Effect Of Hsa_circ₀006602/miR-224/Rac1 Regulatory Axis On The Proliferation And Invasion Of Osteosarcoma Cells

BackgroundOsteosarcoma?OS?is the most common primary malignant tumor of bone tissue in children and young people,which is accompanied by high mortality.Despite advancement strategies such as surgery,adjuvant chemotherapy,and radiotherapy,the 5-year survival rate for patients with osteosarcoma is still low.Accordingly,identifing new molecules involved in tumor progressionmay may provide the novel strategies for diagnosis and therapy of patients with osteosarcoma.Circular RNA?circRNA?is a covalently closed circular RNA produced by the back-splicing process.It does not contain 5 'to 3' polarity and a polyadenylated tail.Some circRNAs are abundant and evolutionarily conserved,and many circRNAs exert important biological functions by acting as microRNA sponges,They have been found to play an important role in cancer progression and have become a hot topic in today's research.Ras-related C3 botulinum toxin substrate 1?Racl?is a member of the RhoGTP enzyme.Racl has been found to be significantly upregulated in malignant tumors,and activation of Racl can increase tumor proliferation and metastasis.Our previous study found that hsa_circ₀006602 is significantly upregulated in osteosarcoma and may be involved in the progression of osteosarcoma through the circRNA-miRNA-mRNA network.This project intends to explore the effect of hsa_circ₀006602/miR-224/Racl regulatory axis on the biological behavior of osteosarcoma cells.PurposeIn this study,we propose to screen for differentially expressed hsa_circ₀006602 by high-throughput sequencing of osteosarcoma tissues and adjacent tissues,and to investigate its effect on the proliferation,invasion,migration and tumorigenic capacity of MG63 cells.At the same time,biological information software was used to predict and screen hsa_circ₀006602 for possible adsorption of miRNA and miRNA targeted binding genes,and explore their interaction pathways.Elucidate the molecular mechanism of hsa_circ₀006602/miR-224/Rac1 regulatory axis in osteosarcoma cells.In order to provide a new theoretical basis for the complex mechanism of circRNA involved in regulating the progression of osteosarcoma and provide new targets and strategies for the prevention and treatment of osteosarcoma.Methods1.Differentially expressed circRNAs?e.g.hsa_circ₀006602?were screened based on high-throughput sequencing of osteosarcoma and adjacent tissues.The Convergent Primer and Divergent Primer were designed to amplify with osteosarcoma cell gDNA and cDNA,respectively,for structural identification and sequencing to verify the presence of hsa_circ₀006602 in osteosarcoma cells.2.The bioinformatics software Circular RNA Interactome database was used to predict and analyze the miRNAs bound to hsa_circ₀006602 sponge?e.g.miR-224?.The TargetScan and miRBase were used to predict miRNA target genes?e.g.Rac1?.3.qRT-PCR was used to detect the expressions of hsa_circ₀006602,miR-224 and Racl mRNA in 30 patients with osteosarcoma tissues and adjacent tissues,and detect the expression in osteosarcoma cell lines?MG63,SAOS-2,U20S?and normal osteoblasts hFOB1.19;The expression of Racl protein were detected by Western blot.4.The effect of knockdown hsa_circ₀006602 on the biological function of MG63 cells was detected.?1?Group:si-circ group as hsa_circ₀006602 knockdown group?transfected with hsa_circ₀006602 siRNA?;si-NC group?transfected with hsa_circ₀006602 negative control?;Blank group?only transfection reagent?.?2?qRT-PCR was used to detect cell transfection efficiency,miR-224 and Rac1 mRNA expression.Western blot was used to detect Racl protein expression in transfected cells.?3?CCK-8,Transwell,and scratch tests were used to test the proliferation,invasion,and migration ability of each group.5.Detecting the effect of overexpress miR-224 on Racl expression in MG63 cells:?1?Group:miR-224 mimics group?transfected with miR-224 mimics?;miR-NC group?transfected with miR-224 negative control?;Blank group?only transfection reagent?.?2?qRT-PCR was used to detect transfection efficiency.?3?qRT-PCR and Western blot were used to detect the expression of Rac1 in transfected cells.6.Wild and mutant recombination vectors of pmirGLO-circ6602 and pmirGLO-Racl were constructed,and verify the binding effect of hsa_circ₀006602 and miR-224,Racl and miR-224 respectively by using luciferase reporter assay.7.The rescue experiments verified that hsa_circ₀006602 can influence the malignant biological behavior of osteosarcoma via Rac1.Divided into three groups:?1?si-circ+Racl group?Transfected hsa_circ₀006602 siRNA and pcDNA3.1-Rac1?;si-circ group?Transfected hsa circ₀006602 siRNA?;Blank group?only liposome?.?2?CCK-8,Transwell and scratch experiments were used to detect cell proliferation,invasion and migration in each group.8.The rescue experiments verified that the regulatory effect of hsa_circ₀006602 on Rac1 depends on miR-224.?1?Group:si-circ+miR-224-in group?Transfected miR-224 inhibitor and hsa_circ₀006602 siRNA?;si-circ group?Transfected hsa_circ₀006602 siRNA?;Blank group?only liposome?.?2?The expression level of Racl in transfected cells was detected by qRT-PCR and Western blot.9.In vivo animal study:hsa_circ₀006602 siRNA lentivirus drip was used to infect osteosarcoma cells.Osteosarcoma cells stably transfected