The Study Of Mechanisms Of Fibulin-3 On Lung Cancer Invasion

Objective:To explore the effect of Fibulin-3 on invasion and metastasis in non-small cell lung cancer,and discuss the possible mechanism of action.Methods:1.Using rt-pcr and immunohistochemistry method system analysis Fibulin-3 changes in the expression of lung cancer compared with normal lung tissue,by methylation specific PCR(MSP)method detecting Fibulin-3 epigenetic inactivation in lung cancer is caused by promoter methylation,systematic analysis of its correlation with tumor metastasis and prognosis.2.Build a Fibulin-3 expression vector transfection into the lung cancer cells A549 H1299 and H460,using cell invasion experiment analysis Fibulin-3 can inhibit tumor cell invasion;Establish Fibulin-3 stable expression cell lines and RNA interference,analysis Fibulin-3 whether inactivation promote tumor cell metastasis.use two Fibulin-3 siRNA transfection H1752 cells(lack of Fibulin-3 promoter methylation and expression of normal levels of protein Fibulin-3)detected using transwell invasion ability.3.Five TIMPS and three kinds of MMPS expression level changes were detected by rt-pcr and Real-Time PCR detection of transfection Fibulin-3 A549 and H1299 cells.Immunohistochemical detection of Fibulin-3 content in lung cancer tissue and MMP-7 express the relationship.4.Using Western Blot detection analysis whether Fibulin-3 through the beta-catenin/TCF 4 pathways of signal transduction influence the expression of MMP-7 proteases.5.Using indirect immunofluorescence test Fibulin-3 for beta-catenin subcellular localization.6.A549 and H1299 cells were cotransfected with Fibulin-3 and the transfection control ?-galactosidase reporter pCMV?,along with TCF-4 reporter plasmid pTOPFlash or the control inactive reporter pFOPFlash or WT MMP-7 reporter(WT)or Mutant MMP-7 reporter(Mutant).7.Parental and stable Fibulin-3-expressing H460/H1299 cells were injected i.v.by tail vein into BALB/c nude mice.For each injection,1 × 106 cells suspended in 200?L PBS were used.Following sacrifice of mice at certain time point,lung metastasis nodules were counted and the body weight was weighed.Results:1.80.4%(37 of 46)of normal lung specimens expressed Fibulin-3,whereas only 30.7%(31 of 101)of NSCLC samples were positive for Fibulin-3.Fibulin-3 expression was detected in the cytoplasm of normal bronchial epithelial cells and Fibulin-3-positive tumor cells.FBL-3 expression was significantly elevated in four cell lines following 5-aza-2'-dC treatment.2.Transfection of Fibulin-3 into A549,H1299 and H460 lung cancer cells significantly suppressed cell proliferation in colony formation assays.Analysis of cell invasion by Matrigel assays revealed that Fibulin-3 expression significantly suppressed invasion of all two cell lines.Transfection with two independent Fibulin-3 siRNA,but not the control siRNA,led to increased H1752 cell invasion,suggesting that Fibulin-3 down-regulation alone is sufficient for stimulating lung cancer cell invasion.3.The expression of MMP-7 was consistently down-regulated by 50%to 80%following Fibulin-3 transfection.immunohistochemistry:51.5%NSCLC samples were positive for MMP-7.Remarkably,a statistically significant inverse correlation between Fibulin-3 and MMP-7 expression was found,with 60.4%of tumors expressing either MMP-7 or Fibulin-3,but only 10.9%expressing both proteins.The relationship between Fibulin-3 promoter methylation and MMP-7 expression.:the loss of Fibulin-3 expression in all of the 12 tumors with promoter methylation,Among the 12 tumors cases where Fibulin-3 promoter methylattion was positive,MMP-7 expression was observed positive in 10(83.3%),but only 8 cases were MMP-7 expression positive in 20(40%)tumors with promoter unmethylation.The correlation between MMP-7 expression and Fibulin-3 promoter methylation was statistically significant.4.The expression of c-Myc and cyclin D1 two well-known TCF-4 downstream targets,was significantly suppressed by Fibulin-3.Knockdown of GSK3?up-regulated sevaral proteins expression including p-Erk,MMP-7,and c-Myc.5.?-catenin,which is normally localized in the nuclei,showed predominant cytoplasmic localization in a significant fraction of cells following Fibulin-3 transfection,and GSK3pmight be involved in Fibulin-3 signaling and can control the amount of ?-catenin which is translocated from cytoplasm to nuclear.6.Transfection of Fibulin-3,significantly inhibited the activity of TCF-4 reporter in A549 and H1299 cells.Fibulin-3 also inhibited transactivation of TCF-4 reporter by the wild-type ?-catenin,as well as that by the mutant ?-catenin(?N).?-catenin/TCF-4 directly binds to TBE sites in the MMP-7 promoter region to drive its transcriptional activation and which can be suppressed by Fibulin-3.7.The mice receiving the parental H460 cells had a number of lung metastasis nodules.In contrast,mice injected with Fibulin-3-expressing H460 cells had much fewer lung metastasis nodules.The mice receiving the parental H460 cells also had significantly lower body weights compared with those injected with Fibulin-3-expressing H460 cells.Conclusion:In this study,we identified fibulin-3 as a frequently silenced gene in lung cancer,and our results indicate that fibulin-3 functions as a metastasis suppressor of lung cancer,and down-regulation of fibulin-3 drives TCF4/catenin-mediated MMP-7 induction,and therefore lung cancer cell invasion.Given its potential tumor-suppressive role,there is an interest to develop fibulin-3 into a new therapeutic agent for patients with lung cancer.