| Objective(s):Osteosarcoma(OS)is a malignant tumor occurring in the bone,which progresses quickly and is easy to metastasize.Chemotherapy is the most important adjuvant therapy,but the resistance of patients to chemotherapy drugs is the main reason leading to poor clinical therapeutic effect.At present,the specific mechanism of drug resistance in osteosarcoma has not been clarified.Therefore,exploring the molecular mechanism of drug resistance in osteosarcoma can provide theoretical basis for clinical development of effective chemotherapy drugs.LncRNA is a class of long-stranded RNA without protein coding ability,which can regulate gene expression at multiple levels.There has been evidence that lnc RNA is involved in the regulation of the development and chemotherapy resistance of osteosarcoma.However,the mechanism of LncRNA PEG3-AS1 in osteosarcoma remains unclear.The purpose of this study was to investigate the effect of LncRNA PEG3-AS1 on the malignant biological behavior of osteosarcoma cells and the regulatory mechanism of cisplatin resistance via the miR-29a-3p/ANXA2 axis.Methods:1.Quantitative real-time PCR(qRT-PCR)was used to detect the expression level of LncRNA PEG3-AS1 in osteosarcoma parental cells U2-OS and drug-resistant cells U2-OS/CDDP;CCK-8 assay,scratch assay,Transwell assay and flow cytometry assay were used to detect the cisplatin resistance of parental cells U2-OS and drug-resistant cells U2-OS/CDDP,as well as cell proliferation,migration,invasion and apoptosis;After transfection of osteosarcoma resistant cells U2-OS/CDDP into si-PEG3-AS1,the cisplatin resistance of U2-OS/CDDP cells and the changes of cell proliferation,migration,invasion and apoptosis were further detected by CCK-8 assay,scratch assay,Transwell assay and flow cytometry.2.Dual luciferase reporter assay and qRT-PCR assay verified the targeting binding relationship between LncRNA PEG3-AS1 and miR-29a-3p;After transfection of si-PEG3-AS1+miR-29a-3p-inhibitor into osteosarcoma resistant cells U2-OS/CDDP,CCK-8 assay,scratch assay,Transwell assay and flow cytometry assay were used to detect the cisplatin resistance of U2-OS/CDDP cells and the cell proliferation,migration,invasion and apoptosis levels.3.Dual luciferase reporter assay,qRT-PCR assay and western blotting assay verified the targeting binding relationship between miR-29a-3p and ANXA2;After co-transfection of si-PEG3-AS1+miR-29a-3p-inhibitor+si-ANXA2 with drug-resistant cells U2-OS/CDDP,cisplatin resistance,proliferation,migration,invasion and apoptosis of U2-OS/CDDP cells were detected by CCK-8 assay,scratch assay,Transwell assay and flow cytometry apoptosis assay.Results:1.qRT-PCR assay: Compared with parental cells U2-OS,the expression level of LncRNA PEG3-AS1 in U2-OS/CDDP was significantly increased in osteosarcoma resistant cells;CCK-8 assay,scratch assay,Transwell assay and flow cytometry assay showed that U2-OS/CDDP had higher cisplatin resistance,higher proliferation,migration and invasion ability,and lower apoptosis level than parental cells;After transfection of si-PEG3-AS1 into osteosarcoma resistant cells U2-OS/CDDP,CCK-8experiment,scratch experiment,Transwell experiment,flow cytometry apoptosis experiment: Compared with the negative si RNA-transfected control group,the cisplatin resistance,proliferation,migration and invasion ability of U2-OS/CDDP cells in the si-PEG3-AS1 group were decreased,and the apoptosis rate was increased.2.Dual luciferase reporter gene assay and qRT-PCR assay showed that miR-29a-3p was the downstream target of LncRNA PEG3-AS1,and LncRNA PEG3-AS1 negatively regulated the expression of miR-29a-3p;After transfection of si-PEG3-AS1+miR-29a-3p-inhibitor into osteosarcoma resistant cells U2-OS/CDDP,CCK-8 assay,scratch assay,Transwell assay and flow cytometry apoptosis assay were performed: Compared with the transfection group of si-PEG3-AS1,transfection group of si-PEG3-AS1+miR-29a-3p-inhibitor U2-OS/CDDP cells showed increased cisplatin resistance,increased cell proliferation,migration and invasion,and decreased cell apoptosis.3.Dual luciferase reporter gene assay,qRT-PCR assay and protein western blotting assay: ANXA2 is the downstream target of miR-29a-3p,which negatively regulates the expression of ANXA2;After transfection of si-PEG3-AS1+miR-29a-3p-inhibitor+si-ANXA2 into osteosarcoma resistant cells U2-OS/CDDP,CCK-8 experiment,scratch experiment,Transwell experiment,flow cytometry apoptosis experiment: Compared with si-PEG3-AS1+miR-29a-3p-inhibitor group,U2-OS/CDDP cell resistance to cisplatin decreased in co-transfection group with si-PEG3-AS1+miR-29a-3p-inhibitor +si-ANXA2 group,the proliferation,migration and invasion of cells were weakened,while the apoptosis level was increased.Conclusion(s):1.Compared with osteosarcoma parental cells U2-OS,LncRNA PEG3-AS1 expression in U2-OS/CDDP was significantly up-regulated.2.LncRNA PEG3-AS1 increased cisplatin resistance of osteosarcoma cells U2-OS,promoted proliferation,migration and invasion of osteosarcoma resistant cells U2-OS/CDDP,and inhibited apoptosis of drug-resistant cells.3.LncRNA PEG3-AS1 regulates cisplatin resistance and malignant behavior of osteosarcoma cells by negatively regulating the expression of miR-29a-3p.4.LncRNA PEG3-AS1 promoted cisplatin resistance of osteosarcoma cells through the miR-29a-3p/ANXA2 axis,and aggravated proliferation,migration,invasion,and apoptosis of osteosarcoma resistant cells U2-OS/CDDP.. |
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