Dysregulated CircRNA₁00876 Suppresses Proliferation Of Osteosarcoma Cancer Cells By Targeting MicroRNA-136

Background:Osteosarcoma?OS?is one of the most common malignant bone tumors of mesenchymal origin,which generally occurs in children and adolescents.Previous studies showed that patients with osteosarcoma commonly progress to metastasis and have a very poor prognosis.Recently,neoadjuvant therapies and adjuvant chemotherapies have been widely used in the clinical setting,but are not able to completely inhibit the tumor growth,leaving the mortality rate of OS still at a high level.Therefore,it is very urgent to explore new bio-markers for OS.Circular RNAs?circRNAs?are a new class of non-coding RNA molecules that extensively exist in the cytoplasm of eukaryotic cells.It is known that circRNAs are different from linear RNAs,lacking 5' to 3' polarity and polyadenylated tail and having great stability.circRNAs have a variety of functions,such as regulating splicing and transcription,binding proteins,and transporting RNAs.Recent studies have shown that the expression of many circRNAs was up-regulated in various cancers and was positively correlated with the progression of the cancer.CircRNA 100876 is one of the typical circRNAs,which takes part in the progression of non-small cell lung cancer and esophageal carcinoma;however,the role of circRNAs 100876 in OS has been rarely reported.In this study,we attempted to explore the relationship between circRNA₁00876 and OS.Moreover,based on the publicly available bioinformatic algorithms?Starbase?,we found that miR-136 might be a downstream gene of circRNA₁00876.Therefore,we predicted that circRNA₁0876 could affect the development of OS through the regulation of microRNA-136?miR-136?,and accordingly,we designed in vivo and in vitro experiments to elucidate the potential underlying molecular mechanism.Part ? Expression characteristics and clinical significance of circRNA₁00876 in osteosarcomaObjectiveTo detect the expression level of circRNA₁00876 both in osteosarcoma tissues and its paracancerous normal tissue,and analysis its clinical implications.Method1.48 cases of OS tissue samples were obtained from patients who underwent surgery from January 1st,2014 to December 31th,2016.2.The expression level of circRNA₁00876 in cancer tissues and paracancerous normal tissues of 48 OS patients was detected by real-time quantitative polymerize chain reaction?RT-qPCR?.Result1.Compared with paracancerous normal tissues,circRNA₁00876 in OS tissues showed higher expression?40/48,83.3%,P<0.001?.2.The high expression of circRNA 100876 was closely association with the tumor size?P=0.004?and the tumor differentiation degree?P<0.001?.3.From the Kaplan-Meier analysis,the median overall survival time of OS patients with high expression level of circRNA₁00876 was significantly shorter than that with low expression level of circRNA₁00876?P = 0.0031?.ConclusionsIn OS cancer tissues,we found that the expression of circRNA 100876 was up-regulated,which was closely correlated to the tumor size,tumor differentiation degree and survival time.Therefore,circRNA 100876 had a promising application for predicting poor prognosis of OS patients.Part ? The effects of circRNA 100876 expression on proliferation and metastasis abilities of OS cancer cell linesObjectiveTo detect the effects of circRNA 100876 on cell proliferation and cell migration of OS cells.Method1.The expression levels of circRNA 100876 in the normal hFOB 1.19 cell line and other six OS cell lines?143B,Saos-2,MG-63,HOS,OS-732 and U2-OS cells?were detected by RT-qPCR.2.We knocked down the expression of circRNA 100876 in MG-63 and HOS cells by lentivirus transfection of siRNA.Then,we screened the cell lines with stable low expression of circRNA₁ 00876 by puromycin.3.Cell Counting Kit-8?CCK-8?and in vitro animal study were applied to detect the cell proliferation ability of MG-63 and HOS cells after the depletion of circRNA 100876.4.After inhibiting the expression of circRNA 100876 by siRNA,cell apoptosis rates of MG-63 and HOS cells were analyzed by flow cytometry.Meanwhile,the expression level of apoptotic regulatory proteins were detected by western blot.5.After inhibiting the expression of circRNA 100876 by siRNA,cell cycle of MG-63 and HOS cells were analyzed by flow cytometry.Meanwhile,the expression level of cyclins regulatory proteins were detected by western blot.6.The migration abilities of MG-63 and HOS cells were analyzed by Transwell assays.Result1.RT-qPCR results showed that,compared with that