| Objective: 1)miR-21-5p was transfected into cisplatin-resistant osteosarcoma cells(U2OS/DDP)to detect the effect of exosomal derived miR-21-5p on osteosarcoma resistance.2)The target genes of miR-21-5p action in osteosarcoma cells were screened and verified,and the regulatory effect of exosomal miR-21-5p target genes on drug resistance in osteosarcoma was detected after transfection into U2OS/DDP.3)The changes of animal serum exosomes miR-21-5p and the expression of related genes in tumor tissues were verified by tumor carrying experiments,and the correlation between exosomes miR-21-5p and target genes in osteosarcoma resistant cells and its possible mechanism of action were explored.Methods: 1)The overexpression vector and knockdown vector of miR-21-5p were transfected into U2OS/DDP,and the effect of miR-21-5p on drug resistance of osteosarcoma cells was detected by cell proliferation and migration experiments.The protein and miRNA expression levels of PDCD4,MDR1,LC3,Beclin1 and Atg5 were detected by Western Blot and q RT-PCR.2)The target genes of miR-21-5p in osteosarcoma cells were screened and verified by Target Scan online tool.m RNA differential expression of programmed cell death 4(PDCD4)in cytogenic exosomes of U2 OS and U2OS/DDP groups was verified by q RT-PCR.PDCD4 overexpression vector and knockdown vector were transfected into U2OS/DDP,and the effect of PDCD4 on drug resistance of osteosarcoma cells was detected by cell proliferation and migration experiments.The protein and miRNA expression levels of PDCD4,MDR1,LC3,Beclin1,Atg5 were detected by Western Blot and q RT-PCR.3)The tumor growth of nude mice with various tumor components was observed,and the particle size of serum exosomes and the protein expressions of CD81 and CD63 were analyzed.The protein and m RNA expression levels of miR-21-5p and PDCD4 in serum exosomes of nude mice were detected by Western Blot and q RT-PCR.Immunohistochemistry was used to detect the expression of related genes in tumor tissue of nude mice.Results: 1)miR-21-5p was transfected into U2OS/DDP through overexpression and knockdown vector,and the transfection efficiency was verified successfully to establish the differential expression system of miR-21-5p.The results showed that compared with U2OS+Exo group,cell proliferation rate and mobility in U2OS+miR-21-5p mimics /Exo group were significantly increased(P<0.0001,P﹤0.01),the protein and m RNA expression levels of PDCD4 were significantly decreased(P<0.05 and P<0.01,respectively).The protein and m RNA expression levels of LC3(LC3 II/ LC3i),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 Target Scan online tool,and there was a binding site of miR-21-5p in the 3’utr of PDCD4 m RNA;PDCD4 overexpression and knockdown vector were transfected into U2OS/DDP cells,and the established differential expression experiment of PDCD4 cells showed that the proliferation rate and mobility of cells in the PDCD4 overexpression group were significantly decreased(P<0.01,P<0.001),the cell proliferation rate and cell mobility of miR-21-5p mimics plus PDCD4 overexpression group were significantly increased(P<0.05,P<0.01).Compared with the control group,the protein expression level of PDCD4 in PDCD4 overexpression group was significantly increased(P<0.001),while the protein expression levels of LC3(LC3II/LC3I),MDR1,Beclin1 and Atg5 were significantly decreased(P<0.01).The Beclin1 protein expression level of miR-21-5p mimics PDCD4 overexpression group was significantly decreased(P<0.05);m RNA expression levels of LC3,MDR1,Beclin1 and Atg5 in PDCD4 overexpression group were significantly decreased(P<0.01).The m RNA expression levels of miR-21-5p,MDR1 and Beclin1 in the overexpression group of miR-21-5p mimics PDCD4 were significantly increased(P<0.0001,P<0.001,P<0.05).3)The results of tumor formation in nude mice showed that after drug intervention,compared with the control group,the tumor volume proliferation rate of nude mice in the miR-21-5p mimics group was significantly accelerated,and the terminal tumor weight was significantly increased.Compared with the control group,m RNA expression level of miR-21-5p in serum exosomes of nude mice in miR-21-5p mimics group was significantly increased(P<0.001),and m RNA expression level of PDCD4 was significantly decreased(P<0.001);The m RNA expression level of miR-21-5p in serum exosomes of nude mice in inhibitor group,PDCD4 overexpression group,miR-21-5p mimics plus PDCD4 overexpression group was significantly decreased(P<0.01,P<0.001,P<0.01).The m RNA expression levels of PDCD4 were significantly increased(P<0.01,P<0.001,P<0.01).Immunohistochemical results showed that compared with the control group,the positive expression of(1)PDCD4 in tumor tissues of nude mice in the miR-21-5p mimics group was decreased.The positive expressions of LC3,MDR1,Beclin1 and Atg5 increased.(2)The positive expression of PDCD4 in tumor tissues increased in inhibitor group,PDCD4 overexpression group,miR-21-5p mimics plus PDCD4 overexpression group.The positive 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. |
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