Effect Of Hsa＿circ＿0000530 On Cell Cycle,Proliferation And Invasion Of Osteosarcoma Cells Through MiR-198/CDK6 Regulatory Axis

Background and purposeOsteosarcoma(OS)is a bone-derived malignant tumor,which prone to recurrence and has a low survival rate.The incidence of osteosarcoma is hidden.Many patients have missed the opportunity for effective treatment when they are discovered.Therefore,it is extremely necessary to explore the development process of osteosarcoma and find biomolecules involved in the development of osteosarcoma to provide new targets for diagnosis and treatment.The dysregulation of circular RNA(circ RNA)expression plays an important role in the development of many cancers.Studies have shown that multiple circ RNAs are differentially expressed in human normal osteoblasts and osteosarcoma cell lines,but the specific mechanism of circ RNAs in the development of osteosarcoma is still poorly understood.Several studies have shown circ RNA that play crucial roles in tumorigenesis and metastasis inhibition by sponging micro RNA(miRNA),while miRNA negatively regulates gene expression via binding the 3’-untranslated region(UTR)of targeted mRNAs at the post-transcriptional level,and participates in biological processes such as cell differentiation,proliferation,autophagy and apoptosis.Cyclin-dependent kinase 6(CDK6)regulates cell-cycle progression.Studies have shown that highly expressed circ RNA inhibited miRNA-mediated CDK6 reduction by acting as miRNA sponges,and promoting the growth and cycle progression of osteosarcoma cells.Tumor cells are blocked in the G1 phase by inhibitors that specifically inhibit the activity of CDK6.These reports suggested that CDK6 plays an important role in the development of tumors and is expected to become a potential target for the treatment of osteosarcoma.In this study,based on the high-throughput sequencing data obtained,preliminary experiments have confirmed that hsa＿circ＿0000530 was highly expressed in osteosarcoma tissues.Bioinformatics software and experiments were used to predict and verify that miR-198 has a binding site with hsa＿circ＿0000530and CDK6.The effect of hsa＿circ＿0000530/miR-198/CDK6 regulatory axis on the cell cycle,proliferation,migration and invasion of osteosarcoma were subsequently explored,providing a new ideas for the diagnosis and treatment of osteosarcoma,and a new theoretical basis for the mechanism of occurrence and development of osteosarcoma.Methods1.Screen and verify the differentially expressed circ RNA in osteosarcoma tissues and cell lines and determine whether hsa＿circ＿0000530 exists in osteosarcoma cells.The differentially expressed circ RNAs(such as hsa＿circ＿0000530)in osteosarcoma and adjacent tissues were screened by high-throughput sequencing analysis,the expression of hsa＿circ＿0000530 in osteosarcoma and adjacent tissues in18 patients with osteosarcoma was detected by Quantitative Real-time PCR(qRT-PCR),and the expression of hsa＿circ＿0000530 in osteosarcoma cell lines MG63,U2 OS,SAOS-2 and human normal osteoblast h FOB1.19 was detected.The Divergent Primer,of hsa＿circ＿0000530 was designed according to the circ Bank database.Sanger sequencing was used to verify whether the reverse splicing site sequence amplified by c DNA from osteosarcoma cells was consistent with the database,which proved the existence of hsa＿circ＿0000530 in osteosarcoma cells.2.To investigate the difference of expression of miR-198 and its corresponding target gene CDK6 in osteosarcoma tissues and osteosarcoma cells.QRT-PCR was used to detect the expression of miR-198 and CDK6 mRNA in osteosarcoma tissue and paracancerous tissue,and osteosarcoma cell lines and human normal osteoblast.At the same time,Western-blot was used to detect the difference of CDK6 protein expression.3.Bioinformatics prediction and dual-luciferase reporter assay were used to verify the existence of binding target sites between miR-198 and hsa＿circ＿0000530and CDK6.Circular RNA Interactome,a bioinformatics software was used to predict miRNAs that might bind to hsa＿circ＿0000530(such as miR-198),Target Scan database to predict the target gene of miRNA(such as CDK6).The wild-type and mutant-type reporter vectors of pmir GLO-hsa＿circ＿0000530 and pmir GLO-CDK6 were constructed,and dual luciferase experiment was used to verify the binding of miR-198 to hsa＿circ＿0000530 and CDK6 3’UTR,further proving that miR-198 has a targeted binding relationship with