in the normal hFOB1.19 cell line,the expression level of circRNA 100876 in OS cell lines?143B,Saos-2,MG-63,HOS,OS-732 and U2-OS cells?was up-regulated,with MG-63 and HOS cell lines showing tme highest expression?P<0.001?.2.The expression of circRNA 100876 in the MG-63 and HOS cell lines decreased significantly after transfection with circRNA 100876 specific siRNA-lentivirus.3.CCK-8 and in vitro tumorigenmcity study consistently all showed that the cell proliferation of MG-63 and HOS cell lines was dramatically inhibited after knocking down the expression of circRNA 100876.4.The down-regulation of circRNA 100876 induced cell apoptosis at a greater extent compared with that in the control group.In addition,the expression levels of cell apoptosis related proteins and anti-apoptosis related proteins were evaluated by western blotting.Consistent with the flow cytometry results,upon circRNA 100876 silencing,the expression of apoptosis-related proteins?cleaved Caspase-3,Bax and p53 protein?were significantly upregulated,while the anti-apoptosis related protein BCL-2 was dramatically inhibited.5.After knocking down the expression of circRNA 100876 by siRNA,MG-63 and HOS cell lines were both arrested in G2/M phase,and cyclin B1 was up-regulated,while cyclin D1 was down-regulated.6.After the expression of circRNA 100876 was down-regulated,no significant differences were observed in the migration and metastasis abilities of MG-63 and HOS cell lines.ConclusionsDown-regulating the expression of circRNA 100876 could inhibit cell proliferation,induce apoptosis and arrested more cells at G2/M phages.Our findings indicate that circRNA 100876 might be considered as a crucial molecular target for OS patients.Part ? The correlation between microRNA-136 and circRNA 100876 expression in osteosarcomaObjectiveTo detect the expression level of circRNA 100876 and microRNA-136?miR-136?in osteosarcoma tissues,and analyze their potential correlation.Method1.Based on the prediction provided by publicly available bioinformatic algorithms?Starbase?,we predicted the potential downstream target for circRNA 100876,and their potential binding site.2.The expression level of miRNA-136 in cancer tissues and paracancerous normal tissues of 48 OS patients was detected by real-time quantitative polymerize chain reaction?RT-qPCR?.Result1.Based on the prediction provided by publicly available bioinformatic algorithms?Starbase?,miR-136 might be a downstream target for circRNA 100876.2.Compared with paracancerous normal tissues,miR-136 in OS tissues showed lower expression?43/48,89.6%,P<0.001?.3.The correlation analysis showed that the expression level of circRNA 100876 was negatively correlated with the expression of miR-136?r=-0.3278,P=0.0229?.ConclusionsIn OS cancer tissues,we found that the expression of miR-136 was down-regulated,which was closely correlated to the higher expression of circRNA 100876.Taken together,these results indicated that miR-136 might be the downstream target for circRNA 100876 in OS.Part ? Down-regulation of miR-136 reverses the anti-cell proliferation effect of silencing circRNA₁00876ObjectiveTo investigate the effect of miR-136 on the proliferation of osteosarcoma cell lines with circRNA₁00876 knockdown.Method1.After constructing the mut-type 293T cell lines,Dual-luciferase reporter assays were applied to detected the binding relationship between circRNA 100876 and miR-136.2.RT-qPCR assay was used to detect the expression of miR-136 in MG-63 and HOS osteosarcoma cell lines after depleting the expression of circRNA₁00876.3.MiR-136-specific inhibitors were applied to knockdown the expression of miR-136 in in MG-63 and HOS osteosarcoma cell lines after depleting the expression of circRNA₁00876.Meanwhile,CCK-8 assay was used to detect the changes of proliferation capacities.Result1.To further explore the association between circRNA₁00876 and miR-136,the luciferase reporter assay was employed and it indicated that the luciferase activity was significantly lower in wild-type cells treated with miR-136 inhibitors.2.RT-qPCR results showed that miR-136 was overexpressed in circRNA 100876 knocked down MG-63 and HOS osteosarcoma cell lines?P<0.001?.3.The results of CCK-8 showed that the silencing of circRNA₁00876 inhibited the cell growth,but this anti-cell proliferation effect could be reversed by miR-136 inhibitors?P<0.001?.ConclusionsCircRNA₁00876 may directly bind miR-136 in osteosarcoma and inhibit the expression of downstream miR-136,thereby promoting the proliferation of osteosarcoma cells.