Effect Of LncRNA XLOC-005950/miR-362-5p/SATB2 Network Regulation On Biological Characteristics Of Osteosarcoma Cells

BackgroundOsteosarcoma?OS?is one of the most common primary bone malignant tumors.The incidence of osteosarcoma is high in children and adolescents.Early invasive metastasis can make osteosarcoma progress rapidly and cause poor prognosis,resulting in lower overall survival rate.In recent years,targeted therapy has shown great potential in the treatment of osteosarcoma,but more effective therapeutic targets still need to be explored.Long noncoding Ribonucleic Acid?lncRNA?plays an important role in cancer biology.It can be used as a key regulator of tumor metastasis,and can also directly or indirectly regulate the expression of target proteins by interacting with miRNA.However,the role of lncRNA in osteosarcoma still requires more exploration and research.SATB2?special AT-rich binding sequence protein 2?is a DNA binding protein that can specifically bind to the nuclear matrix attachment region and act as a regulator of chromatin high-level domain transcription.And it is related to invasion,metastasis and apoptosis in a variety of malignancies.Our previous experimental studies have preliminarily shown thatthereisapotentialconnectionbetweenthe regulationof lncRNA-miRNA-mRNA network and osteosarcoma,which provides a new idea for exploring the molecular mechanism of osteosarcoma occurrence and development.Our group plans to further study the targeting effect of lncRNA XLOC-005950 and miR-362-5p and the effect of the targeting effect between miR-362-5p and target gene SATB2 in the regulation of osteosarcoma invasion,migration and apoptosis and its biological characteristics.PurposeIn this study,the targeting effect of lncRNA XLOC-005950 with miR-362-5p,miR-362-5p with SATB2 was verified by bioinformatics software to predict the miRNA,lncRNA XLOC-005950 and SATB2 interaction with each other.At the same time,the expressions of lncRNA XLOC-005950,miR-362-5p and SATB2 in normal osteoblast cell line hFOB1.19,osteosarcoma cell line MG63 and knock-out lncRNA XLOC-005950 cell line MG63^-/-were detected,as well as the differences in cells biology.MiR-362-5p mimics was transfected into MG63 cells to verify the regulation of miR-362-5p on target genes and its effect on cell biological traits.In order to find the mechanism of osteosarcoma and new prognostic indicators and therapeutic targets for osteosarcoma.Methods1.Use bioinformatics software Diana Tools to predict miRNAs that binding to lncRNA XLOC-005950;Bioinformatics software such as TargetScan and microRNA.org were used to predict miRNA targeting SATB2.2.The 26 cases of osteosarcoma patients'tissues and adjacent tissues used in this experiment were collected from the First Affiliated Hospital of Zhengzhou University from September 2016 to June 2018 and then stored in liquid nitrogen.Real-time quantitative PCR?qPCR?was used to detect the expression of lncRNA XLOC-005950,miR-362-5p and SATB2 mRNA in the paired tissues.The expression of SATB2 protein was detected by Western blot.3.RT-qPCR detects the expression of lncRNA XLOC-005950,miR-362-5p and SATB2 mRNA in osteosarcoma cell line MG63,knocked-out lncRNA XLOC-005950 osteosarcoma cell line MG63^-/-and normal osteoblast cell line hFOB1.19;Western blot was used to detect the expression of SATB2 protein in the three cell lines.CCK-8 assay was used to detect the ability of proliferation in the three cell lines.Transwell assay was used to detect the ability of invasive in the three cell lines;and scratch test was used to detect the ability of migration in the three cell lines;Flow cytometry Annexin V/PI test was used to detect the apoptosis in the three cell lines.4.Liposome transfected miR-362-5p mimics and miR-362-5p negtive control in MG63 cells and divided into three groups:miR-362-5p mimic group?transfected liposome and miR-362-5p mimics?;NC group?transfected liposome and miR-362-5p negative control?;Blank group?only transfected liposome?.5.qPCR was used to detect cell transfection