| Lung cancer is the most mortality of malignant tumor.At present,the incidence of lung cancer is gradually rising.Among lung cancers,85%patients are diagnosed as non-small cell lung cancer(NSCLC).Lung adenocarcinoma is the dominant subtype of NSCLC.Usually when diagnosed,about 70%patients have already progressed to late stage.In recent years,although the applications of surgery,radiotherapy,chemotherapy,molecular targeted drugs and biological treatment make some patient's survival period to be extended,however,the prognosis of NSCLC is still worse than that of the other malignant tumors.Thus,there is an urgent need to identify novel predictive and therapeutic targets for lung adenocarcinoma.The epidermal growth factor receptor(EGFR)gene is one of the best characterized driver genes for lung adenocarcinoma.Previous studies have shown that Dys-activation of EGFR might lead to uncontrolled cell proliferation,differentiation,metastasis,and survival.EGFR is usually overexpressed in patients with lung adenocarcinoma,which can affect the prognosis of patients with lung cancer.The strategies to target EGFR,especially by using EGFR tyrosine kinase inhibitors(TKIs),have achieved good results in treatment of lung adenocarcinoma patients with somatic mutations.Although most patients exhibit good response to these TKIs,acquired resistance to TKIs usually occurs.Thus,there is an urgent need to characterize novel molecular mechanisms to intervene EGFR signaling pathway,to identify novel therapeutic targets so as to overcome both innate and acquired resistance.Filamin A(FLNa)is a actin cross-linking proteins,which functions as a scaffold and interacts with over 90 binding partners.Through interactions with these proteins,FLNa is involved in the regulation of multiple cellular functions,especially migration,invasion,and adhesion.In recent years,the functions of FLNa in cancers have been paid much attention to.Numerous studies have demonstrated that the contribution of FLNa to cancer proliferation,progression and metastasis.FLNa is initially identified as a cancer promoting gene,which promotes cancer cell migration,invasion,and metastasis.However,recent studies indicated that FLNa could play negative roles in cancer progression depending on its cellular localization.The contribution of FLNa to cancer migration and invasion depends on its modification of signaling pathways.FLNa has been reported to play both positive and negative roles in the regulation of ERK1/2 and PKB/Akt signaling pathways,and suppression of FLNa could attenuate the K-ras-induced ERK and Akt pathway activation.In melanoma cells,FLNa has been reported to interact with integrin and modulate phosphorylation of PKB/Akt/ERK kinases.However,the effects of FLNa on EGFR signal transduction in lung adenocarcinoma have seldom been studied.Based on the decisive role of EGFR signaling pathway in lung adenocarcinoma and the important role of FLNa in cancer migration and invasion,we speculate that FLNa may play an important role in lung cancer formation and development.To confirm this hypothesis,the lung adenocarcinoma A549 stable cell line A549(KD)was constructed,in which,FLNa expression was knocked down,moreover,the control cell line A549(ctrl)was constructed,in which,FLNa expression was not affected.The effects of knockdown FLNa expression on proliferation,migration and invasion in A549 cell lines were detected by MTT assay,wound healing assay and invasion assay,respectively.The effects of knockdown FLNa expression on EGFR phosphorylation and ERK1/2 phosphorylation were investigated by western blot.After A549(KD)and A549(ctrl)cells were inoculated into nude mice,respectively,the effects of knockdown FLNa expression on the growth and metastasis of A549 cells in nude mice were observed.Finally,clinical datas were collected to observe the relationship between FLNa expression and clinicopathological features in human lung adenocarcinoma.In this study,the effects of FLNa expression on the biological behavior of A549 cells were analyzed at molecular,cellular and animal experimental levels.The mechanism of FLNa in regulating EGFR signaling pathway activation was explored.The results would provide valuable molecular indicators for the individualized treatment and prognosis of lung cancer,and provide an new target for treatment of lung cancer.This study consists of the following four parts:Part one Effects of FLNa on proliferation,migration and invasion in lung adenocarcinoma A549 cellsObjective:To investigate the effects of FLNa on proliferation,migration and invasion in lung adenocarcinoma A549 cells.Methods:1 A549(KD)stable cell line and A549(ctrl)stable cell line were cultured in vitro.The total RNA were extracted from cells,and the levels of FLNa m RNA were detected by real-time PCR(RT-PCR).The total proteins were extracted from cells,and levels of FLNa protein were measured by western blot.2 Cell proliferation was determined by MTT proliferation assayA549 cells were added into 96-well plates,and the cells were starved by serum deprivation for 4h,then treated with EGF at a concentration of 0 nM,4nM,and 20 nM for 48h,respectively.A total of 20?l MTT reagent(5 mg/ml)was added to