with hsa_circ₀006602 siRNA were screened by puromycin,and then inoculated subcutaneously in nude mice.The volume and weight of tumor-forming tissues were measured and recorded;ki-67 protein changes were detected by immunohistochemical staining;qRT-PCR was used to detect hsa_circ₀006602,miR-224 and Racl mRNA expression in tumor tissues,and Western blot was used to detect Racl protein expression.Results1.hsa_circ₀006602 was selected for study by high-throughput sequencing,which revealed significant differential expression?log2FC=5.4 P=1.83E-10?in osteosarcoma tissues and adjacent tissues.PCR amplification and Sanger sequencing results showed that only the corresponding size band of hsa_circ₀006602 could be detected by Divergent Primer amplification in the cDNA.The PCR amplification product sequence showed BackSplice Junction,indicating that hsa_circ₀006602 exists in osteosarcoma cells.2.Detection of osteosarcoma patient tissues and osteosarcoma cell lines?MG63,SAOS-2,U20S?by qRT-PCR:Compared with adjacent tissues and normal human osteoblast hFOB1.19,the expression of hsa_circ₀006602 and Racl in osteosarcoma tissue and cell lines was up-regulated?P<0.05?;The expression of miR-224 was decreased?P<0.05?.3.Detection results of hsa_circ₀006602 siRNA transfected cells:?1?Compared with the Blank group,hsa_circ₀006602 expression in the transfected si-circ group cells decreased significantly?P<0.05?,while there was no difference in hsa_circ₀006602 expression in the transfected si-NC group.MG63 cells transfected with hsa_circ₀006602 siRNA successfully.?2?Compared with the Blank and si-NC group,the expression of miR-224 in the si-circ group was Significantly elevatedand?P<0.05?,and Rac1 mRNA and protein in the si-circ group was significantly reduced?P<0.05?.This indicates that knockdown hsa_circ₀006602 promotes miR-224 expression and inhibits Racl expression.?3?CCK-8,Transwell and cell scratch experiments showed that Compared with the Blank group and the si-NC group,the proliferation activity of MG63 cells in the si-circ group was significantly suppressed?P<0.05?,and the cell invasion and migration ability were reduced?P<0.05?.This indicates that down-regulating hsa_circ₀006602 can inhibit the proliferation,invasion and migration ability of osteosarcoma cells.4.Detection results of MG63 cells transfected with miR-224 mimics:?1?Compared with the Blank group,there was no significant change in the expression level of miR-224 in the transfected miR-224-NC group,while the expression of miR-224 in miR-224 mimics group increased significantly?P<0.05?.This indicates that MG63 cells were successfully transfected.?2?qRT-PCR and Western Blot showed that compared with the Blank group and miR-224-NC group,the mRNA and protein expression of Racl in the miR-224 mimics group was reduced?P<0.05?,indicating that up-regulating miR-224 can inhibit the expression of Racl,which is consistent with the trend of down-regulating hsa_circ₀006602.5.Bioinformatics software analysis and dual luciferase reporting experiments showed that hsa_circ₀006602 and Racl both had targeted binding sites for miR-224.hsa_circ₀006602 can bind to miR-224 and down-regulate its expression.miR-224 can target Racl 3 'UTR region and negatively regulate its expression.The results suggest that hsa_circ₀006602/miR-224/Racl may constitute an adjustment axis in MG63 cells.6.The results of rescue experiments showed that the decrease of Racl expression caused by knocking down hsa circ₀006602 can be partially restored by miR-224 inhibitor?P<0.05?.This result indicates hsa_circ₀006602 regulate Racl through miR-224.The results of CCK8,Transwell and scratch experiments showed that in cells that knocked down hsa_circ₀006602,The promotion effect of hsa_circ₀006602 on the proliferation,metastasis and invasion of MG63 cells can be partially restored by overexpression of Racl?P<0.05?.It suggested that the effect of hsa_circ₀006602 on the malignant biological behavior of osteosarcoma depends on Rac1.This also further suggests that there may be hsa_circ₀006602/miR-224/Rac1 regulatory axis in osteosarcoma cells.7.In vivo animal study:Compared with the negative control group,tumor volume,weight,and growth rate were significantly lower in the tumor tissue that knocked down hsa_circ₀006602?P<0.05?,ki-67 protein expression was significantly lower in the tumor tissue that knocked down hsa_circ₀006602?P<0.05?,The expressions of hsa circ₀006602 and Racl were significantly lower in the tumor tissue that knocked down hsa_circ₀006602?P<0.05?and the expressions of miR-224 were higher in the tumor tissue that knocked down hsa_circ₀006602?P<0.05?.The results of in vivo experiments are consistent with the trend of in vitro cellular experiments.Conclusions1.hsa_circ₀006602 and Rac1 are overexpressed in osteosarcoma tissue and osteosarcoma cell line,while miR-224 is underexpressed.2.hsa_circ₀006602 binds miR-224,while miR-224 targets Rac1 3'UTR and inhibits its expression.3.Down-regulating hsa_circ₀006602 or over-expressing miR-224 can reduce Racl expression in MG63 cells.At the same time,knocking down hsa_circ₀006602 can reduce cell proliferation,invasion,migration and subcutaneous tumorigenicity in nude mice.4.The rescue experiment further verified that hsa_circ₀006602 regulates osteosarcoma cell proliferation,invasion,and migration through the miR-224/Rac1 regulatory axis.