efficiency.6.Detection of transfected cells:?1?CCK-8 assay detects the ability of proliferation in transfected cells;?2?Transwell assay and scratch test respectively detect the ability of invasion and migration in transfected cells;?3?Flow cytometry Annexin V/PI test was used to detect the apoptosis in transfected cells.?4?RT-qPCR was used to detect the expression of SATB2 mRNA and VEGF-B mRNA in transfected cells,and Western blot was used to detect the expression of SATB2 and VEGF-B protein.7.Construct wild-type and mutant-type recombinant vectors of pmirGLO-SATB2and p-GL3-promoter-lncRNA XLOC-005950 respectively,and verify the binding effect of lncRNA XLOC-005950 and miR-362-5p,SATB2 and miR-362-5p respectively by using dual luciferase reporter assay.Results1.Detection of osteosarcoma tissues and adjacent tissues showed that the expression of lncRNA XLOC-005950,SATB2 mRNA and protein in osteosarcoma tissues was significantly higher than that in adjacent tissues?P<0.01?;however,the expression of miR-362-5p in osteosarcoma tissues was lower than that in the corresponding adjacent tissues?P<0.05?.2.Detection of osteosarcoma cell line MG63,knocked-out lncRNA XLOC-005950cell line MG63^-/-and normal osteoblast cell line hFOB1.19,the results showed that the expression of lncRNA XLOC-005950,SATB2 mRNA and protein in osteosarcoma cell line MG63 is higher than that of knocked-out lncRNA XLOC-005950 cell line MG63^-/-and normal human osteoblast cell line hFOB1.19?P<0.01?;while the expression of miR-362-5p in MG63 cells was significantly lower than that in MG63^-/-and hFOB1.19 cells?P<0.05?.3.The results of CCK-8 assay showed that compared with MG63 cells,the ability of growth and proliferation in MG63^-/-cells decreased?P<0.05?;the results of transwell assay and scratch test respectively showed that compared with MG63 cells,the ability of invasion and migration in MG63^-/-cells decreased?P<0.05?;the results of flow cytometry Annexin V/PI test showed that compared with MG63 cells,the apoptosis rate in MG63^-/-cells increased?P<0.01?.4.QPCR detection of transfection efficiency showed thatMG63 cells successfully transfected miR-362-5p mimics.5.The results of transfected cells:?1?Results of CCK-8 assay showed that compared with NC group and Blank group,the ability of growth and proliferation in miR-362-5p group decreased?P<0.05?;?2?results of transwell assay and scratch test showed that the ability of cell invasion and migration in miR-362-5p mimic group decreased compared with NC group and Blank group?P<0.05?;?3?the results of flow cytometry Annexin V/PI test showed that the apoptosis rate of miR-362-5p group was increased compared with NC group and Blank group?P<0.01?;?4?the results of qPCR showed that compared with NC group and Blank group,the expression of SATB2 mRNA?P<0.05?and VEGF-B mRNA?P<0.01?both decreased.And the results of western blot also showed that the expression of SATB2 and VEGF-B proteins decreased.6.Bioinformatics analysis predicted that there were interaction binding sites between lncRNA XLOC-005950 and miR-362-5p and between SATB2 and miR-362-5p.The dual luciferase reporter assay were respectively verified,and the results were consistent with the bioinformatics analysis prediction,indicating that there were binding sites between lncRNA XLOC-005950,miR-362-5p and SATB2.Conclusions1.In osteosarcoma tissues and osteosarcoma cell line MG63,lncRNA XLOC-005950 and SATB2 are both highly expressed and the expression of miR-362-5p is lower compared with their corresponding adjacent tissues and normal osteoblast cell line hFOB1.19.2.The expression of SATB2 in MG63^-/-cells knocked-out lncRNA XLOC-005950and MG63 cells transfected with miR-362-5p isdecreased,and the ability of proliferation,invasion,migration in the two types cells are decreased and apoptosis rate is increased.3.MiR-362-5p can specifically bind to the 3'UTR region of SATB2,which is the target gene of miR-362-5p,and inhibit the expression of SATB2;lncRNA XLOC-005950 can specifically bind to miR-362-5p and play a role of negative regulatory.