| Objective:Colorectal cancer is one of the most common malignant tumors, the incidence of colorectal cancer increasing in most countries over the past 20 years. Colorectal cancer is often diagnosed at an advanced stage, leading to a poor prognosis. Critical cancer hallmarks include enhanced cell proliferation, apoptosis resistance, and acquired ability to migrate and invade foreign tissues. The initiation and progression of colorectal cancer is a complex cellular process. The elucidation of this mechanism may advance the development of new therapies and improve survival rates.Filamins are a family of actin-binding proteins composed of filamin A, B and C. Filamin A(FLNa), identified in macrophages in 1975, previously known as ABP-280, was characterized initially as a protein that could cross-link actin filaments and form rigid gels. The capacity of cross-link couples extracellular signaling to the cellular cytoskeleton. It forms a homo-dimer and cross-links cortical actin filaments into a dynamic three-dimensional structure. By interacting with transmemberane receptor complexs, adaptor molecules, and second messengers, it regulates signaling events involved in cell shape change and motility. The molecular function of FLNa in cell chemotaxis remains debated and seems to vary depending on FLNa expression levels and its interacting partners. Abnormal expression of FLNa is associated with a wide spectrum of human disorders which are caused by impaired interactions between FLNa and binding partners. Zhu considered that FLNa regulated the activation of EGFR in human melanoma cells.Epidermal growth factor receptor(EGFR) is a major regulator of cell proliferation, growth, survival, metabolism and motility. Epidermal growth factor receptor(EGFR) is a tyrosine kinase active receptor, together with Erb B2(HER2), Erb B3(HER3) and Erb B4(HER4) composed of EGFR family. EGFR is a transmembrane receptor composed of extracellular ligands binding domain, transmembrane anchoring domain and intracellular tyrosine kinase activity domain. Homo-dimerization or hetero-dimerization of ligand activates EGFR results in receptor tyrosine kinase autophosphorylation and activates a number of important signaling molecules such as Ras/Raf/MEK/ERK, PI3K/Akt pathways, and then promote cancer proliferation, invasion, metastasis and cancer angiogenesis, its over-expression or inappropriately activated plays important role in many cancers. In cell signaling network, PI3K/Akt pathway is a typical anti-apoptotic pathways, Akt also known as PKB, which is an important downstream target kinase of PI3 K signaling pathway. Extracellular signal-regulated kinases(extracellular SIGNA-l regulated kinase, ERK) is a significant part of extracellular signaling pathways, containing 3 typical levels of activation processes.In this study, we constructed and cultured colorectal cancer cells(SW480) stabling transfected with plasmid pc DNA3.1 and FLNa. We adopted MTS, Wound healing and Transwell cell invasion assays to observe the effect of FLNa on the proliferation, migration and invasion in colorectal cancer cells SW480. Secondly observed transformations of EGFR and p-EGFR, Ak tand p-Akt, ERK and p-ERK by Western blot. At last, we detected the protein expression of FLNa, EGFR and Ki-67 in colorectal cancer, analyzed the correlation. Therefore, the study is divided into three Parts listed below: Part One FLNa impacts on biological behaviours of SW480 cellsMethods:1 Protein expression in different colorectal cellsFLNa protein expression in different kinds of colorectal cell lines(HCT116, T84, HT29, SW837, SW620, SW480) were detected by Western blot.2 Construction of stably transfected cell linesThe expression plasmid of FLNa sh RNA gene vector was transfected into SW480 cells. Puromycin was used to screen transfected cells. After getting the stably transfected knockdown of FLNa group(SW480/KD) and control group(SW480 /ctrl), the levels of FLNa protein were examined by Western blot.3 MTS assay was used to examine the proliferation abilities of stably transfected cell linesSW480/KD and SW480/ctrl cells were seeded in 96-well plates respectively. After stimulating with different concentrations of EGF(0n M, 4n M, 20 n M, 100 n M) for 48 hours, MTS assay was used to measure the OD values, and then examine the proliferation ability of stably transfected cell lines.4 Wound healing assay was used to examine the migration abilities of stably transfected cell linesWound healing assay was used to examine the migration abilities of stably transfected cell lines. SW480/KD and SW480/ctrl cells were seeded in 24-well plates respectively. Cells were serum-starved for 4 hours, and then scratched with a 10μl pipette tip and washed with serum-free medium(SFM) to remove floating cells. Cells then were divided into two groups: EGF(20n M) stimulated group and no EGF group. Images of cells at the same field were taken until closure of the scratch.5 Transwell cell invasion assay was used to examine the invasion abilities of stably transfected cell linesThe invasion assays were carried out using transwell chamber coated with matrigel. After starved with serum-free 1640 medium for 4 hours, SW480/KD and SW480/ctrl cells were seeded into upper chamber with serum-free 1640 medium, respectively. Lower chamber were added into 10% serum 1640 medium. After incubated for 24 hours, the invaded cells were fixed with 95% ice-cold ethanol and then stained with hematoxylin and eosin(HE).Results:1 Levels of FLNa expression in different colorectal cell linesWestern blot was used to detect FLNa levels in different kinds of colorectal cell lines. HCT116,T84, HT29, SW837, SW620, SW480(0.02±0.00),(0.02±0.00),(0.00±0.00)0.05±0.00),(0.02±0.00),(0.09±0.000), max in SW480 cells.