ANKRD49 Promotes The Invasion And Metastasis Of Lung Adenocarcinoma Via A P38/ATF-2 Signaling Pathway

Objective:Lung adenocarcinoma(LUAD)accounts for over 40% of lung cancers,and it is is the most difficult cancer to treat.Ankyrin repeat domain 49 protein(ANKRD49)is highly expressed in several carcinomas,however,its pattern of expression and role in LUAD are not known.Methods:In order to study the role of ANKRD49 in the development of LUAD,we constructed stable A549 cells with LUAD A549 cell line,which could overexpress ANKRD49(ANKRD49 OE)or knock down ANKRD49(ANKRD49 KD)by lentivirus transfection.Using these cells,the effects of ANKRD49 on proliferation,invasion and migration of lung cancer cells in vitro were investigated by CCK-8,clonal formation,wound healing and transwell assay.We also identified the potential role of ANKRD49 in the development of LUAD in mice by injecting these ANKRD49 overexpressed A549 cells into nude mice.The expression of MMP-2/MMP-9 was detected by gelatinase spectrometry,Western blot and immunohistochemistry.Western blot and/or immunohistochemical analysis were performed to verify the total expression of MAPK pathway proteins and transcription factor ATF-2 and its expression level in cytoplasmic nucleus.Western blot was used to analyze the effect of ATF-2 knockdown in A549 of overexpress ANKRD49 on MMP-2/MMP-9.Results:A549 cells with overexpression and knockdown of ANKRD49 were successfully constructed.CCK-8 and clone formation experiments showed that ANKRD49 had no effect on the proliferation of A549 cells in vitro.Wound healing and transwell assay showed that ANKRD49 could promote the invasion and migration of A549 cells in vitro.In nude mice,ANKRD49 promoted lung metastasis and invasion of A549 cells.The results of real-time q PCR,Western blotting,immunohistochemistry,and gelatinase spectrum showed that ANKRD49 promoted the expression and enzyme activity of MMP-2/9 in A549 cells.After using MMPs inhibitor,the migration ability of A549 cells overexpressing ANKRD49 was decreased.Western blot and immunohistochemistry results showed that after ANKRD49 was overexpressed,the phosphorylation level of P38 and ATF-2 increased.Western blot results of cytoplasm and nucleoprotein showed that after overexpression of ANKRD49,the expression of ATF-2 increased.After knocking down ATF-2 in A549 cells of overexpressing ANKRD49,the expression of MMP-2/MMP-9 decreased.Conclusion:Our findings suggest that ANKRD49 is a latent biomarker for evaluating LUAD prognosis,and promotes the metastasis of A549 cells via upregulation of MMP-2 and MMP-9 in a P38 /ATF-2 pathway-dependent manner.