Mechanism Of CXCR4 Mediated PI3K/AKT Signaling Pathway On Blood-Brain Barrier Permeability And Brain Metastasis Of Lung Cancer

AIMLung cancer is the most common malignant tumor worldwide,and it is also the malignant tumor with the highest mortality rate in men and the second highest mortality rate in women.According to statistics,there are more than 2 million new lung cancer cases(accounting for 11.6%of the total cancer cases)and more than 1.7 million deaths(accounting for 18.4%of the total cancer deaths)every year.With the continuous improvement of treatment methods,the 5-year survival rate of lung cancer patients has increased from 15.6%in 2011 to the current 19.4%,but it is still less than 20%.Studies have shown that most lung cancer-related deaths are caused by metastasis,among which the brain is the most common site for distant metastasis of lung cancer,and up to 50%of lung cancer patients will eventually develop brain metastasis(BM)during the course of the disease,due to the low overall survival rate in patients with lung cancer brain metastasis(LCBM),the median survival time is merely 6 months,so a deep understanding of the molecular mechanism of LCBM is necessary to prevent the occurrence of BM,develop new targets for BM treatment,improve the prognosis of BM patients.According to the basis of previous work and previous literature reports,we put forward the following hypothesis:CXCR4 overexpressed on the lung cancer cell membrane binds to its ligand and activates the intracellular PI3K/AKT signaling pathway,resulting in lung cancer cell proliferation,reduced apoptosis,and reduced invasiveness.It destroys the integrity of blood-brain barrier(BBB),increases the permeability of BBB and promotes the occurrence of LCBM.In this study,we aimed to clarify two key questions:(1)whether CXCR4 mediates brain metastasis of lung cancer cells through PI3K/AKT signaling pathway;(2)how does CXCR4 destroy the integrity of BBB,resulting in increased permeability.Methods(1)The brain tissue of newborn mice(1-2 days)was taken,and astrocytes were extracted and identified by microscopic morphological observation and flow cytometry;astrocytes and mouse brain microvascular endothelial cells(b End.3)were co-cultured in a Transwell chamber to build an in vitro BBB model,and evaluate the stability of in vitro BBB through a 4-hour leakage test and a fluorescein sodium permeability test;(2)Knock down the expression of CXCR4 in lung cancer cells(A549 and PC9)by sh RNA technology,and verify the knockdown level by PCR technology;construct lung cancer cells stably transfected with CXCR4-sh RNA by combining fluorescence and antibiotic screening;(3)The migration ability of lung cancer cells after CXCR4 knockdown was detected by cell scratch assay and Transwell assay;the proliferation ability of lung cancer cells after CXCR4 knockdown was detected by CCK-8 assay;the apoptosis of lung cancer cells after CXCR4 knockdown were detected by flow cytometry;Transwell assay was used to detect the invasive ability of lung cancer cells after CXCR4knockdown;the above experiments were repeated with CXCR4 inhibitor AMD3100,in order to verify the effect of CXCR4 on the migration,proliferation,apoptosis and invasion of lung cancer cells;the CXCR4 agonist NUCC-390 were added to perform a recovery experiment,in order to further verify the effect of CXCR4 on the migration,proliferation,apoptosis and invasion of lung cancer cells;(4)The protein expression levels of PI3K,p PI3K(phosphorylated PI3K),AKT and p AKT(phosphorylated AKT)on the PI3K/AKT signaling pathway were detected by Western blot in CXCR4-sh RNA lung cancer cells and negative control(NC)lung cancer cells.The above experiments were repeated with CXCR4 inhibitor AMD3100to verify the effect of CXCR4 on PI3K/AKT signaling pathway-related proteins in lung cancer cells;CXCR4 agonist NUCC-390 was used to perform recovery experiments to further verify the effect of CXCR4 on PI3K/AKT signaling pathway-related proteins in lung cancer cells;(5)The effect of CXCR4-sh RNA on the expression of tight junction proteins(Claudin-5,Occludin,ZO-1)in the in vitro BBB model was detected by Western blot;the above experiments were repeated using the CXCR4 inhibitor AMD3100 to verify the effect of CXCR4 on BBB tight junction protein;the CXCR4 agonist NUCC-390was used to perform a recovery assay to further verify the effect of CXCR4 on BBB tight junction protein;(6)The lung cancer brain metastasis model was constructed in nude mice by injecting tumor cells into the left ventricle,and the formation of intracranial metastases was determined by small animal CT to evaluate the intracranial metastatic tumor.The tumorigenic ability was calculated and the following experiments were carried out(1)Evans blue was used to detect the effect of CXCR4-sh RNA on the permeability