The Preliminary Study Of ANKRD49 On Promoting Metastasis And Invasion Of A549 And NCI-H1299 Cells

Objective:Previous studies on tissue chips in NSCLC patients showed that the expression of ANKRD49 in cancer tissues was higher than that in para-cancer normal tissues,and its high expression was found in poorly differentiated and highly metastatic cancer tissues.The recombination eukaryotic expression vector of p3 × Flag-CMV-14/ANKRD49 was successfully constructed and then was transfected into A549 cells.The results showed that the proliferation of A549 cells transfected with ANKRD49 was not statistically different from that of the control group.Therefore,we speculate that the function of ANKRD49 maybe involved in regulating cell metastasis and invasion.To further study the function of ANKRD49 in NSCLC,the ANKRD49-lentiviral recombinant plasmid was constructed and then infected into A549 and NCI-H1299 cells.The function of ANKRD49 on cell metastasis and invasion in A549 and NCI-H1299 cells was studied and the underlying mechanism was preliminary expored.NSCLC cell lines,exploring the related mechanism of ANKRD49 to promote metastasis and invasion,and providing new ideas for in-depth study of NSCLC invasion and metastasis.The present study paves the pathway for further research the function of ANKRD49 in NSCLC.Methods:1.Construction of a lentiviral vector LV5-ANKRD49 that highly expresses ANKRD49;2.Construction of ANKRD49 stably overexpressed A549 and NCI-H1299 cells by transfecting the ANKRD49-lentiviral vector;3.The expression of ankrd49 mRNA and ANKRD49 protein in ANKRD49 stably overexpressed A549 and NCI-H1299 cells were tested using RT-PCR and Western blotting;4.CCK-8 assay was used to detect whether ANKRD49 high expression has effect on the proliferation of A549 and NCI-H1299 cells;5.The effect of ANKRD49 overexpression on the migration ability of A549 and NCI-H1299 cells was detected via cell scratch test and transwell migration test;6.The effect of ANKRD49 overexpression on the invasion ability of A549 and NCI-H1299 cells was investigated through transwell invasion assay;7.Western blotting was used to detect the effect of ANKRD49 overexpression on the expression of MMP-2/9 in A549 and NCI-H1299 cells.Results:1.RT-PCR and Western blotting results showed that ANKRD49 was highly expressed in ANKRD49-lentiviral vector transfected A549 and NCI-H1299 cells,and the A549 and NCI-H1299 cell models which stably overexpressed ANKRD49 were successfully constructed;2.CCK-8 assay showed that overexpressed ANKRD49 did not affect the proliferation of A549 and NCI-H1299 cells;3.The results of cell scratch test and transwell migration experiment showed that high expression of ANKRD49 could promote the migration ability of A549 and NCI-H1299 cells;4.Transwell invasion experiment results showed that high expression of ANKRD49 could promote the invasion ability of A549 and NCI-H1299 cells;5.Western blotting test results showed that compared with the naked cell group and the empty vector group,the expression of MMP-2/9 in A549 and NCI-H1299 cells with high expression of ANKRD49 was significantly increased.Conclusion:1.A549 and NCI-H1299 cell models with stable and high expression of ANKRD49 were successfully constructed;2.High expression of ANKRD49 does not affect the proliferation of A549 and NCI-H1299 cells;3.ANKRD49 overexpression may promote the migration and invasion of A549 and NCI-H1299 cells through increasing MMP-2/9 expression.