Study On Expression Of Filamin A In Colorectal Adenocarcinoma And Its Inhibitory Effect On Tumor

Colorectal cancer is one of the most common malignant tumor,and its incidence increased year by year.However,the final effect of the disease is still not optimistic,and the 5-year survival rate rangs from 50% to 60%.The study of tumor suppressor gene is an important strategy for cancer gene therapy, so it is very important to screen and discover new tumor suppressor gene for therapy.Filamin A (FLNa), a member of the non-muscle actin-binding protein family, is widely expressed in mammals.Recent genetic evidence suggests that FLNa is essential for mammal development.FLNa is large cytoplasmic proteins,and was originally identified as actin-binding protein that stabilizes delicate three-dimensional actin webs.Recently,many researches have suggested that the rod domain repeats of the filamin molecule potentially form sandwiches ofβ-sheets resembling the immunoglobulin domain, and they apparently can also function as interfaces for protein-protein interactions.By interacting with transmembrane receptor complexes, adaptor molecules, and second messengers, it regulates signaling events involved in cell shape change and motility.FLNa is associated with tumor progression.In the study, we used immunohistochemistry, reverse transcription polymerase chain reaction (RT-PCR), and Western blot to analyze the expression of filamin A (FLNa) in 60 specimens of colorectal adenocarcinoma tissues and colorectal normal tissues.We also analyzed the correlation between its expression and the clinicopathologic features, and explore the correlation between the the expression level of FLNa and the pathogenesis of colorectal adenocarcinoma.Nextly, expressing vector pcDNA3.1/V5-His-TOPO/FLNa was transfected into human colon carcinoma cell line SW480 by lipofectamine protocols. After G418 selection,the cells SW480/FLNa expressing FLNa stably were obtained.Expression of FLNa in tumor cells was confirmed by RT-PCR and Western blot..Cell proliferation in vitro were assessed by MTT assay.Inhibitory effect of filamin A on SW480 cells were observed by Wound healing assay,Transwell chamber experiment, and Matrigel invasion assay .SW480/FLNa cells,SW480/pcDNA3.1 cells and SW480 cells were injected into nude mice to observe the formation of tumors. After 8 weeks, weight and inhibitory rate of the tumor were measured.The morphology of tumor were observed under optical microscope.Finally, SW480 cell line and SW480/FLNa cell line that were treated by EGF and U0126,respectively. We observed the expression of FLNa, ERK,p-ERK,and MMP-9 in SW480 cells and SW480/FLNa cells to explore the inhibitory effect of FLNa gene on human colon carcinoma cell line SW480 in ERK signaling pathway.PartⅠExpression of filamin A in colorectal adenocarcinoma and its clinical significance.Objective: To study the expression of filamin A(FLNa) in human colorectal adenocarcinoma tissues and explore their correlation with the pathogenesis of colorectal adenocarcinoma.Methods:1 specimens collection: All specimens were taken from 60 patients who underwent colorectal resection in second department of surgery ,the Fourth Hospital of Hebei Medical University.Carcinoma tissues were taken from tumor center, while the site of normal tissue were more than at least 10 centimeters from the edge of tumor. All cases were confirmed by pathological diagnosis. After drawn,Some specimens were placed in liquid nitrogen, then were stored in the refrigerator of -80℃;some specimens fixed in 10% formalin, then embedded in paraffin for immunohistochemical staining.2 The expression of FLNa in 60 specimens of colorectal tumor tissues and colorectal normal tissues was detected by immunohistochemistry.3 The correlation between clinicopathologic features and expression level of FLNa were analyzed.4 The expression of FLNa mRNA in colorectal tumor tissues and colorectal normal tissues was detected by RT-PCR.5 The expression of FLNa protein in colorectal tumor tissues and colorectal normal tissues was detected by Western blot.Results:1 The positive expression of FLNa localized in the cytoplasm. The positive rate of FLNa was 50.0%(30/60)in colorectal adenocarcinoma tissues,while the positive rate of FLNa was 91.7%(55/60)in colorectal normal tissues(P=0.000<0.05).2 Expression level of FLNa was positively correlated with the TNM staging,liver metastasis,lymph node metastasis,and the depth of tumor invasion,but not with the patient's age,gender,lesion site,tumor size,the grade of tumor differentiation,gross type,and histological type.3 The expression of FLNa mRNA was lower in adenocarcinoma tissues than that in normal tissues[(0.24±0.03) vs (0.95±0.04), P=0.017<0.05].4 The expression of FLNa protein was lower in colorectal adenocarcinoma tissues than that in colorectal normal tissues[(0.15±0.02) vs (0.76±0.04), P=0.013<0.05].Conclusions:1 The expression of FLNa may be an integral part in the pathogenesis of colorectal adenocarcinoma.2 The expression level of FLNa was positively correlated with the TNM staging,liver metastasis,lymph node metastasis,and the depth of tumor invasion.PartⅡThe effect of filamin A on invasion and metastasis of human colon carcinoma cell line SW480Objective: By transfecting FLNa cDNA into human colon carcinoma cell line SW480,and overexpression of intracellular FLNa gene,we explored the