Effect And Mechanism Of FHL2 On Angiogenesis And Permeability Of Non-small Cell Lung Cancer

ObjectiveNon-small cell lung cancer(NSCLC)has been the leading cause of cancer-related death in the word.Anti-angiogenetic therapy is one of the effective strategies for NSCLC treatment.However,NSCLC patients ultimately develop resistance to anti-angiogenetic drugs in the clinical,promoting us to continuously explore the specific mechanism of angiogenesis and find new therapeutic targets.Four-and-a-half LIM-domain protein 2(FHL2)could directly interacted with a number of proteins and is involved in various biological progress including cell adhesion,proliferation,apoptosis,invasion and differentiation.It is reported that FHL2 could promote multiple cancer progression such as colorectal cancer,ovarian cancer and cervical cancer and is associated with poor prognosis.However,the role of FHL2 in NSCLC has not been further studied.This study will further explore the role and specific mechanism of FHL2 in NSCLC,providing potential therapy target of NSCLC treatment.Methods1.TCGA database was used to analyze the expressions of FHL2 in NSCLC samples and the correlation between FHL2 and prognosis of patients.2.The expression of FHL2 in NSCLC cell lines was detected by qPCR.3.FHL2 was overexpressed or knockdown in A549 and H226 cells and then CCK-8 assay and EdU assay were performed to determine the cell viability.4.TCGA database was used to analyze the correlation between FHL2 and angiogenesis-related protein CD 146 in NSCLC samples.5.CCK-8 assay was performed to determine the cell viability of HUVECs after the treatment of conditioned medium from FHL2-overexpressing(oeFHL2)or FHL2knockdown(siFHL2)NSCLC cells.6.Wound-healing assay was performed to investigate the migration ability of HUVECs after the treatment of conditioned medium from oeFHL2 or siFHL2 NSCLC cells.7.Transwell invasion assay was performed to investigate the invasion ability of HUVECs after the treatment of conditioned medium from oeFHL2 or siFHL2 NSCLC cells.8.Tube formation assay was performed to investigate the tube formation ability of HUVECs after the treatment of conditioned medium from oeFHL2 or siFHL2 NSCLC cells.9.The downstream targets of FHL2 in NSCLC cells was detected by qPCR and then ELISA kit was used to investigate the effect of FHL2 on the secretion of VEGFA.10.Western blot analysis was performed to investigate the effect of conditioned medium from oeFHL2 NSCLC cells on the Akt/mTOR signaling pathway.11.Rescue assay was performed to verify the role of Akt/mTOR signaling pathway in the FHL2-induced NSCLC angiogenesis by using MK-2206,an Akt inhibitor.12.Permeability assay was performed to investigate the permeability of the HUVEC monolayers after the treatment of conditioned medium from oeFHL2 or siFHL2 NSCLC cells.13.Rescue assay was performed to investigate the roles of VEGFA and Akt/mTOR signaling pathway in the FHL2-induced vascular permeability by using siVEGFA and MK2206.14.Xenograft model was used to investigate the antitumor effect of FHL2 in vivo.Results1.Based on the TCGA database,FHL2 mRNA level in NSCLC tissues were significantly higher than that in normal tissues.In addition,the prognosis analysis of TCGA database supposed that the high expression of FHL2 was closely associated with low overall survival(OS)and disease-free survival(DFS).2.The expression of FHL2 was higher in NSCLC cell lines,compared with that in normal bronchial epithelial(HBE)cells.3.The results of CCK-8 assay and EdU assay showed that FHL2 markedly increased cell viability in A549 and H226 cells.4.The TCGA database showed that the FHL2 expression was correlated with the expression of CD 146.5.The results of CCK-8 assay showed that the conditioned medium from oeFHL2 NSCLC cells notably increased HUVECs viability.6.The results of wound-healing assay showed that the conditioned medium from oeFHL2 NSCLC cells observably promoted HUVECs migration.7.The results of transwell invasion assay showed that the conditioned medium from oeFHL2 NSCLC cells significantly promoted HUVECs invasion.8.The results of tube formation assay showed that conditioned medium from oeFHL2 NSCLC cells obviously increased the number of meshes.9.The results of qPCR and ELISA kit showed that FHL2 could increase the secretion of VEGFA in NSCLC cells.10.Western blot analysis showed that FHL2 could active the Akt/mTOR signaling pathway.11.The results of rescue assay showed that FHL2 facilitated NSCLC angiogenesis through VEGFA/Akt/mTOR signaling pathway.12.The results of permeability assay showed that the FHL2 could induce NSCLC vascular leakiness.13.The results of rescue assay showed that FHL2 induced NSCLC vascular leakiness through VEGFA/Akt/mTOR signaling pathway.14.The results of xenograft model showed that FHL2 could promote NSCLC tumor growth in vivo.ConclusionIn this study,we firstly demonstrated the role and specific mechanism of FHL2 in NSCLC progression.Firstly,we found that FHL2 was significantly upregulated in NSCLC tissues,which was associated with poor prognosis.In addition,FHL2 was also highly expressed in NSCLC cell lines and could promote NSCLC cell proliferation,supposing that FHL2 might be an oncogene in NSCLC.Meanwhile,we found that the conditioned medium from oeFHL2 NSCLC cells significantly promoted proliferation,migration,invasion and tube formation ability of HUVECs,indicating that FHL2 could induce angiogenesis of NSCLC.Furthermore,we determined that FHL2 activated the Akt/mTOR signaling pathway in HUVECS via promoting VEGFA secretion from NSCLC cells,thereby inducing angiogenesis.Moreover,FHL2 could induce vascular leakiness through VEGFA secretion.Finally,we also found that FHL2 could promote NSCLC tumor growth in vivo.Collectively,FHL2 could promote NSCLC proliferation,angiogenesis and vascular permeability,resulting in NSCLC tumor growth both in vitro and in vivo.Our study revealed the role of FHL2 in NSCLC and the mechanism by which FHL2 promoted NSCLC tumorigenesis,providing a new sight on targeted therapy for NSCLC.