Preliminary Study Of ANKRD49 Overexpression In Promoting Metastasis And Invasion Of Non-small Cell Lung Cancer

Objective:Lung adenocarcinoma(LUAD)accounts for more than 40% of lung cancers and is the most challenging tumor in clinical practice.Identifying new proteins that play a role in the development of LUAD will help elucidate pathogenesis and provide new prognostic biomarkers.The research group constructed the lentiviral vector LV5-ANKRD49 with high expression of ANKRD49 in the early stage.After infecting H1299 cells,it was found that it does not participate in regulating cell proliferation,but can promote the migration and invasion of H1299 cells.In order to further study the role of ANKRD49 in migration and invasion,Real-time q PCR and Western blotting were used to detect the expression of migration and invasion-related proteins to further explore the mechanism of stable overexpression of ANKRD49 affecting the migration and invasion ability of NCI-H1299 cells.The next step is to study the function of ANKRD49 in NSCLC to lay the foundation.Methods:Analyze ankyrin repeat domain 49(ANKRD49)protein in LUAD and paired nontumor lung tissues by tissue microarray and immunohistochemistry technology;Analysis the correlation between the expression of ANKRD49 and the clinical pathological characteristics of patients by χ2 test and Spearman correlation;The association between ANKRD49 and patient survival rate was estimated by Kaplan-Meier method and log-rank test;Evaluate the prognostic value of ANKRD49 by Cox proportional hazards model;Detect the m RNA and Protein levels of MMP-2/9 and TIMP-1/2 in the NCI-H1299 cell line overexpressing ANKRD49 by Real-time q PCR and Western blotting;Detect MMP-2/9enzyme activity in ANKRD49 overexpressing NCI-H1299 cell line by gelatin zymography test;Detect the expression of related proteins in the SAPK/JNK signaling pathway in the ANKRD49 overexpression NCI-H1299 cell line by Western blotting;Study the nuclear translocation of related transcription factors by extract cytoplasm and nuclear proteins.Results:The increase of ANKRD49 in LUAD is related to the stage of tumor lymph node metastasis,lymph node metastasis,distant metastasis and differentiation.The survival rate of patients with high-level ANKRD49 is significantly lower than that of patients with lowlevel ANKRD49.Cox(Cox)model shows that higher ANKRD49 in lung tissue can be used as an independent prognostic indicator for LUAD patients.Real-time q PCR and Western blotting results showed that compared with the control group,the m RNA level and protein level of MMP-2/9 in the NCI-H1299 cells in the ANKRD49 high expression group were significantly increased,while the m RNA level of TIMP-1/2 And protein levels have dropped significantly.Gelatin zymography experiment results show that high expression of ANKRD49 can promote the enzymatic activity of MMP-2/9.Western blotting results showed that after ANKRD49 overexpression,the total protein levels of JNK,ERK,and P38 did not change significantly,but the phosphorylation levels of JNK and P38 were significantly increased.The results of cytoplasm and nucleus extraction showed that the nuclear translocation of transcription factors ATF2 and c-jun increased after ANKRD49 was overexpressed.Conclusion:Our results show that ANKRD49 can be used as a potential biomarker for evaluating the prognosis of LUAD,and as a potential therapeutic target for LUAD treatment.The ANKRD49 high expression group can increase the expression of MMP-2/9 by activating the NF-κB signaling pathway to promote the migration and invasion of NCI-H1299 cells.The ANKRD49 high expression group can activate the SAPK/JNK signaling pathway,and the transcription factor ATF2 can regulate MMP-2/9 to promote the migration and invasion of NCI-H1299 cells.