inhibitory effect of FLNa on invasion and metastasis of human colon carcinoma cell line SW480.Methods:1 Expressing vector pcDNA3.1/V5-His-TOPO/FLNa was transfected into SW480 cells by lipofectamine protocols.After G418 selection,the cells SW480/FLNa expressing FLNa stably were obtained.2 Expression of FLNa in tumor cells was confirmed by RT-PCR and Western blot.3 Cell proliferation in vitro were assessed by MTT assay.4 The migration ability of tumor cells were observed by Wound healing assay.5 Inhibitory effect of filamin A on SW480 cells were observed by Transwell chamber experiment,and Matrigel invasion assay.6 SW480/FLNa, SW480/pcDNA3.1 and SW480 cells were injected into nude mice to observe the formation of tumors.7 After 8 weeks, weight and inhibitory rate of the tumor were measured.8 The morphology of tumor were observed under optical microscope.Results:1 FLNa gene was stably expressed in SW480 cells.2 MTT assay showed the proliferation of different SW480 cells were similar in vitro.3 Wound healing experimental results showed that SW480/FLNa cells slowly migrated to the gap,while SW480 group migrated to the scratched area significantly faster than SW480/FLNa group.4 The results of Transwell chamber experiment and matrigel invasion assay showed that the number of SW480/FLNa cells penetrating through membrane or matrigel significantly reduced .5 Compared with SW480 group, growth rate of the tumors in SW480/FLNa group were significantly restrained.6 Compared with SW480 group, weight of the tumors in SW480/FLNa group were significantly restrained.7 Degeneration and necrosis were found in the tissues of SW480/FLNa group.Conclusions:FLNa has significant inhibitory effect on the invasion and metastasis of human colon carcinoma cell line SW480 in vitro ,and may inhibit the tumor formation activity of SW480 cells in nude mice. PartⅢInhibitory effect of filamin A on invasion of human colon carcinoma cell line SW480 in vitro correlates with ERK signaling pathway Objective: To explore the effect of FLNa on invasion of human colon carcinoma line SW480 cells in ERK signaling pathway, and investigate its mechanisms of the action.Methods:1 To observe the effect of inhibitor U0126 on the expression of ERK in human colon carcinoma cell line SW480.2 To observe the effect of activator EGF on the expression of ERK in human colon carcinoma cell line SW480.3 To measure the expression of ERK, p-ERK,and MMP-9 in SW480 cells, SW480 cells incubated with U0126 for 20 minutes,and SW480/FLNa cells detected by Western blot.4 To measure the expression of ERK, p-ERK,and MMP-9 in SW480/FLNa cells, SW480/FLNa cells incubated with EGF for 20 minutes,and SW480 cells detected by Western blot.5 To explore the ability of invasion of SW480 cells, SW480 cells incubated with U0126 for 20 minutes,and SW480/FLNa cells.6 To explore the ability of invasion of SW480/FLNa cells, SW480/FLNa cells incubated with EGF for 20 minutes,and SW480 cells.Results:1 After incubated with U0126 for 5,10,20,and 30 minutes,the expression of ERK in SW480 cells reached 0.936±0.030,0.826±0.029,0.130±0.007, and 0.124±0.001,respectively(F=1974.56,P=0.00).After incubated with U0126 for 20 minutes,the expression level of ERK in SW480 cells reached the maximum inhibitory effect.2 After incubated with EGF for 5,10,20,and 30 minutes,the expression of ERK in SW480/FLNa cells reached 0.154±0.018,0.140±0.026,0.832±0.039,and 0.796±0.034, respectively(F=796.488,P=0.000).After incubated with EGF for 20 minutes,the expression level of ERK in SW480/FLNa cells reached the maximum activation effect.3 The expression level of ERK in SW480 cells, SW480 cells incubated with U0126 for 20 minutes,and SW480/FLNa cells was 0.500±0.016,0.082±0.013,and 0.090±0.016, respectively(F=1279.433,P=0.000);The expression level of p-ERK was 0.439±0.014,0.003±0.001,and 0.010±0.002, respectively(F=5025.294,P=0.000);The expression level of MMP-9 was 0.450±0.036, 0.004±0.002,and 0.004±0.001, respectively(F=766.091, P=0.000).4 The expression level of ERK in SW480/FLNa cells, SW480/FLNa cells incubated with EGF for 20 minutes,and SW480 cells was 0.071±0.006, 0.758±0.075,and 0.808±0.058, respectively ( F=279.252,P=0.000 ); The expression level of p-ERK was 0.021±0.002,0.260±0.004,and 0.269±0.010, respectively (F=2325.506,P=0.000);The expression level of MMP-9 was 0.012±0.005, 0.615±0.036,and 0.594±0.018,respectively (F=1086.979, P=0.000).5 The number of penetrating through matrigel of SW480 cells, SW480 cells incubated with U0126 for 20 minutes,and SW480/FLNa cells was 214±5, 81±2,and 77±3, respectively(F=1340.740,P=0.000); Compared with SW480 group, the number of SW480 cells incubated with U0126 for 20 minutes was les(sP=0.000); The number of SW480/FLNa cells was less than that of SW480 cells(P=0.000).6 The number of penetrating through matrigel of SW480/FLNa, SW480/FLNa cells incubated with EGF for 20 minutes,and SW480 cells was 54±3,126±2, and 129±3, respectively(F=731.379,P=0.000);Compared with SW480/FLNa group, the number of SW480/FLNa cells incubated with EGF for 20 minutes was more(P=0.000);The number of SW480 cells was more than that of SW480/FLNa cells(P=0.000).Conclusions:FLNa has significant inhibitory effect on the invasion and metastasis of human colon carcinoma cell line SW480.The mechanism of action may be ralated with the suppressive effect of ERK signaling pathway,and the synthesis of MMP-9.