hsa＿circ＿0000530 and CDK6.4.To explore effects of down-regulation of hsa＿circ＿0000530 on miR-198 and CDK6 expression,proliferation,migration and invasion of osteosarcoma cells and subcutaneous tumorigenicity of MG63 cells.hsa＿circ＿0000530 siRNA was synthesized in vitro and transfected into osteosarcoma cells MG63 and U2 OS.Experimental groups: blank group(transfection reagent only,Blank),hsa＿circ＿0000530 negative control group(transfection hsa＿circ＿0000530 negative control,si-NC),hsa＿circ＿0000530knockdown group(transfection hsa＿circ＿0000530 siRNA,si-circ).The expression of hsa＿circ＿0000530,miR-198 and CDK6 in the transfected cells was detected by qRT-PCR and Western-blot,and the effects of down-regulated hsa＿circ＿0000530 on the proliferation,cycle progression,migration and invasion of osteosarcoma cells were observed by CCK-8,flow cytometry,wound healing and Transwell assay.MG63 cells stably low expressing hsa＿circ＿0000530 were constructed by hsa＿circ＿0000530 siRNA lentivirus infection.The transplanted tumor model of nude mice was established by tumor formation experiment in nude mice.The effect of hsa＿circ＿0000530 on tumor growth in vivo was observed through murine xenograft model.Immunohistochemical staining method was used to detect the expression level of Ki-67 in tumor tissues.5.To explore effects of up-regulation of miR-198 on CDK6 expression,proliferation and cycle,migration and invasion of osteosarcoma cells.MG63 and U2 OS cells were transfected with miR-198 mimics.Experimental groups: miR-198 negative control group(transfected miR-198 negative control,miR-NC),miR-198 overexpression group(transfected miR-198 mimics,miR-198mimics).The expression of miR-198 and CDK6 in the transfected cells were detected by qRT-PCR and Western-blot.Cell function experiments were performed to observe the changes in the proliferation,cell cycle,migration and invasion capabilities of osteosarcoma cells after miR-198 overexpression.6.To explore the regulatory relationship between hsa＿circ＿0000530/miR-198/CDK6 axis,which has an impact on the proliferation and cycle,migration and invasion of osteosarcoma cells through rescue experiments.To clarify whether hsa＿circ＿0000530 knockdown inhibits the proliferation,migration and invasion of MG63 and U2 OS cells through miR-198/CDK6,we conducted functional rescue experiments.miR-198 inhibitor was transfected into MG63 and U2 OS cells knocking down hsa＿circ＿0000530,and the experiment was divided into: Blank,si-circ and si-circ+miR-198 inhibitor groups.MG63 and U2 OS cells were co-transfected with miR-198 inhibitor and hsa＿circ＿0000530 siRNA,qRT-PCR and Western-blot were used to detect the effect of down-regulation of miR-198 on CDK6 expression when hsa＿circ＿0000530 is knocked down,and to further explore the regulatory relationship between hsa＿circ＿0000530/miR-198/CDK6.The changes of proliferation and cycle progression,migration and invasion of osteosarcoma cells after transfection were observed by rescue experiments.Results1.The differentially expressed circ RNA in osteosarcoma tissues and cell lines has been verified,proving that hsa＿circ＿0000530 actually exists in osteosarcoma cells.The high expression of hsa＿circ＿0000530 in osteosarcoma tissues was found to be analyzed by high-throughput sequencing.qRT-PCR detection showed that compared with the corresponding adjacent tissues and human normal osteoblasts,hsa＿circ＿0000530 was highly expressed in osteosarcoma tissue and osteosarcoma cell lines MG63,U2 OS and SAOS-2(P<0.01).Sanger sequencing results showed that hsa＿circ＿0000530 actually exists in osteosarcoma cells.hsa＿circ＿0000530 was selected as the research object.2.Differential expression of miR-198 and its corresponding target gene CDK6 in osteosarcoma tissues and cells.qRT-PCR detection showed that compared with the corresponding adjacent tissues and human normal osteoblasts,miR-198 expression was low in osteosarcoma tissue and cell lines(P<0.01,P<0.05),and CDK6 mRNA expression was high(P<0.05),while Western-blot detection showed that CDK6 protein was also highly expressed.The results indicate that the expression of hsa＿circ＿0000530 may be related to the expression level of miR-198 and its corresponding target gene CDK6 in osteosarcoma cells.3.Bioinformatics prediction and dual-luciferase reporter assay verify the existence of binding sites between miR-198 and hsa＿circ＿0000530 and CDK6.The database predicted that there was a miR-198 binding