FBW7 Inhibits Invasion And Metastasis Of Human Non-small Cell Lung Cancer By Regulating The Expression Of CCL2

Objective:In this study,we investigated the effect of FBW7 on the expression of CCL2 in human non-small cell lung cancer(NSCLC)and observed the possible role of CCL2 derived from tumor cells in invasion and metastasis of tumors through mouse Lewis lung cancer LLC and gene knockout mice models.After knocking down and overexpressing FBW7 in non-small cell lung cancer cells A549 and H1299,the effect of FBW7 on the expression of CCL2 gene and protein was detected.To explore whether FBW7 can exert tumor inhibition by negatively regulating Notch pathway to regulate the level of CCL2 derived from cancer cells,and to validate the role of CCL2 from tumor cells in inhibiting invasion and metastasis of NSCLC by animal models.providing theoretical basis and new ideas for cancer treatment strategies targeting FBW7/Notch/CCL2 axis.Methods:1.Real-time PCR and Western Blot immunoblotting were used to detect the expression of FBW7 in four different human non-small cell lung cancer cell lines.Real-time PCR and enzyme-linked immunosorbent assay(ELISA)were used to detect the expression of CCL2 in four different NSCLC cells.2.The FBW7 Low expression NSCLC cells A549 Sh FBW7 and H1299 Sh FBW7 and their empty control cell lines A549 Sh Ctrl and H1299 Sh Ctrl were constructed by lentiviral transfection technology,The FBW7-Overexpressing NSCLC cells A549-FBW7 and H1299-FBW7 and their empty control cells A549-EV and H1299-EV were constructed by liposome transient transfection method.Real-time PCR and Western Blot immunoblotting were used to detect the interference and overexpression efficiency.Real-time PCR and enzyme-linked immunosorbent assay(ELISA)were used to detect the effect of interference or overexpression of FBW7 on the expression of CCL2.3.Western Blot immunoblotting was used to detect the expression of FBW7 and Notch signaling pathway related proteins such as Notch1,Notch3,C-Myc,C-Myb,Cyling E1,HES1,and MAML-1 in FBW7 knockdown or overexpression cells.4.Cell scratch test and Transwell migration test were used to detect the migration ability of NSCLC cells with low expression of FBW7 and control cells cultured in different conditions.5.LY3039478,an inhibitor of Notch pathway,was used to treat A549,H1299 cells with low expression of FBW7 and control cells.Then use Real-time PCR and ELISA to detect the expression of CCL2.Scratch test and transwell migration test were used to determine whether Notch inhibitors could inhibit the migration ability induced by conditioned medium of cells with FBW7 low expression.6.Lentiviral transfection technique was used to construct Luciferase-labeled mouse Lewis lung cancer cell: LLC-luc-Sh FBW7,a mouse Lewis lung cancer cell with low expression of FBW7 and its control cell LLC-luc-Sh Ctrl,which were injected into wild-type and CCL2 knockout mice by tail vein injection.Dynamic detection of tumor growth and metastasis using small animal in vivo imaging techniques.Results:1.Real-time PCR and Western blot results showed that FBW7 was expressed highest in H1299 cells and lowest in H441 cells;Real-time PCR and ELISA results showed that CCL2 was expressed highest in H441 cells and lowest in H1299 cells.2.The FBW7 Low expression NSCLC cells A549 Sh FBW7 and H1299 Sh FBW7 and their empty control cell lines A549 Sh Ctrl and H1299 Sh Ctrl were successfully constructed by lentiviral transfection technology.Highly expressed NSCLC cells A549-FBW7 and H1299-FBW7,as well as their empty control cells A549-EV and H1299-EV were successfully constructed by transient transfection with liposome.3.The results of Real-time PCR and ELISA showed that FBW7 in knockdown or overexpression of A549 and H1299 cells showed a trend of increasing or decreasing CCL2 expression in tumor cells(P<0.05).4.Western blot showed that down-regulate FBW7,Notch signaling pathway-related proteins Notch1,Notch3,C-Myc C-Myb,Cyling E1,MAML-1and so on increased in A549 and H1299 cells,the expression of HES1 and RBPSu H decreased.While over-expression of FBW7,Notch signaling pathway-related proteins Notch1,Notch3,C-Myc,C-Myb,and Cyling E1 decreased in A549 and H1299 cells and the expression of HES1 increased.5.The cell scratch test and Transwell migration test showed that the cell migration ability of A549 and H1299 cells with low FBW7 expression was significantly enhanced compared with the control cells(P<0.05),and there was no difference in the cell migration ability between the blank control cells cultured in the conventional medium and cultured in the conditioned medium(the secreted supernatant of the control cells).The migration ability of A549 and H1299 cells with low expression of FBW7 was enhanced in conditioned medium(supernatant secreted from FBW7 low-expression cells)compared with that in conventional medium culture(P<0.05);CCL2neutralizer could reverse or inhibit the migration of NSCLC cells induced by CCL2 from tumor cells regulated by FBW7(P<0.05).6.Real-time PCR and ELISA results showed that different concentrations of Notch pathway inhibitor LY3039478 showed no difference in CCL2 expression level in empty control cells,but inhibited the increase of CCL2 level in FBW7 low expression cells(P<0.05);the Scratch test and the transwell migration assay showed that used the Notch pathway inhibitor LY3039478 can inhibited the migration of FBW7 low expression cells induced by conditioned medium(P<0.05).7.Animal experiments showed that the metastasis ability of the four groups of mice was LLC-luc-Sh FBW7 WT mice > LLC-luc-Sh Ctrl WT mice >LLC-luc-Sh FBW7 CCL2 KO mice > LLC-luc-Sh Ctrl CCL2 KO mice,with significant difference(P<0.05).Tumor cells with low expression of FBW7 were more able to grow and metastasize in mice than control cells.Knockout mouse CCL2 gene can inhibit the growth and metastasis ability of FBW7 low expression cells in mice.The increase of tumor cell-derived CCL2 mediated by FBW7 downregulated can promote metastasis in mice,that is,FBW7 negatively regulates tumor cell-derived CCL2 and inhibits the growth and metastasis of tumors in mice.Conclusion:FBW7 inhibits the expression of CCL2 and the invasion and metastasis of non-small cell lung cancer(NSCLC)by negatively regulating Notch pathway.This study provides theoretical basis and new ideas for cancer therapy targeting the FBW7/Notch/CCL2 axis.