each well and incubated for 4h.Then the culture medium was discarded and 150?l DMSO per well was added.The absorbance at 490 nm wavelength was measured by using a microplate reader.3 Cell migration assay3.1 Cell migration was determined by scratch wound-healing assayA549(KD)and A549(ctrl)cells were cultured in 24-well plates for24h,and the cells were cultured with serum deprivation for 4h,the confluent cell monolayers were scratched with a pipette tip and the plates were washed with PBS buffer before addition of fresh medium containing EGF(20nM)or without EGF.Then cell migration was observed and photographed at 0h,4h,8h,12h and 14h.3.2 Cell migration was determined by transwell chamber assayA549(KD)and A549(ctrl)cells(1×10~6/ml)were collected and transferred to the top chamber in serum-free medium,respectively.The bottom chamber contained medium with 10%FBS as a chemoattractant.The cells were cultured in a humidified incubator at 37°C for 24h.The noninvasive cells were removed from the upper surface of the transwell membrane by using a cotton swab.After washing with PBS,the cells that migrated to the underside of the filter were fixed with 95%ethanol and stained with HE and counted by using bright-field microscopy.4 Cell invasion was determined by transwell chamber invasion assayA549(KD)and A549(ctrl)cells(1×10~6/ml)were collected and seeded onto filters of a 24-well transwell chamber that was coated with matrigel.Invasion of the cells through the matrigel to the underside of the filter was assessed 24h later by fixation and staining with HE.The cells were counted by using bright-field microscopy.Results:1 Real-time PCR and western blot were used to detect the levels of FLNa mRNA and proteinAs demonstrated by RT-PCR,the relative quantitive values of FLNa mRNA in A549(KD)cells(0.21±0.09)were significantly lower than those of A549(ctrl)cells(1.00±0.00),(P<0.01).Western blot was used to detect FLNa protein levels of A549 stably cells.As the results indicated,the relative quantitive values of FLNa protein in A549(KD)cells(0.31±0.01)were significantly lower than those of A549(ctrl)cells(0.86±0.00),(P<0.01).2 The proliferation ability of A549 stably cell linesOD value of each well was detected by MTT assay and statistically analyzed.After stimulating by 0nM,4nM,20nM EGF,OD values of A549(KD)cells(0.57±0.05,0.66±0.03,0.70±0.01)were significantly higher than those of A549(ctrl)cells(0.37±0.02,0.44±0.02,0.45±0.03)(P<0.01).3 The migration ability of A549 stably cell lines3.1 Cell migration was determined by scratch wound-healing assayMigration rate of A549(KD)cells without EGF stimulating for 4h(10.42±2.87)was hihger than that of A549(ctrl)cells,but there was no statistical significance(P>0.05).Migration rates of A549(KD)cells without EGF stimulation for 8h,12h and 14h(26.80±4.01%,37.95±1.68%,40.19±2.90%)were significantly higher than those of A549(ctrl)cells(P<0.05).Migration rate of A549(KD)cells(16.97±4.47%)after 20nM EGF stimulation for 4h was higher than that of A549(ctrl)cells(8.50±2.98%),but there was no statistical significance(P>0.05).Moreover the migration rates of A549(KD)cells(29.73±2.17%,44.74±4.81%,50.00±0.00%,respectively)after 20nM EGF stimulation for 8h,12h and 14h were significantly higher than those of A549(ctrl)cells(18.69±1.06%,29.46±3.66%,37.75±2.71%,respectively),(P<0.05).3.2 Cell migration was determined by transwell chamber assayThe migration cell numbers of A549(KD)cells(636.33±28.02)were significantly more than those of A549(ctrl)cells(471.00±49.12)without EGF stimulation(P<0.05).The migration cell numbers of A549(KD)cells(697.67±109.89)were significantly more than those of A549(ctrl)cells(474.33±50.36)after 20nM EGF stimulation(P<0.05).4 Cell invasion was determined by transwell chamber invasion assayThe invasion cell numbers of A549(KD)cells(401.00±12.00)were significantly more than those of A549(ctrl)cells(184.33±15.82)without EGF stimulation(P<0.01).Part two Effects of FLNa on the activation of epidermal growth factor receptor in lung adenocarcinoma A549 cellsObjective:To investigate the effects of FLNa on the activation of epidermal growth factor receptor in lung adenocarcinoma A549 cells.Methods:A549(KD)and A549(ctrl)cells were seeded in 35mm dishs for48h.The serum-starved cells were left untreated or treated with EGF(20 n M)for 5min,10min,and 30 min,then the cells were lysed with cold lysis buffer.After determining the protein concentration,the proteins were separated by using SDS-PAGE and transferred to polyvinylidene difluoride(PVDF)membranes.The membranes were blocked in 5%nonfat milk or 3%bovine serum albumin,and incubated with various primary antibodies that included FLNa,EGFR,phosphotyrosine,ERK1/2,phospho-ERK1/2 and?