The Function And Mechanism Research Of Glypican-5Regulating Invasion And Metastasis Of Lung Adenocarcinoma

Background and Objective:Non-small cell lung cancer (NSCLC) is one of the most malignant and highly metastatic cancers worldwide. Glypican-5(GPC5) may be a potential lung cancer suppressor gene. The present study aimed to investigate the function and mechanism research of Glypican-5regulating invasion and metastasis of lung adenocarcinoma (LAC).Methods:1. GPC5mRNA expression was examined by real-time RT-PCR in134paired human LAC samples. The data were further analyzed according to clinicopathologic characteristics. An additional75human LAC samples were examined by tissue microarrays (TMAs) for GPC5immunohistochemical (IHC) staining and further survival analysis.2. GPC5expression was also examined in NSCLC cell lines by real-time PCR and western blot analyses. Lentivirus-induced GPC5overexpression was used in LAC cell lines (A549and H1975) for the in vitro analyses of functions including migration (Monolayer wound healing assay), invasion (Transwell invasion assay), and proliferation (CCK8), as well as the cell cycle (PI single staining). We established two animal models of LAC orthotopic implantation and metastasis in vivo.3. We selected relative genes in Wnt/β-catenin signaling pathway by Phalanx OneArray6.1whole genome microarray expression analysis on the overexpression of GPC5in LAC cell lines, including E-cadherin, wnt3a, wnt5a/b, p-GSK-3β, cyclinD1, MMP2, MMP9and MMP7. Western blot was engaged for determining the protein levels of these genes. We also detected the expression of β-catenin and E-cadherin by immunofluorescence. The TCF reporter assay (TOP/FOP) was used to detect the activation of the Wnt canonical pathway. Immunoprecipitation was used to explore the relationship between GPC5and wnt3a.4. We performed an association study on GPC5-wnt3a-E-cadherin-MMP9pathway in clinical LAC samples by Spearman rank correlation, Pearson correlation and Cox regression model analysis.Results:1. The GPC5expression in the lymph node metastasis group (stage N1-2) was remarkably lower than that in the non-metastasis group (stage NO).2. The TMA study found that the overall survival rate of the GPC5-positive group was significantly higher than that of the GPC5-negative group.3. Overexpressing GPC5in LAC cell lines significantly suppressed their migration, invasion, and proliferation activities and also induced G1phase arrest of the cells in vitro.4. GPC5could significantly inhibit LAC cells growth rate, metastasis, proliferation and tumorigenicity in vivo.5. The expression levels of Wnt/β-catenin signaling pathway related proteins wnt3a, cyclinD1, MMP2, MMP9, MMP7were significantly down-regulated, while the expression levels of p-GSK-3βand E-cadherin were significantly up-regulated.6. GPC5could combine with wnt3a, and then inhibited Wnt/β-catenin signaling pathway on the upper level.7. There was a positive correlation between the expression levels of GPC5and E-cadherin, while a negative correlation between the expression levels of GPC5and MMP9, wnt3a by correlation analysis.8. GPC5might be one of independent prognostic factors in LAC by Cox regression model analysis. Conclusions:1. GPC5may be a novel metastasis suppressor gene in LAC.2. GPC5may be a potential biomarker that predicts LAC metastasis.3. GPC5is able to regulate LAC invasion and metastasis by Wnt/β-catenin signaling pathway.4. GPC5may be one of independent prognostic factors in LAC.