Effects of cisplatin resistant osteosarcoma cell derived exosomes on proliferation and migration of osteosarcoma cells through delivery of miR-21-5p

SUN Chao; MENG Chen-yang; ZHAO Jia-li; DOU Rui; FENG Wei; GUO Shi-bing

doi:10.3969/j.issn.2095-252X.2025.02.006

Abstract：Objective To verify the effect of miR-21-5p and its target genes on drug resistance of osteosarcoma cells transfected with miR-21-5p and PDCD4,respectively.Tumor-bearing experiments were carried out to verify the changes of serum exosomes miR-21-5p and the expression of related genes in tumor tissues,and to explore the relationship between exosomes miR-21-5p and target genes in drug-resistant osteosarcoma cells and its possible mechanism of action.Methods(1)Proliferation and migration experiments were performed to assess the proliferation and migration levels of osteosarcoma cells by co-culturing osteosarcoma cells with exosomes from cisplatin-resistant cells overexpressing or knocked down with miR-21-5p.Western Blot and qRT-PCR were used to detect the expression of PDCD4,MDR1,LC3,Beclin1,Atg5 protein and miRNA.(2)Target genes of miR-21-5p were screened and validated by TargetScan online tool.The differential expression of PDCD4 mRNA in U2OS and its drug-resistant cell-derived exosomes were detected by qRT-PCR.The effect of PDCD4 on cell proliferation and migration was detected by cell proliferation and migration assay.The protein and miRNA expression levels of PDCD4,MDR1,LC3,Beclin1,Atg5 were detected by Western Blot and qRT-PCR Assay.(3)To observe the growth of tumor in nude mice,analyze the expression of CD81 and CD63 protein in serum exosomes,and detect the expression of miR-21-5p and PDCD4 protein and mRNA in serum exosomes by Western Blot and qRT-PCR.Immunohistochemistry was used to detect the expression of related genes in tumor tissues of nude mice.Results(1)miR-21-5p overexpression or knockdown vector-transfected cisplatin-resistant cell differential expression system results indicated that the cell proliferation rate and migration rate of the U2OS+miR-21-5p mimics/EXO group were significantly increased compared with the U2OS+Exo Group(P＜0.0001,P＜0.01).The protein and mRNA expression levels of PDCD4 were significantly decreased(P＜0.05 and P＜0.01,respectively).The protein and mRNA expression levels of LC3(LC3 Ⅱ/LC3 Ⅰ),MDR1,Beclin1 and Atg5 were significantly increased(P＜0.01 and P＜0.001,respectively).(2)PDCD4 was predicted to be a potential target gene of miR-21-5p by TargetScan online tool,and there was a binding site of miR-21-5p in the 3'UTR of its mRNA.The differential expression experiments of U2OS/DDP cells transfected with PDCD4 over-expression or knockdown vector showed that the cell proliferation rate and migration rate were significantly decreased in PDCD4 over-expression group compared with the control group(P＜0.01,P＜0.001),while the cell proliferation rate and migration rate were significantly increased in the miR-21-5p mimics plus PDCD4 over-expression group(P＜0.05,P＜0.01).The protein and mRNA expression levels of PDCD4,LC3(LC3 Ⅱ/LC3 Ⅰ),MDR1,Beclin1,Atg5 in PDCD4 over-expression group were significantly higher than those in control group(P＜0.001).The protein expression of Beclin1,the mRNA expression levels of miR-21-5p,MDR1 and Beclin1 were significantly increased in the group of miR-21-5p mimics plus PDCD4 over-expression(P＜0.05,P＜0.0001,P＜0.001,P＜0.05).(3)Compared with the control group,the tumor volume of nude mice in the miR-21-5p mimics group proliferated faster and the final tumor weight increased significantly.The mRNA expression level of miR-21-5p in the serum exosomes of nude mice was significantly increased(P＜0.001),while that of PDCD4 was significantly decreased(P＜0.001).The mRNA expression levels of miR-21-5p in serum exosomes of nude mice in the inhibitor group,PDCD4 overexpression group,miR-21-5p mimics plus PDCD4 over-expression group were significantly decreased(P＜0.01,P＜0.001,P＜0.01),and the mRNA expression levels of PDCD4 were significantly increased(P＜0.01,P＜0.001,P＜0.01).Compared with the control group,the expression of PDCD4 in the tumor tissues of the miR-21-5p mimetic group was decreased,while the expression of LC3,MDR1,Beclin1 and Atg5 was increased.The expressions of PDCD4 in the tumor tissues of the inhibitor group,PDCD4 over-expression group,miR-21-5p mimetic plus PDCD4 over-expression group were increased,while the expressions of LC3,MDR1,Beclin1 and Atg5 were decreased.Conclusions(1)miR-21-5p can promote the proliferation and invasion of osteosarcoma cells;(2)PDCD4 is the target gene of miR-21-5p;(3)miR-21-5p could target the expression of PDCD4 and promote the expression of MDR1 and autophagy related factors;(4)Targeting PDCD4 by miR-21-5p regulates proliferation,invasion and drug resistance of cisplatin resistant cells in osteosarcoma.