site in hsa＿circ＿0000530 and a matching site between miR-198 and CDK6 3’UTR.The wild type and mutant reporter vectors of pmir GLO-hsa＿circ＿0000530 and pmir GLO-CDK6 were successfully constructed,and dual-luciferase reporter experiments showed that hsa＿circ＿0000530 could bind to miR-198,and miR-198 could target the binding region of CDK6 3’UTR.The results suggest that CDK6 is a direct target gene of miR-198 and that there may be a hsa＿circ＿0000530/miR-198/CDK6 regulatory axis in osteosarcoma cells.4.hsa＿circ＿0000530 down-regulation in osteosarcoma cells can affect the expression of miR-198 and CDK6,proliferation,migration and invasion of osteosarcoma cells,and subcutaneous tumorigenicity of MG63 cells.hsa＿circ＿0000530 siRNA was transfected into MG63 and U2 OS cells.In si-NC group there was no significant difference in the expression of hsa＿circ＿0000530 in osteosarcoma cells,compared with Blank group.In si-circ group,the expression of hsa＿circ＿0000530 and CDK6 decreased(P<0.01,P<0.05),while the expression of miR-198 increased(P<0.05).In si-circ group,hsa＿circ＿0000530 was down-regulated,the proliferation of MG63 and U2 OS cells was inhibited,the cell cycle was blocked in G1 phase,the proportion of cells in S phase was decreased(P<0.01),and the ability of cell migration and invasion was decreased(P<0.05),compared with si-NC group.Xenograft formation experiment showed that hsa＿circ＿0000530 knockdown inhibited the subcutaneous tumorigenesis of MG63 cells and decreased the expression of Ki-67 protein in tumor tissue(P<0.01).The results suggest that down-regulation of hsa＿circ＿0000530,could upregulate the expression of miR-198 and down-regulate the expression of CDK6 in osteosarcoma cells,inhibit the transition of cell cycle G1 and S phase,keep the cells in G1 phase,inhibit cells proliferation in vivo and vitro,and inhibit the migration and invasion of osteosarcoma cells.5.miR-198 up-regulation in osteosarcoma cells can affect the expression of CDK6,proliferation and cycle,migration and invasion of osteosarcoma cells.miR-198 mimics was transfected into MG63 and U2 OS cells,the expression level of miR-198 mimics group was significantly increased(P<0.01),and the expression of CDK6 was decreased(P<0.05),compared with the miR-NC group.miR-198 overexpression inhibited the proliferation,migration and invasion of MG63 and U2OS cells,and the cell cycle stayed in the G1 phase.The above results indicate that both miR-198 overexpression and hsa＿circ＿0000530 knockdown could down-regulate the expression of CDK6 and inhibit osteosarcoma cell proliferation and cell cycle progression,and reduce cell migration and invasion capabilities.6.It is further proved that there is a regulatory relationship between hsa＿circ＿0000530/miR-198/CDK6,which has an effect on the proliferation and cycle,migration and invasion of osteosarcoma cells through rescue experiments.miR-198 inhibitor was transfected into hsa＿circ＿0000530 knockdown MG63 and U2OS cells.The reduction of CDK6 expression caused by hsa＿circ＿0000530knockdown could be partially restored by inhibiting miR-198 expression(P<0.05).Knocking down hsa＿circ＿0000530 can down-regulate the expression of CDK6 by up-regulating miR-198 expression,indicating that there is an axial regulation relationship between miR-198,hsa＿circ＿0000530 and CDK6.Inhibition of miR-198 expression can partially offset the inhibitory effects of hsa＿circ＿0000530knockdown on the proliferation,migration and invasion of MG63 and U2 OS cells(P<0.05),and promot part of the cell cycle from G1 phase to S phase.These results suggest that hsa＿circ＿0000530 can regulate the proliferation,cycle,migration and invasion of osteosarcoma cells through the miR-198/CDK6 axis Conclusionshsa＿circ＿0000530 could promote the proliferation and invasion of osteosarcoma cells,and miR-198 has the role of tumor suppressor in osteosarcoma cells.Knockdown of hsa＿circ＿0000530 and overexpression of miR-198 can inhibit CDK6 expression in MG63 and U2 OS cells,prevent the cell cycle from G1 phase to S phase,and inhibit the proliferation,migration and invasion of osteosarcoma cells.hsa＿circ＿0000530 regulates the expression of CDK6 by sponging miR-198 to affect the proliferation and cycle,migration and invasion of osteosarcoma cells.The effect of hsa＿circ＿0000530/miR-198/CDK6 regulatory axis on the proliferation and invasion of osteosarcoma cells may provide a new idea for the diagnosis and treatment of osteosarcoma.