Affect On Lung Adenocarcinoma Cell Proliferation And Metastasis Through Regulating The Activation Of EGFR By Filamin A

Lung cancer is a malignant carcinoma with most morbidity and mortalityworldwide, developing in more than a million new patients annually.Non-small cell lung cancer (NSCLC) represents approximately80%total lungcancer cases. Lung cancer is a systemic disease, its development is a complexprocedure involved multifactor. There are many signal pathways that relatedwith tumorigenesis, development, metastasis and recurrence, such as receptortyrosine kinase mediated Ras/MAPK signal pathway, PI3K/Akt path way et al.The research to tumor signal transduction pathway contributes to understandthe pathogenesis of NSCLC, and provides a novel molecule target for NSCLCtreatment.Epidermal growth factor receptor (EGFR) is a tyrosine kinase activereceptor (RTK), with a molecular weight of170kDa (1186Amino acid),located in7q12chromosomes. There are four structure similarly receptormolecules of EGFR family: ErbB1(EGFR), ErbB2(HER2), ErbB3(HER3)and ErbB4(HER4). EGFR is a transmembrane receptor composed withextracellular ligands binding domain, transmembrane anchoring domain andintracellular tyrosine kinase activity domain. Upon ligand binding, EGFRfamily proteins dimerize by receptor homo-dimerization or hetero-dimerization and subsequently activate tyrosine kinase activity. ActivatedEGFR family receptors then trigger a myriad of downstream signalingpathways, such as Ras/Raf/MEK/ERK, PI3K/Akt pathways, which promotecancer proliferation, invasion, metastasis and cancer angiogenesis. Uponepidermal growth factor (EGF) binding, activated EGFR regulates normal andmalignancy epithelial cell growth, differentiation and survival. EGFRoverexpress in40-80%NSCLC, and its expression is correlated with a bad prognosis. Therefore, EGFR and its family members had become maincandidate target molecules for targeted therapy development. EGFRoverexpression in NSCLC patients may prompt that the higher TNM stages,the more opportunity lymph nodes metastasis and a bad progression prognosis.Filamin A (FLNa) is a non-muscle actin binding protein, its genestructure is highly conservation and generally expressed, which is important tothe growth and development of mammalian. FLNa protein includes one actinbinding domain and24tandem repeats, research findings that FLNa repeatsforme a immunoglobulin like protein structure with β-sheet strands whichprovide a surface for protein and protein interaction. FLNa is involved inmultiple signal transduction pathways related with tumorigenesis, such asMAPK/ERK, PI3K/Akt, TGF-β, which may affect the behavior of tumor cellproliferation, adherence, migration and invasion. Many researches indicatedthat FLNa had an increased expression in many human epithelium tumors,such as hepatic carcinoma, melanoma, breast cancer, lung cancer andcolorectal cancer. Zhu considered that FLNa regulated the activation of EGFRin human melanoma cells. There is no report that whether FLNa regulates theactivation of EGFR in NSCLC cells.In the current studies, different lung adenocarcinoma cells were culturedin vitro.(1)The levels of FLNa and EGFR protein expression in different lungadenocarcinoma cells were detected by western blot.(2)The proliferationabilities in different lung adenocarcinoma cells were detected by MTT assay.(3) FLNa was silenced through plasmid transfection and stably transfectedcells were constructed. MTT assay, Wound healing assay and Transwell cellinvasion assay were used to examine the proliferation, migration and invasionabilities in stably transfected cells.(4)Western blot and Immunoprecipitation(IP) were used to examine EGFR phosphorylation levels and variety of itsdownstream signal molecules.Objective: To observe FLNa and EGFR expression levels in the differentlung adenocarcinoma cells, and analysis the correlation of FLNa, EGFR andproliferation abilities in lung adenocarcinoma cells. pSIF1-FLNa siRNA plasmid was transfected into GLC-82cells to reduce the levels of FLNaexpression and observe the proliferation, migration and invasion abilities inGLC-82stably transfected cells. To explore the mechanism of EGFRphosphorylation and its downstream signaling molecules regulated by FLNa.Methods:1The levels of FLNa and EGFR protein expression in different kinds oflung adenocarcinoma cell lines were detected by western blot.2The proliferation abilities in different kinds of lung adenocarcinomacells were detected by MTT assay.3Inhibit FLNa expression through plasmid transfection and constructstably transfected cells. MTT assay, Wound healing assay and Transwell cellinvasion assay were used to examine the proliferation, migration and invasionabilities in stably transfected cells.4Western blot and Immunoprecipitation were used to examine EGFRphosphorylation levels and variety of its downstream signal molecules.Results:1Protein expression and proliferation abilities of different lungadenocarcinoma cells.1.1Protein