-actin,and incubated with HRP-conjugated secondary antibodies subsequently,and visualization with enhanced chemiluminescence(ECL)detection reagents.Signals were quanti-tated by densitometry by means of Image J software.Results:Western blot was used to detect the protein expression after EGF stimulating for 0min,5min,10min and 30min.The results were analysed,the levels of FLNa protein in A549(KD)cells(0.38±0.02,0.43±0.02,0.35±0.02,0.21±0.02,respectively)were significantly lower than those of A549(ctrl)cells(0.68±0.03,0.74±0.02,0.72±0.02,0.55±0.02,respectively),(P<0.01).The levels of p-EGFR after EGF stimulating for 5min,10min and 30min in A549(KD)cells(1.05±0.00,1.05±0.01,0.60±0.02,respectively)were signific-antly higher than those of A549(ctrl)cells(0.90±0.00,0.65±0.01,0.16±0.00,respectively),(P<0.01).The levels of p-ERK1/2 after EGF stimulating for0min,5min,10min and 30min in A549(KD)cells(0.05±0.00,1.01±0.00,1.09±0.00,0.82±0.00,respectively)were significantly higher than those of A549(ctrl)cells(0.04±0.00,0.57±0.00,0.66±0.01,0.66±0.01,respectively),(P<0.01).Part three Effects of FLNa on growth and metastasis of lung adenoc-arcinoma A549 cells in nude miceObjective:To investigate the effects of FLNa on growth and metastasis of lung adenocarcinoma A549 cells in nude mice.Methods:A total of 1×10~6 cells in 100?l PBS A549(KD)cells or A549(ctrl)cells were injected subcutaneously into the backs of the BALB/c nude mice(5-week age).There are 6 mice in each group.After inoculation,the body weight of the nude mice was weighed at an interval of every 3 days,and the tumor length(a)and the tumor short diameter(b)were measured by caliper.Tumor volume was determined according to the formula V=length×width~2×0.52.After 28 days of inoculation,all the mice were sacrificed,and tumor tissues,lungs and liver were removed.The tumor tissues were dissected into two parts.One part was for detection of the expression of FLNa,p-EGFR and Ki67 in tumor tissues by using immunohistochemical examination after fixed by 4%paraformaldehyde,and the other part was for detection of the expression of FLNa,p-EGFR,EGFR,p-ERK1/2 and ERK1/2in tumor tissue lysates by western blot.Results:1.The effects of knockdown FLNa expression on growth and metastasis of A549 cells in nude miceAfter A549(ctrl)and A549(KD)cells were inoculation for 1 week,a tumor(3mm in size)appeared in each nude mice.The tumor volume of nude mice was increased with time.The growth rate of A549(KD)cells was increased significantly in the nude mice,as compared with that of A549(ctrl)cells.After 28 days of inoculation,all the mice were sacrificed,and tumor tissues were dissected and weighed.The weight of the tumor tissue in A549(KD)cells group was significantly greater than that of A549(ctrl)cell group(P<0.01).No metastatic tumor was found in the liver and lungs in both groups.2.Proteins expression levels in xenografts tumor tissues were detected by western blotWestern blot was used to examine the protein expression levels in A549xenografts tumor tissues,it was found that the expression levels of FLNa protein in A549(KD)group(0.14±0.02)were significantly lower than those in A549(ctrl)group(1.45±0.03),(P<0.01).The levels of p-EGFR and p-ERK1/2in A549(KD)group(1.72±0.25,1.59±0.03)were significantly higher than those in A549(ctrl)group(0.32±0.52,0.52±0.01),(P<0.01).However there were no significant differences in the total levels of EGFR and ERK1/2between two groups.3.The proteins expression levels in xenografts tumor tissues were detected by immunohistochemistryThere were FLNa,p-EGFR,Ki67 expression in both tumor tissue groups.The Ki67 positive staining signal was mainly located in the nucleus.The FLNa and p-EGFR positive staining signals were mainly located in the membrane and cytoplasm.The expression levels of FLNa,p-EGFR and Ki67in A549(KD)group were significantly higher than those in A549(ctrl)group.Part four Expression of FLNa in lung adenocarcinoma tissues and its correlation with clinicopathological featuresObjective:To investigate the expression of FLNa in lung adenocarcinoma tissues and its correlation with clinicopathological features.Methods:Twenty-eight specimens of adenocarcinoma tissues were collected from Department of Patholody of Bethune International Peace Hospital,and the expression levels of FLNa were detected by immunohistochemistry.Moreover,the correlation between the expression of FLNa and prognosis of patients was analyzed.Results:1.Expression of FLNa in lung adenocarcinomaIn lung adenocarcinoma,FLNa expressed in cell membrane and cytoplasm with brownish yellow or brown granules.2.The relationship between FLNa and clinical pathological features of lung adenocarcinomaIn 28 cases of lung adenocarcinoma tissues,it seemed that patients with lower FLNa expression displayed larger tumor size than that in patients with higher FLNa expression.What is more,patients with lower FLNa expression exhibited more lymphnode metastasis numbers and lower clinical stage,although threre are no significant difference(P>0.05).Conclusion:1.Knockdown FLNa expression can promote the proliferation,invasion and migration in human lung adenocarcinoma A549 cells.2.Knockdown FLNa expression can promote the growth of human lung adenocarcinoma A549 cells by enhancing EGF-induced EGFR tyrosine phosphorylation and downstream ERK signaling pathway.3.Knockdown FLNa expression can promote the growth of adenoca-rcinoma A549 cells in nude mice.4.The expression of FLNa in lung adenocarcinoma with different pathological features is different,FLNa may play a negative role in the occurrence and development of lung adenocarcinoma. |
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