The Study On The Correlation Between NOK With EGFR Expression In NSCLC And On The Biological Characteristics To SPC-A-1-NOK Cells With High NOK Expression

Background:Receptor tyrosine protein kinase RPTKs (Receptor protein tyrosine kinases)plays an important regulatory role in cell growth process including cell migration,cell proliferation and inhibition of cell apoptosis. In2004, Liu Li reported a novelprotein NOK (Novel Oncogene with Kinase-domain) with RPTKs alike structure.NOK has been considered as a cancer gene which transformates NIH3T3and BaF3cells, promotes tumor formation, induces tumor cell invasion and metastasis to otherorgans. NOK is capable of promoting the transformation of cells, tumor formationand metastasis as novel cancer genes both in vivo and in vitro.There is30%NOKgene being homology to FGF/PDGF receptor superfamily. NOK activates MAPkinase and phosphatidylinositol3-kinase (PI3K) signaling pathway simultaneously.Therefore, NOK-mediated oncogenesis may represent an example of cancerdevelopment involving RPTK.The effects and mechanisms of NOK on lung cancer cell biologicalcharacteristics have not yet been reported. NOK protein is lack of extracellular ligandbinding domain. The means of which NOK protein tyrosine kinase is activated and how to regulate downstream signaling pathway of NOK are still not clear.Objective:To investigate the colocalization of NOK expression with EGFR protein inNSCLC and identify the correlation of NOK expression with EGFR gene in NSCLCtissue; to establish the lung adenocarcinoma cell line (SPC-A-1-NOK) with stableNOK expression and to investigate the changes of the biological properties such asproliferation, apoptosis, invasion and migration for the SPC-A-1-NOK;all which isin order to provide a platform for the further study on the correlation of NOKexpression with EGFR and signal pathways with NOK.Methods:1. Whether there is colocalization of NOK expression with EGFR protein inNSCLC tissue was detected by immune laser confocal. RT-PCR was used to detectNOK and EGFR genes expression in NSCLC tissue.2. The recombinant NOK eukaryotic expression plasmid(pcDNA3.1/NOK)was trasfected into SPC-A-1cells by electroporation apparatus. The cloned strainwith stable expression of NOK was screened by G418and named as SPC-A-1-NOK,and identified by RT-PCR、Real Time-PCR and Western blot. Immune laserconfocal was used to detecte whether there is colocalization between NOK withEGFR protein in the SPC-A-1-NOK.3. The cell cycle and cell apoptosis were detected by flow cytometry and thecell growth curve was determined by MTT. Cell migrating was tested by cell scratchtest and transwell migrating assay; meanwhile cell invasion was tested by matrigelinvasion assay.Results:1. Immune laser confocal result showed that there is colocalization between NOK with EGFR protein in NSCLC tissue. RT-PCR result displayed that there iscorrelation of NOK and EGFR genes in different TNM stagings（rs=0.720，P<0.001）.2. RT-PCR、Real Time-PCR and Western blot demonstrated the stable highexpression level of NOK in SPC-A-1-NOK cells.Immune laser confocal resultdisplayed the presence of colocalization phenomenon between NOK with EGFRprotein in SPC-A-1-NOK cells.3. MTT showed the growth curve of SPC-A-1-NOK was steeper than thoseof SPC-A-1and blank control.Detected by flow cytometry, the number ofSPC-A-1-NOK cells in S stage was larger than those of SPC-A-1cells and blankcontriol(28.00±1.42VS23.75±1.20、24.93±1.57，both P<0.05); The proliferationindex (PI) in SPC-A-1-NOK was higher than those in SPC-A-1and blankcontriol(56.70±1.43VS46.89±1.19、46.47±1.32，both P<0.05); the number ofSPC-A-1-NOK cells in Q2and Q4stage was lower than those of SPC-A-1cells andblank contriol (4.0±0.2VS8.4±0.5、7.9±0.4，both P<0.05).4. Cell scratch test demonstrated the migration speed of SPC-A-1-NOK wasfaster than those of normal control and blank control cells. Transwell migration assayshowed that the number of transfected cells that passed the Transwell membrane wasmore than those of normal control and blank control cells(68.6±7.3VS36.7±4.3、38.4±5.9，both P<0.05). Matrigel invasion assay showed that the number oftransfected cells that passed the Transwell membrane was more than those of normalcontrol and blank control cells(46.8±6.21VS17.5±3.28、16.23±4.1， bothP<0.05).Conclusion:1. The colocalization phenomenon between NOK with EGFR protein is observed in lung adenocarcinoma and squamous cell carcinoma. There is correlationof NOK with EGFR expression in different TNM staging in lung adenocarcinoma,which suggests that there may be interactions between NOK and EGFR.2. The SPC-A-1-NOK cell line with stable high expression level of NOK wassuccessfully established in this study, which provides the platform for the furtherresearch of NOK.3. NOK may improve the proliferation, migration, invasion of SPC-A-1cellsand inhibit the apoptosis of SPC-A-1cells, so it might play an important role in thedevelopment of human lung cancer.