| Objective:Lung cancer is the leading cause of cancer-related death worldwide.More than 4/5 of lung cancer cases are Non-small cell lung cancer(NSCLC).The 5-year survival rate of patients with metastatic lung cancer is only 19.7%.Therefore,there is an urgent need to discover and study new and sensitive biomarkers for the detection of early NSCLC and develop new therapeutic targets.Previous studies showed that Ankyrin repeat domain 49(ANKRD49)gene promoted the migration and invasion of lung adenocarcarcinoma cell A549 through p38 MAPK/ATF2-MMP-2/9 signaling pathway.However,ANKRD49 promotes migration and invasion in lung adenocarcinoma cell H1299 through JNKATF2/c-Jun-MMP-2/9,different cells show different molecular mechanisms.Therefore,this study intends to further study the effect of ANKRD49 on NSCLC migration and invasion and its molecular mechanism by using ANKRD49 overexpressed and knockdown lung cancer cell lines.Methods:Lentivirus infection combined with antibiotic screening was used to construct lung adenocarcinoma cell lines carrying ANKRD49 overexpression H1299.Lentivirus infection combined with antibiotic screening were used to construct lung adenocarcinoma cell lines carrying ANKRD49 knockdown H1299 and lung squamous cell lines carrying H1703.The expression level of ANKRD49 was detected by Real-time PCR and Western blot.The effects of ANKRD49 on cell proliferation were detected by cck-8 and cell clonal formation assay.The effects of ANKRD49 on cell migration and invasion were detected by cell scratch assay,transwell migration and invasion assay.Real-time PCR and Western blot were used to detect the m RNA and protein expressions of matrix metalloproteinase 2/9(MMP-2/-9)and EMT(Epithelial-mesenchymal transformation)related markers in the control group and the experimental group,respectively.The enzyme activity level of MMP-2/-9 was detected by gelatin enzyme assay.Western blot was used to detect the expression of related molecules in MAPK signaling pathway in H1299 and H1703 cell lines.H1299 cells with stable ANKRD49 overexpression and H1703 cells with stable ANKRD49 knockdown were injected into the tail vein of BALB/c nude mice to observe the lung metastasis of non-small cell lung cancer cells in mice.Results:RT-q PCR and Western blot results showed that H1299 and H1703 cells with stable knockdown of ANKRD49 were successfully constructed.Cell scratch and Transwell assay showed that ANKRD49 knockdown inhibited the migration and invasion of lung adenocarcinoma and lung squamous cell carcinoma.RT-q PCR,Western blot and gelatin enzyme assay showed that ANKRD49 knock-down significantly inhibited the m RNA,protein expression and enzyme activity of MMP-2/-9,and significantly increased the m RNA and protein level of TIMP-1/-2.Western blot results showed that the total protein levels of JNK and P38 did not change after ANKRD49 knockdown,but the phosphorylation levels were significantly decreased,and the phosphorylation levels of downstream transcription factors ATF2 and c-jun were also significantly decreased.The protein level of MMP-2/-9 was recovered by adding a JNK activator(Anisomycin)in H1703 cells,while the expression of MMP-2/-9 protein was not changed by P38 activation.Western blot results showed that overexpression of ANKRD49 promoted the expression of TGF-β1,Vimentin,α-SMA,Snail-1 and Slug,and decreased the expression of E-cadherin in H1299 cells,while the above proteins showed an opposite trend in knockdown ANKRD49 cells.The results of nude mice showed that compared with the negative control group,metastatic nodules on the lung surface of mice in the ANKRD49 overexpression group were significantly increased,while nodules on the lung surface of mice in the knockdown group were significantly decreased.HE results showed that ANKRD49 overexpression significantly enhanced the migration and invasion ability of H1299 cells,while ANKRD49 knockdown inhibited the migration and invasion of H1703 cells.Immunohistochemical results showed that H1299 cells overexpressing ANKRD49 expressed more Napsin A,NKX2-1,MMP-2,MMP-9,p-cjun,p-ATF2 and p-JNK in mouse lung tissue.However,H1703 cells with ANKRD49 knockdown showed less p63,MMP-2,MMP-9,p-cjun,pATF2 and p-JNK in mouse lung tissue.Conclusion:ANKRD49 regulates the expression of MMP-2/-9 in H1299 and H1703 cells through the JNK-c-Jun/ATF2 signaling pathway to promote the migration and invasion of lung cells.ANKRD49 may also increase migration and invasion of non-small cell lung cancer by promoting EMT. |
