using Matrigel(BD Bioscience,NJ,USA).(2)The effect of mi R-21 and PP2 A on biological behavior of osteosarcoma cells1)mi R-21 mimic and mi R-21 inhibitor were transfected into osteosarcoma cell MG63 and cultured,and after 1,2,3 and 4 days culture,the expression of m RNA and protein of PP2 A were evaluated by real-time RT-PCR and Western blotting.2)mi R-21 mimic with PP2A-wt and PP2A-mt separately were transfected into osteosarcoma cell MG63,luciferase assays were performed to determine whether mi R-21 binds to 3'UTR of PP2 A.3)Osteosarcoma cell line MG63 was cultured and Cell transfection was performed using Lipofectamine 3000 reagent.Cells was divided into four groups:group Control,group sh-NC+NC,group mi R-21 inhibitor+ sh-NC,group mi R-21inhibitor+sh-PP2 A.Then after 1,2,3 and 4 days culture,real-time RT-PCR and Western blotting were used to evaluate the expression of m RNA and protein of PP2 A.cell viability was detected using trypan blue exclusion assay.Cell migration was measured using cell transwell assay with a pore size of 8 mm.The rate of cell invasion was measured similar with the rate of cell migration except that the transwell membrane was pre-incubated using Matrigel(BD Bioscience,NJ,USA).4)Osteosarcoma cell line MG63 was cultured and Cell transfection was performed using Lipofectamine 3000 reagent.Cells was divided into four groups:group NC,group mi R-21 mimic,group mi R-21 inhibitor,group mi R-21inhibitor+sh-PP2 A.Western blotting was used to analyze the effects of mi R-21 on PI3K/AKT/GSK3? and MEK/ERK signaling pathways in osteosarcoma cells.[Result]1 expression profile of lnc RNA-ASBE in osteosarcoma and its effects on proliferation,migration,invasion of osteosarcoma cells.(1)Compared to h FOB1.19 cells,ASBEL had higher expression levels in MG63 and OS-732 cells.These results indicated that ASBEL may play important roles in tumorigenicity of osteosarcoma cells.(2)The relative ASBEL expression was obviously decreased after transfection with sh-ASBEL.Compared to sh NC transfection group,sh-ASBEL transfection significantly inhibited cell viability of MG63.In addition,the relative migration and invasion of MG63 cells were remarkably decreased after transfection with sh-ASBEL.These above results suggested that suppression of ASBEL notably inhibited osteosarcoma cell proliferation,migration and invasion2 The molecular mechanism of lnc RNA-ASBE involving biological behavior of osteosarcoma cells by targeting mi R-21.(1)The effect of lnc RNA-ASBEL on expression of mi R-21 in osteosarcoma cell MG631)mi R-21 expression in MG63 cells was noticeably down-regulated after sh-ASBEL transfection,which implied that mi R-21 maybe involved in the regulatory effects of lnc RNAASBEL on osteosarcoma cell proliferation,migration and invasion.2)Relative mi R-21 expression in MG63 cells was significantly increased after transfection with mi R-21 mimic and markedly decreased after transfection with mi R-21 inhibitor?3)Compared to single sh-ASBEL transfection,sh-ASBEL+mi R-21 mimic transfection remarkably enhanced MG63 cell viability.Moreover,sh-ASBEL transfection-induced cell migration and invasion inhibition were markedly reversed by mi R-21 overexpression.These finding pointed out that mi R-21 was participated in the regulatory effects of ASBEL on osteosarcoma cell viability,migration and invasion.(2)The effect of mi R-21 and PP2 A on biological behavior of osteosarcoma cells1)mi R-21 mimic transfection down-regulated both the m RNA and protein expressions of PP2 A,However,the m RNA and protein levels of PP2 A were up-regulated after transfection with mi R-21 inhibitor,which suggested that PP2 A was a potential direct target of mi R-21 in MG63 cells.2)Luciferase activity assay showed that co-transfection with mi R-21 mimic and PP2A-wt remarkably decreased the relative luciferase activity.3)Both m RNA and protein levels of PP2 A were up-regulated by mi R-21 inhibitor transfection,while these increases were reversed by transfection of sh-PP2 A.4)Besides,we found that knockdown of PP2 A significantly alleviated the mi R-21 inhibitor-induced decreases of cell viability,migration and invasion.The aforementioned data indicated that knockdown of PP2 A reversed the mi R-21inhibitor-induced cell viability,migration and invasion inhibition,and further suggested that mi R-21 promoted osteosarcoma cell viability,migration and invasion by down-regulation of PP2 A.5)mi R-21 mimic transfection up-regulated the expression levels of p-PI3 K,p-AKT,p-GSK3?,p-MEK and p-ERK in MG63 cells.Transfection with mi R-21 inhibitor had an opposite effect.More importantly,suppression of PP2 A alleviated mi R-21 inhibitor-induced down-regulations of these proteins.These findings indicated that mi R-21 activated PI3K/AKT/GSK3? and MEK/ERK signaling pathways in osteosarcoma cells by suppression of PP2 A.[Conclusion]In conclusion,our findings revealed that lnc RNA-ASBEL as an acceleratory effect of tumorigenicity plays a great role in the cell viability,migration,invasion of osteosarcoma cell.The expression level of mi R-21 were suppressed by knockdown of ASBEL.PP2 A was a direct target of mi R-21,which was down-regulated after mi R-21 overexpression and played an important role in ASBEL and mi R-21-induced the activation of PI3K/AKT/GSK3? and MEK/ERK signaling pathways as well as osteosarcoma cell proliferation,migration and invasion.ASBEL-mi R-21-PP2 A pathway may play critical effects on the tumorgenesis of osteosarcoma and could be as the potential therapeutic target and biomarker for osteosarcoma treatment.[Innovation]We found that lnc RNA-ASBEL expressed increasingly in osteosarcoma cell lines for the first time.This is the first time the molecular mechanism of lnc RNA-ASBE involving in viability,migration and invasion of osteosarcoma cell was explored,and which is helpful for further understand the tumor promoting effect of ASBEL and may provide a possible therapeutic target for osteosarcoma treatment.