| Background:As one of the most threatening malignant tumors to human health and life,lung cancer has received much attention in recent years.With the progress of science and technology and the development of medical technology,lung cancer’s incidence and mortality in many developed countries has been declining,but the overall incidence and mortality of lung cancer in many developing countries,including China,is still not optimistic,and even increased.Among many categories of lung cancer,lung adenocarcinoma,as the most common cancer with high incidence,has gradually become the focus of research.Although the current clinical treatment methods include radiotherapy,chemotherapy,surgery and gene targeted therapy,and good results have been achieved,the overall prognosis of patients with lung adenocarcinoma is poor,and the long-term survival rate is low.Therefore,it is urgent for us to further explore the pathogenesis of lung adenocarcinoma and seek new diagnostic markers and therapeutic targets.AHNAK(AHNAK nucleoprotein)is a family of giant cellular scaffold proteins,widely expressed in cardiomyocytes and a variety of tissue cells.It is related to cell structure and migration,cardiac calcium channel signal transduction and other processes,and participates in physiological and pathological activities such as fat metabolism,cell membrane repair and tumor migration.The AHNAK2 gene is a member of the AHNAK family and has significant homology and the same triad protein structure with AHNAK,so it is highly likely to have similar functions to AHNAK.Current studies have confirmed that AHNAK2 gene is a potential new oncogene in some cancers,but the role of AHNAK2 gene in lung cancer is limited.In this study,Oncomine database analysis showed that AHNAK2 gene was abnormally high in lung adenocarcinoma tissues.Therefore,this study took AHNAK2 gene as the research target to explore the role and mechanism of AHNAK2 in migration,invasion and epithelial-mesenchymal transformation in lung adenocarcinoma cells,in order to find new targets and provide theoretical and experimental basis for the diagnosis and treatment of lung adenocarcinoma.Objective:First,the Oncomine database was used to analyze the expression differences of AHNAK2 in normal lung tissues,lung adenocarcinoma tissues and paracanceroustissues,and the expression of the gene was verified in related cell lines.Secondly,the effect of TGF-β1 on cell migration and invasion ability with different expression levels of AHNAK2 was observed by cell transfection technique,and the effect of AHNAK2 on the biological function of lung adenocarcinoma cells was explored.Finally,the effect of AHNAK2 on epithelial mesenchymal transformation of lung adenocarcinoma cells was studied in lung adenocarcinoma lines to explore the possible carcinogenic mechanism.Materials and methods:1.Oncomine database was used to analyze the expression of AHNAK2 in normal lung tissues,lung adenocarcinoma tissues and paracancerous tissues.The expression of the gene in human lung adenocarcinoma cell lines A549 and H1299 in vitro was further verified by qPCR and Westernblot.2.Si-AHNAK2 and si-NC were transfected into A549 and H1299 cell lines with lipoposomes to construct low-expression and normal-expression cells of AHNAK2.The effects of AHNAK2 on proliferation,invasion and migration of lung adenocarcinoma cells were detected by cell cloning,Transwell assay and cell scratch assay.The expression of EMT markers and the levels of phosphorylated Smad3(pSmad3)and Smad3 were detected by Westernblot to evaluate the effect of AHNAK2 on epithelial mesenchymal transformation of lung adenocarcinoma cells.3.Smad3 phosphorylation inhibitors were added to human lung adenocarcinoma cell lines A549 and H1299.Cell migration and invasion ability were detected by cell scratch assay and cell invasion assay.Westernblot was used to detect the expression of EMT markers in si-NC and si-AHNAK2 transfected cells and cells with or without TGF-β1,to further verify the role of AHNAK2 in lung adenocarcinoma.Results:1.AHNAK2 geneexpression was significantly up-regulated in lung adenocarcinoma tissue samples.The expression in human lung adenocarcinoma cell line A549 and H1299 was significantly higher than that in normal lung epithelial cell line BEAS-2b.2.When cells were stimulated by TGF-β1,AHNAK2 gene knockout inhibited cell migration,invasion and epithelial mesenchymal transformation,and inhibited TGF-β-induced Smad3 signal transduction.3.When p-Smad3 was inhibited,AHNAK2 gene knockout had no effect on the two cell lines with or without TGF-β1.ConclusionAHNAK2 is an oncogenic gene that is upregulated in lung adenocarcinoma.AHNAK2 genepromotes the migration,invasion and epithelial-mesenchymal transformation of lung adenocarcinoma cells mainly through TGF-β/Smad3 pathway.AHNAK2 gene knockout inhibited cell migration,invasion and epithelial mesenchymal transformation,and inhibited TGF-β-induced Smad3 signal transduction.Therefore,AHNAK2 may become a new target and prognostic marker for the treatment of lung adenocarcinoma. |
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