Timosaponin A-Ⅲ Suppressing NSCLC Growth Through Regulation Of The ASAP1-IT1/DNMT3b/YAP1 Axis

Objective: To explore the effect and mechanism of Timosaponin A-Ⅲ(TAⅢ)on NSCLC growth.Methods: A549 cells was processed with different concentrations of TAIII(5,10,15,20,25,30 μmol/L),the cell viability was measured by CCK-8.q RT-PCR was used to detect the expressions of Adenosine diphosphate ribosylation factor guanylate kinase1-Intronic Transcript 1(ASAP1-IT1)and Yes-associated Protein 1(YAP1)in tissues and cells.EDU,flow cytometry and Western blot were used to detect ASAP1-IT1,DNA(cytosine-5-)-methyltransferase 3β(DNMT3b),YAP1 and their effects on cell proliferation,apoptosis rate and the influence of apoptosis related genes(Bax and caspase-3)and anti-apoptotic gene Bcl2.Bisulfite sequencing PCR(BSP)was used to analysis the relationship between ASAP1-IT1 and YAP1.RNA immunoprecitation(RIP)and q RT-PCR assays were used to analyze the interaction between ASAP1-IT1 and DNMT3 b.Co-Immunoprecipitation(Co-IP)was used to confirm the interaction between YAP1 and DNMT3 b.q RT-PCR method was used to analyze the effect of TAⅢon the expression of ASAP1-IT1/DNMT3b/YAP1 axis-related genes.Results:(1)ASAP1-IT1 and YAP1 was up-regulated in NSCLC tissues and cancer cells,and DNMT3 b was down-regulated in NSCLC tissues and cancer cells,the levels of ASAP1-IT1,YAP1 and DNMT3 b were reversed after using TAⅢ.(2)Silencing of ASAP1-IT1 and YAP1,overexpression of DNMT3 b or taking TAⅢ significantly increased apoptosis rate of A549 cells and apoptosis related indexes.(3)TAⅢ reversed the silencing or overexpression of ASAP1-IT1,DNMT3 b and YAP1.(4)Furthermore,ASAP1-IT1 was found to interact with DNMT3 b,and DNMT3 b and YAP1 were interacted.Conclusion: ASAP1-IT1/DNMT3b/YAP1 axis promotes NSCLC progression by regulating cell proliferation and apoptosis.TAⅢ suppresses proliferation and improve apoptosis of lung cancer cells by regulating ASAP1-IT1/DNMT3b/ YAP1 axis.