The Study Of The Effects Of ADAM23Gene On Invasion And Metastasis Of Lung Adenocarcinoma Cell SPC-A-1and Its Mechanisms

Background:Lung cancer is one of the most common malignant tumors.Invasion and metastasis are the major causes for death of patients with lung cancer.Therefore,it is important for clinical therapy to further investigate the mechanism of lung cancer invasion and metastasis,look for new diagnostic markers.provide strong theoretical basis for the development of new treatment strategies.A Disintegrin And Metalloprotease23gene, a member of the ADAM protein family, is a transmembrane glycoprotein modulating cell-cell and cell-extracellular marix interactions.At present, it has been reported that ADAM23as a tumor suppressor gene.its downregulation has closely correlated with malignant tumors. Our previous studies have shown that ADAM23gene low expression has negative correlation to lung cancer invasion and metastasis duing to promoter methylation;ADAM23can promote the invasion and metastasis of lung cancer by negative regulation of αvβ3. ADAM23plays an important role in the invasion and metastasis of lung cancer, but the exact mechanism is unclear.EMT is the phenomenon that epithelial cells transfer to mesenchymal cell under specific physiological and pathological conditions. Studies have shown that EMT plays a key role in the process of invasion and metastasis of malignant tumors, and is closely related with the clincal stage.But the relationship of ADAM23and EMT has not been reported in the invasion and metastasis of lung cancer.The human fascinl gene belongs to the fascin family,encoding a55KD cytoskeletal protein which is able to bind with F-actin proteins.Fascinl localizes in the core actin bundles of microspikes,filopodia and actin-based protrusions underneath the plasma menbrane and that has benn implicated in the cell motility, invasion and metastasis. Studies have shown that Fascinl involved in the formation of liver cancer EMT;high level of Fascinl expression was reported in many carcinomas,it is closely related to tumor regional lymph node metastasis, distant metastasis, recurrence and patient prognosis. Recently, studies have reported fascin1plays an important role in invasion and metastasis of lung cancer.microRNA(miRNA) is endogenous non-coding RNA about20-25nucleotides, which inhibit the expression and translation of the target gene in the post-transcriptional level.It is closely related to individual development, proliferation, differentiation and tumorigenesis. Studies have shown that miRNA can be used as oncogenes or tumor suppressor genes involved in the development and progression of tumors, play an important role in tumor invasion and metastasis.This research intends to study the effect of ADAM23gene on invasion and metastasis of lung adenocarcinome cell SPC-A-1,to detect the miRNA differentially expressed spectrum in lung adenocarcinoma cell with stable suppression of ADAM23expression, to explore the function and mechanism of ADAM23in invasion and metastasis of lung cancer, and their mutual relationship with EMT and Fascinl,pro vide a theoretical basis and experimental evidence for ADAM23as a target of lung cancer gene therapy.Methods:(1)Using the liposome-mediated transfection techniques transiently transfect ADAM23-shRNA-1、ADAM23-shRNA-2、 ADAM23-shRNA-3and blank plasmid (pYr-3.1-CON) into SPC-A-1cell,the CON group was served as negative control and the blank transfection group as the blank control; real time PCR and western blot were used to investigate the inhibition efficiency of the three plasmids,in order to filter out the best interference plasmid;(2) Using the liposomemediated transfection techniques transfect ADAM23-shRNA-3、 pYr-3.1-CON into SPC-A-1cell,positive colonies were selected with G418.Expression of ADAM23protein and mRNA in the transfected and non-transfected SPC-A-1cell(NT) were examined by real time PCR and western blot,respectively;(3)The TWIST、E-cadherin、viment、α-SMA protein levels of shADAM23cell lines were investigated by western blot;(4) The fascinl mRNA and protein levels of shADAM23cell lines were investigated by real time PCR and western blot,and the correlation between ADAM23and fascinl was analyzed;(5)The invasive ability of shADAM23cell lines were detected by transwell chamber assay,transwell migration assay;(6)The miRNAs differentially expressed spectral of shADAM23cell lines were detected by miRNA microarray assay.Results:(1)ADAM23-shRNA-3can availably inhibit the expression of ADAM23protein and mRNA;(2)The cell SPC-A-1model stable suppression ADAM23expression was successfully constructed;(3)Compared with CON and NT cells,the E-cadherin protein expression was decreased,the TWIST、vimentin、α-SMA protein expressions were increased;(4)Compared with CON and NT cells, shADAM23showed lower level of fascinl mRNA and protein;(5)Compared with CON and NT cells, shADAM23showed lower invasive and metastatic abilities (P<0.05);(6)Compared with CON cells,Chip results showed34miRNAs were upregulated,36miRNAs were downregulated.Conclusions:(1)ADAM23gene downregulation promote the invasion and metastasis of lung adenocarcinoma cell;(2)ADAM23gene downregulation can induce EMT by raising TWIST to promote the invasion and metastasis of lung adenocarcinoma cancer;(3)There seem some relationship of invasion and metastasis betwwen ADAM23gene and fascinl in lung adenocarcinoma cancer;(4) Chip results showed34miRNAs were upregulated,36miRNA were downregulated in lung adenocarcinoma cell with stable suppression of ADAM23expression18figures,12tables,86references.