expression in different lung adenocarcinoma cells.The application ImageJ software with protein bars to scan，and SPSS13.0analysis. The levels of FLNa and EGFR expression in different lungadenocarcinoma cells respectively: GLC-82(1.02±0.18,0.86±0.01), H1975(1.15±0.05,0.92±0.01), A549(0.64±0.03,0.95±0.01), H1650(0.22±0.01,0.69±0.01) and H1299(0.80±0.02,1.13±0.01), the levels of FLNa and EGFRin H1650cell were both significantly lower compare with other cells(P<0.01).1.2Proliferation abilities in different lung adenocarcinoma cells.The OD value of each well was measured by MTT colorimetric assay,after EGF stimulating for20h or44h, respectively. OD values of different cellsafter EGF stimulating for20h and44h: GLC-82(0.42±0.01,0.64±0.09),H1975(0.40±0.03,0.54±0.06), A549(0.30±0.02,0.65±0.06), H1650 (0.12±0.01,0.20±0.03) and H1299(0.40±0.02,0.91±0.00), OD value ofH1650cell with low level of FLNa was significantly lower compare withother cells according to both time point (P<0.01).2Real-time quantitative PCR，Western blot were used to verify the effect ofplasmid transfection.2.1Real-time quantitative PCR was used to detect FLNa mRNA levels ofstably transfected cells.Real-time quantitative PCR was used to detect the FLNa mRNA levels ofGLC-82stably cells, the relative quantitive values of FLNa mRNA in GLC-82/FLNa siRNA cells (0.38±0.23) were significantly lower compare withGLC-82/Control (P<0.05).2.2Western blot was used to detect levels of FLNa expression in stablytransfected cells.Western blot was used to detect levels of FLNa expression in GLC-82stably transfected cells. The relative levels of FLNa expression in GLC-82/FLNa siRNA cells (0.39±0.00) were significantly lower compare withGLC-82/Control (0.66±0.00)(P<0.05).3Proliferation, migration and invasion abilities in GLC-82stably transfectedcells.3.1Proliferation abilities of GLC-82stably transfected cells.GLC-82stably transfected cells were seeded in96-well plates, OD valueof each well was detected by MTT assay and statistical analyzed. Afterstimulating by0nM,4nM,20nM EGF, OD values of GLC-82/FLNa siRNAgroup (0.48±0.02,0.64±0.03,0.70±0.04) were significantly lower comparewith control group (0.55±0.04,0.73±0.05,0.81±0.04) according to EGFconcentration (P<0.05).3.2Migration abilities of GLC-82stably transfected cells.Migration rates of GLC-82/FLNa siRNA cells without EGF stimulatingfor8h,16h,24h, and32h (8.11±2.45%,15.78±0.84%,16.23±2.27%,19.89±2.91%) were significantly lower compare with control group (15.47±0.74%,21.38±1.66%,25.00±2.17%,30.43±0.11%)(P<0.05). Migration rates of GLC-82/FLNa siRNA cells after20nM EGF stimulating were significantlyhigher compare with no EGF stimulating group. Migration rates of GLC-82/Control cells after20nM EGF stimulating for more than24h weresignificantly higher compare with no EGF stimulating group (P<0.05).3.3Invasion abilities in GLC-82stably transfected cells.Invasion abilities in GLC-82stably transfected cells were detected byTranswell assay, the invasion cell numbers of GLC-82/FLNa siRNA group(192±70) were significantly less than control group (368±73)(P<0.05).4The mechanism of FLNa regulating EGFR activation4.1Effects of FLNa regulating EGFR activation of GLC-82stably cells.The expression of different proteins in GLC-82stably cells were detectedby Western blot After stimulating by EGF (20nM) for0min,5min,10min and30min, the levels of FLNa expression in GLC-82/FLNa siRNA group (0.10±0.00,0.28±0.00,0.25±0.00,0.18±0.00) were significantly lower comparedwith GLC-82/Control group (0.52±0.00,0.72±0.00,0.71±0.00,0.73±0.04)(P<0.01). The levels of pY-EGFR expression in GLC-82/FLNa siRNA groupafter stimulating by EGF(20nM) for5min,10min,30min (0.74±0.00,0.53±0.00,0.23±0.00) were significantly lower compare with GLC-82/Controlgroup (0.81±0.00,0.68±0.00,0.60±0.02)(P<0.01). The levels of p-ERKexpression in GLC-82/FLNa siRNA group after stimulating by20nM EGF for5min,10min,30min (0.32±0.00,0.40±0.00,0.31±0.00) were significantlylower compare with GLC-82/Control group (0.37±0.01,0.63±0.00,0.98±0.01)(P<0.01).4.2IP confirmed that FLNa affects EGFR activation.After immunoprecipitated by anti-EGFR antibody, we used anti-phosphotyrosine antibody4G10blot, the levels of EGFR phosphorylationwere observed. After EGF stimulating for5min, the levels of pY-EGFR inGLC-82/FLNa siRNA cells (0.95±0.03) were significantly lower comparewith GLC-82/Control group (1.16±0.01)(P<0.01).Conclusion:1The levels of FLNa expression are closely related with lung adenocarcinoma cell proliferation, migration and invasion. Inhibition of FLNacan reduce the abilities of proliferation, migration and invasion in lungadenocarcinoma cells, indicating that FLNa can promote lung adenocarcinomacell proliferation and metastasis.2The level of EGFR phosphorylation is a key to affect the developmentof lung adenocarcinoma. FLNa may affect lung adenocarcinoma cellproliferation and metastasis through EGFR activation and regulating its downsteam Ras/Raf/MRK/ERK signaling pathway.