The MiR-34a-5p Promotes The Multi-chemoresistance Of Osteosarcoma Via Repression Of The AGTR1 Gene

BackgroundChemotherapy is one of main treatments of cancers,while chemoresistance is one of the main reasons for treatment failure in chemotherapy.MicroRNAs(miRNAs)are key players in diverse biological processes including the chemoresistance of cancers.ObjectiveTo investigate the role of mir-34a-5p and AGTR1 in chemoresistance of osteosarcoma cells.Methods:1.Based on RNA-Seq and miR-omic genomic analysis of osteosarcoma cells,the target genes that could be regulated by miR-34a-5p was predicted by miRBase,Targetscan and CHIPBase.2.The expression levels of mir-34a-5p and AGTR1 in chemosensitive G-292 and chemoresistant SJSA-1 cells were detected by qRT-PCR and western blot.3.Double luciferase reporter assay system was used to show whether mir-34a-5p could bind to the 3'UTR sequence of AGTR to confirm whether AGTR1 was a target gene of mir-34a-5p.4.Mimic of mir-34a-5p(si-AGTR1)was transfected into G-292 cells and antagomir,of mir-34a-5p(GFP-AGTR1)was transfected in SJSA-1 cells.The expression level of AGTR1 in the transfected cells was checked by qRT-PCR analysis.5.The mechanism of mir-34a-5p and AGTR1 in the chemoresistance of osteosarcoma cells was confirmed by chemotherapy sensitivity test(MTT),apoptosis,cell proliferation and nude mice transplanted osteosarcoma xenograft model.Results:1.RNA-Seq and miR-omic data showed that the expression level of mir-34a-5p in the resistant cell line SJSA-1 was significantly higher than that in the sensitive cell line G-292,and the expression level of AGTR1 is just the opposite,it is noted that the angiotensin ?type 1 receptor(AGTR1)may be one of the mir-34a-5p target genes.2.The expression level of mir-34a-5p in SJSA-1 was significantly higher than that in G-292 cells,while the expression level of AGTR1 was opposite,expression level was significantly lower in SJSA-1 than that in sensitive cell line G-292 cells.3.The results of double luciferase reporter system showed that mir-34a-5p could bind to the 3'UTR sequence of AGTR and decrease the intracellular fluorescence activity.It was proved that AGTR1 was the target gene of mir-34a-5p.4.The AGTR1 mRNA and protein expression level in G-292 cells transfected with mir-34a-5p mimic(si-AGTR1)was down-regulated.The AGTR1 mRNA and protein expression level in SJSA-1 cells transfected with mir-34a-5p antagomir(GFP-AGTR1)was up-regulated.5.The results of MTT showed that the mimic of mir-34a-5p could increase the chemoresistance of osteosarcoma cells,while the antagomir of mir-34a-5p could reduce the chemoresistance.The results of MTT by si-AGTR1 interference was consistent with the mimic results of mir-34a-5p,which could improve the chemosensitivity of osteosarcoma cells.The results of GFP-AGTR1 interference were consistent with the antagomir of mir-34a-5p,which could reduce the sensitivity of osteosarcoma cells to chemotherapy.These results were consistent with those in cell apoptotic experiments and nude mice xenograft model.Conclusionsmir-34a-5p regulates chemoresistance of osteosarcoma cells by targeting AGTR1 gene.It is suggested that mir-34a-5p may be used as a biomarker in chemotherapy of osteosarcoma patients.