of nude mice BBB;(2)The serum cytokines(VEGF,IL-1α,IL-3,IL-6,IFN-γ,TNF-α,TGF-βand PDGF-BB)in the serum of mice of different intracranial tumorigenesis groups were detected by ELISA kit;(3)Immunohistochemical technique was used to detect the difference in the expression of molecules(VEGF,Ki-67,p53 protein and CXCR4)in the brain tissue of CXCR4-sh RNA and NC lung cancer cells;(4)Immunofluorescence technique was used to detect the difference of CD3+T,CD4+T and CD8+T expression between CXCR4-sh RNA and NC lung cancer cell intracranial tumor-forming tissues;(5)RNA sequencing was performed on CXCR4-sh RNA and NC lung cancer cell intracranial tumor tissue,in order to explore gene expression differences in different intracranial tumor tissues from the perspective of genomic level,and the enrichment analysis was investigated to determine the biological pathways in which genes may be enriched.Results(1)The in vitro BBB model was successfully constructed by co-culturing astrocytes and b End.3 cells;(2)After knocking down CXCR4 in lung cancer cells(A549 and PC9),the migration,proliferation and invasion abilities of lung cancer cells were significantly decreased,and the apoptosis was significantly increased;after using AMD3100,the migration,proliferation and invasion abilities of lung cancer cells were significantly decreased,and the apoptosis was significantly decreased.The NUCC-390 could significantly increase the migration,proliferation and invasion abilities of CXCR4-sh RNA lung cancer cells,and decrease apoptosis;(3)After knocking down CXCR4,the protein content of PI3K and AKT in lung cancer cells did not change significantly,while the protein levels of p PI3K and p AKT decreased significantly;after using AMD3100,the protein levels of PI3K and AKT in lung cancer cells did not change significantly,while p PI3K and p AKT protein levels were significantly decreased;after using NUCC-390,the protein levels of PI3K and AKT in lung cancer cells did not change significantly,while the protein levels of p PI3K and p AKT increased significantly;(4)Knocking down of CXCR4 in lung cancer cells could increase the expression of BBB tight junction proteins(Claudin-5,Occludin,ZO-1);after using AMD3100,lung cancer cells can increase the expression of Claudin-5,Occludin and ZO-1 proteins;After adding NUCC-390,lung cancer cells can reduce the expression of Claudin-5,Occludin and ZO-1 proteins;(5)The nude mouse lung cancer brain metastases model showed that compared with NC lung cancer cells,the ability of CXCR4-sh RNA lung cancer cells to form brain metastases was reduced,and the intracranial tumor formation rate of CXCR4-sh RNA PC9 lung cancer cells was 50%.The tumor formation rate of NC cells was 66.66%;(6)The results showed that the level of Evans blue in the brain tissue of nude mice in the CXCR4-sh RNA group was significantly lower than that in the untreated lung cancer cell group,suggesting that the BBB permeability was reduced;(7)ELISA results showed that the levels of IFN-γand TNF-αin the CXCR4-sh RNA group were significantly higher than those in the NC lung cancer cell group,and the difference was statistically significant(P<0.05).There were no significant differences in the other cytokines between the two groups(P>0.05);(8)Immunohistochemical results showed that compared with the CXCR4-sh RNA cell tumorigenic group,the expression of VEGF in brain metastases formed by untreated lung cancer cells was significantly increased,and the expression of CXCR4was also increased compared with the CXCR4-sh RNA group.There were no significant differences about the p53 protein and Ki-67 expressions;(9)Immunofluorescence results showed that there were no significant differences in the expression of CD3~+T and CD8~+T in CXCR4-sh RNA cell tumorigenic group and NC untreated lung cancer tumorigenic group,while CD4~+T was expressed in NC lung cancer PC9 cells group increased significantly;(10)RNA sequencing results showed that compared with the NC lung cancer cell group,there were a total of 464 significantly different genes in the brain tissue of CXCR4-sh RNA cells,including 103 up-regulated and 361 down-regulated genes(FDR<0.05,log FC≥1 were set as the thresholds);enrichment analysis showed that the differential genes were mainly related to biological processes such as cytokine chemotaxis,cell adhesion,and extracellular matrix remodeling.ConclusionsOur experimental results show that:(1)after knocking down CXCR4 in lung cancer cells,the ability of tumor cell migration,proliferation and invasion decreased,apoptosis increased,the expression of BBB tight junction protein increased,the permeability decreased,and the ability to cross the BBB decreased;(2)CXCR4 in lung cancer cells mediates the occurrence of brain metastases from lung cancer by activating the PI3K/AKT signaling pathway.