Methyltransferase like 3 N6-methyladenosine modified circSAFB2 regulates invasion and apoptosis of osteosarcoma cells
Objective To observe the effect of methyltransferase like 3(METTL3)N6-methylade-nosine(m6A)modified circSAFB2 on invasion and apoptosis of osteosarcoma cells.Methods The ex-pression levels of METTL3 mRNA and circSAFB2 in osteosarcoma tissues and MG63 cells were detected by qRT-PCR from 16 cases of osteosarcoma and MG63 cell line.The m6A methylation site in circSAFB2 was predicted by SRAMP prediction server online prediction software,and the m6A methylation modification of circSAFB2 in osteosarcoma tissues and MG63 cells was verified by m6A MeRIP kit.The methylation level of circSAFB2 m6A in osteosarcoma tissues and MG63 cells was detected by MeRIP-qPCR.Osteosarcoma cells were infected with METTL3 siRNA recombinant lentivirus and METTL3 NC recombinant lentivirus,and the RNA stability,invasion ability and apoptosis of cells in each group were detected by actinomycin D,Transwell and flow cytometry,respectively.T-test was used for comparison between two samples and two groups.One-way analysis of variance was used for comparison among groups,and LSD method was used for comparison among groups.P＜0.05 was considered statistically significant.Results The results of qRT-PCR showed that the expression levels of METTL3 mRNA(2.354±0.224)and circSAFB2(2.072±0.264)in osteosarcoma tissues were significantly increased as compared with those in adjacent tissues(1.000±0.089,1.000±0.104,P＜0.05).Compared with normal osteoblasts(1.000±0.100,1.000±0.136),mRNA expression levels of METTL3(2.429±0.211)and circSAFB2(2.271±0.214)in MG63 cells were significantly increased(P＜0.05).SRAMP online m6A methylation site prediction re-sults showed that the m6A methylation modification sites of circSAFB2 existed at 115 and 234 sites,and verified the presence of M6A methylation modification sites in osteosarcoma tissues and MG63 cells.The circSAFB2 abundance of m6A antibody immunocoprecipitation(18.397±1.627,23.338±2.225)was significantly higher than that of IgG antibody(1.000±0.130,1.000±0.060,P＜0.05),indicating the existence of m6A methylation modification on circSAFB2.MeRIP-qPCR results showed that the methylation level of circSAFB2 m6A RNA in osteosarcoma tissues(3.264±0.217)was significantly higher than that in adjacent tissues(1.000±0.070,P＜0.05).Compared with normal osteoblasts(1.000±0.064),the methylation level of circSAFB2 m6A RNA in MG63 cells was significantly increased(3.379±0.205,P＜0.05).Compared with those transfected with METTL3 NC recombinant lentivirus(si-NC group),the ex-pression level of circSAFB2 in MG63 cells transfected with METTL3 siRNA recombinant lentivirus were significantly decreased(87.540±5.539,71.131±4.320;72.570±4.809,52.195±3.081)after treat-ment with actinomycin D for 6 h and 12 h(P＜0.05).The number of invasive cells(124.667±13.577,54.333±6.429)was significantly decreased and the apoptosis rate[(7.241±0.533)％,(25.105±1.638)％]was significantly increased(P＜0.05).Conclusion METTL3 regulate the invasion and apop-tosis of osteosarcoma cells through m6A modification of circSAFB2.
NETL
NSTL
万方数据