(P<0.01)2 The expression efficiency of FLNa in stably transfected cell linesThe relative levels of FLNa expression in SW480/KD cells(0.48±0.00)were significantly lower compare with and SW480/ctrl(1.13±0.03)(P<0.01).3 Proliferation abilities of stably transfected cellsSW480 cells stably transfected cells were seeded in 96-well plates, OD value of each well was detected by MTS assay and statistical analyzed. After stimulating by 0n M, 4n M, 20 n M, 100 n M EGF, OD values of SW480/KD(0.99±0.03, 1.23±0.04, 1.52±0.01,1.90±0.01) were significantly higher compare with SW480/ctrl group(0.49±0.00,0.70±0.00,0.98±0.01,1.31±0.04) according to EGF concentration(P<0.05 or P<0.01).4 Migration abilities of stably transfected cellsMigration rates of SW480/KD cells without EGF stimulating(0h, 8h, 16 h, 24h)(0.00±0.00,5.67±1.03%,19.85±0.95%,32.42±1.71%)were significantly higher compare with SW480/ctrl group(0.00±0.00%, 4.95±0.37%, 9.94±0.44%,19.34±0.23%)(P<0.01). Migration rates of SW480/KD(0.00± 0.00%, 11.56±1.47%, 21.57±0.47%, 50.00±0.00%) after 20 n M EGF stimulating were significantly higher compare with SW480/ctrl group(0.00± 0.00%, 6.19±0.51%, 9.94±0.44%, 22.27±0.26%)(P<0.01).5 Invasion abilities in stably transfected cells Invasion abilities in stably transfected cells were detected by Transwell assay, the invasion cell numbers of SW480/KD group(62±3) were significantly higher than SW480/ctrl group(18±1)(P<0.01). Part two Effect of FLNa on activation of EGFR in colorectal cancer Western blot was assessed the expression of FLNa, EGFR, p-EGFR p-Akt, Akt, p-ERK, ERK and β-actin in stably transfected colorectal cancer cells SW480. Samples were divided into two groups: SW480/KD group and SW480/ctrl control group. After 20 n M EGF stimulating for 0min, 5 min, 10 min, 30 min, extracted proteins respectively. Determined the expression of FLNa, EGFR, p-EGFR, EGFR, p-Akt, Akt, p-ERK, ERK and β-actin in stably transfected colorectal cancer cells by Western blot.Results:Effect of low expression of FLNa on activation of EGFRThe results of statistics demonstrated that levels of FLNa expression in the SW480/KD group(0.38±0.05,0.41±0.03,0.32±0.02,0.34±0.06) is significantly lower than the SW480/ctrl group(0.92±0.08, 0.89±0.07,0.88±0.06,0.90±0.05) at every tested point; The levels of p-EGFR expression in SW480/KD group after stimulating by EGF(20n M) for 5min, 10 min, 30min(0.40±0.09,0.33±0.06,0.25±0.04)were significantly higher compare with SW480/ctrl group(0.27±0.08,0.13±0.02,0.05±0.04)(P<0.01). The levels of p-ERK expression in SW480/KD group after stimulating by 20 n M EGF for 5min, 10 min, 30min(0.39±0.02,0.49±0.08,0.34±0.06) were significantly higher compare with SW480/ctrl group(0.30±0.01,0.16±0.01,0.11±0.01)(P<0.01). The levels of p-Akt expression in SW480/KD group after stimulating by EGF(20n M) for 5min, 10 min, 30min(1.36±0.12,1.02±0.15,0.85±0.09) were significantly higher compare with SW480/ctrl group(0.60±0.03,0.21±0.02,0.05±0.01)(P<0.01). Part Three The expression of FLNa in colorectal cancer tissue andrelationships with clinicopathological featuresMethods:To explore FLNa of biological behavior and its significance for prognose in colorectal cancer, 82 cases, including different levels of differentiation invasive colorectal cancer, were obtained from the surgical pathology files at Bethune International Peace Hospital with the approval from the Institutional Review Board, have been detected by Non-biotin enzyme II(PV) immunohwastochemical method. PV Immunohistochemistry was applied to detect the expression of FLNa, EGFR and Ki-67 in 82 cases. And the relationship was analysed with clinicopathological features.Results:1 In colorectal cancer,FLNa and EGFR expressed in cell membrane and cytoplasm with brownish yellow or brown granules. The positive expression rate of FLNa was 51.2%(42/82), the positive rate of EGFR expression was 52.4%(43/82), Ki-67 distributed in nucleus with brownish yellow or brown granules, the positive rate of Ki-67 was 53.7%(44/82).2 The relationship between FLNa/Ki-67 and clinical pathological features of colorectal cancerIn 82 cases of colorectal cancer tissue, there were significant relationship between expression of FLNa and the lymph node metastasis and TNM stage, the differences were statistically significant(P<0.01), but not with age, sex, tumor size and degree of tumor differentiation.3 The correlation between FLNa and Ki-67 in colorectal cancerCoexpression of FLNa and Ki-67 were 16 cases in 82 while 12 cases were completely negative, they were negatively correlated(r=-0.320, P<0.01).Conclusion:1 Low expression of FLNa may promote colorectal cancer cells(SW480) proliferation, migration and invasion abilities.2 FLNa may affect biological behaviour of colorectal cancer cells through EGFR activation and regulating its down steam MAPK/ERK and PI3K/Akt signaling pathways.3 The expression of FLNa on colorectal cancer has a relationship with proliferation and metastasis of tumor. |
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