Functional Study On The Mechanism Of IGFBP3 Overexpression In Brain Metastasis Of Lung Adenocarcinoma

Background and objective: Brain metastasis is the main cause of lung cancer-related mortality.Among all the types of non-small cell lung cancer(NSCLC),lung adenocarcinoma(LUAD)is especially associated with metastatic disease in the brain,and can be an independent prognostic factor of LUAD.Therefore,an in-depth understanding of the mechanism underlying NSCLC brain metastasis is crucial to identify new therapeutic targets and improve the prognosis of lung cancer.Insulin-like growth factor binding protein-3(IGFBP3)is the main carrier of insulin growth factors(IGFs)in the circulation.Recent studies had found that abnormal expression of IGFBP3 has been linked to the pathogenesis of cancers.While,the effects of IGFBP3 on brain metastasis of NSCLC is still unclear.Therefore,this study aimed to investigate the role of IGFBP3 in brain metastasis of lung adenocarcinoma and the potential molecular mechanisms.Methods: The supernatants of human astrocyte cells(HA1800)and human lung adenocarcinoma cells(A549)cultured in serum-free medium were collected and labeled as HA1800-CM,A549-CM,separately.Human astrocyte-conditioned medium to simulate the brain microenvironment.A549 cells were exposed to HA1800-CM,A549-CM,respectively.Through the wound healing experiments,the changes of the scratch healing ability of the two experimental groups at different time points were observed.Microarray data analysis was used to identify differentially expressed genes in A549 cells(exposed to HA1800-CM,A549-CM,respectively),which were confirmed by quantitative real-time polymerase chain reaction(qRT-PCR)and Western blot.A549 cells were infected with lentivirus to build stable up-regulated IGFBP3 cell lines.The effects of up-regulation IGFBP3 on epithelial-mesenchymal transition(EMT)markers were observed by qRT-PCR and Western blot.After treatment of A549 cells with transforming growth factor-β1(TGF-β1),the morphological changes were observed,and the changes of EMT markers were observed by qRT-PCR and Western blot.The A549 cells were infected with the short interfering RNA(si-RNA)vector to construct a knockdown IGFBP3 cell line.A549 cells knocked down IGFBP3 were treated with TGF-β1 again,qRT-PCR and Western blot were used to investigate the effect of knockdown of IGFBP3 on EMT after exposure to TGF-β1.Next,the effect of overexpression or knockdown of IGFBP3 on the migration and invasion of A549 cells was observed by transwell assay.The most significant change in the mothers against decapentaplegic homolog(Smad)family was screened for overexpression in IGFBP3 by qRT-PCR.Then,in A549 cells overexpressing IGFBP3,Smad4 was knocked down to explore the effect of Smad4 on the effect of IGFBP3.The state of IGFBP3 in 63 human lung and brain tissues was observed by immunohistochemistry.The experiment was divided into 5 groups: a,normal lung tissue specimens(15);b,non-metastatic lung adenocarcinoma tissue specimens(15);c,other metastatic lung adenocarcinoma tissue specimens(15);d,brain-metastatic lung adenocarcinoma tissue specimens(10);e,brain metastatic lesion specimens(8).Results: 1.Through wound healing experiments,we found that the A549 cells exposed to HA1800-CM(experimental group)had significantly higher scratch healing ability than A549-CM(control group).Microarray analysis screened the expression of genes in A549 cells in the two groups.IGFBP3 is the most significantly up-regulated gene.According to recent studies by researchers,IGFBP3 plays an important role in tumorigenesis and development,so we choose IGFBP3 for further research.The expression of IGFBP3 at day 4 and 5 was significantly increased by qRT-PCR and Western blot in A549 cells exposed to HA1800-CM.2.Up-regulation of IGFBP3 promoted A549 cell EMT.The morphology of A549 cells treated with TGF-β1 changed from cobblestone to elongated shape.Consistent with the morphological changes,the EMT ability was enhanced and the expression of IGFBP3 was increased.Knockdown of IGFBP3 significantly reduced TGF-β-induced EMT.3.Transwell experiments showed that overexpression of IGFBP3 enhanced the migration and invasion ability of A549 cells,while,knocked down IGFBP3,decreased the migration and invasion of A549 cells.4.qRT-PCR was used to observe the changes of Smad family when IGFBP3 was overexpressed.The results showed that the expression of Smad1,Smad4,Smad6 and Smad7 was increased in the IGFBP3 overexpression group compared with the control group.Studies have shown that Smad4 promotes bone metastasis in breast cancer through TGF-β-induced EMT.Therefore,we chose Smad4 for further research.The expression of Smad4 was increased in A549 cells overexpressing IGFBP3 by qRT-PCR and Western blot.In contrast,down-regulation of IGFBP3 is accompanied by down-regulation of Smad4.After transfection of A549 cells with Smad4-siRNA(si-Smad4),even if IGFBP3 was overexpressed,migration and invasion of A549 cells were significantly inhibited.In the immunohistochemical results,the expression of IGFBP3 in the primary tumor of lung adenocarcinoma was higher than that in the control group.Lung cancer tissues with distant metastasis,especially lung cancer tissues with brain metastasis,have higher expression levels of IGFBP3 than lung cancer tissues without metastasis.High expression of IGFBP3 was also confirmed in brain metastases compared to expression in non-metastatic lung tissues.Conclusion:(1)Up-regulation of IGFBP3 promoted the EMT,migration and invasion ability of A549 cells.(2)IGFBP3 plays an important role in EMT induced by TGF-β/Smad4.(3)IGFBP3 expression was significantly increased in lung cancer tissues and intracranial metastatic tissues.Up-regulation of IGFBP3 may mediate brain metastasis of lung adenocarcinoma,making it a potential therapeutic target.