Effect Of Id4 On Proliferation,Invasion And Metastasis Of Colon Cancer Cell HCT116

Objective To explore the effects of differentiation inhibitory factor 4(Id4)on the proliferation,invasion and metastasis of colorectal cancer cells and mechanisms of it.Methods Id4 m RNA level and protein expression of colorectal cancer cells was detected by real-time PCR and western blot.Lentivirus system was used as a carrier to transfer Id4 gene colorectal cancer cell lines HCT116,and an Id4 expressed HCT116 cell line was established.WST test detection,real-time cell analysis system(RTCA),clone formation experiment and both FACS cell cycle we used to detect cell proliferation ability;Scratch test and the transwell migration and invasion detection were used to detect ability of cell invasion and metastasis;The morphological changes of HCT116 cells with Id4 over-expression were observed under microscope.The molecules involved in the proliferation,metastasis,EMT were detected with western blot assay.The protein what interacts with Id4 was detected with Co-IP and mass spectrometry.Results 1.According to PCR assay and Western blot detection,there is not found Id4 expression in the four colorectal cancer cells HCT116,SW480,SW620 and HCT-8.2.Id4 over-expression in HCT116 cells suppressed the proliferation and clone formation ability(p < 0.05),and result in,the cell cycle arrest in G0 / G1 phase(p <0.05),with the down-regulation of survivin,PCNA,BCL-2,cyclin D1,cyclin E,p-PI3 K,PI3K,p-AKT,AKT,and up-regulation of p21 and p27(p < 0.05).3.The scratch healing ability and migration or invasion ability was reduced in HCT116 cells with Id4 expressed,with down-regulation of MMP2 and MMP7,and up-regulation of TIMP1 and TIMP2(p < 0.05).4.The morphology of Id4 expressed HCT116 cells became round instead of long shuttle,with up-regulation of E–cadherin,and down-regulation of ?-catenin,twist,slug and snail(p < 0.05).5.It was found that Id4 could interact with keratin K18 and down-regulate the expression of K18,p-K18(S33)and p-K18(S52)in HCT116 cells,according to Co-IP assay and mass spectrometry.Conclusion 1.Id4 suppressed the proliferation of HCT116 cells by inhibiting the expression of survivin,PCNA,Bcl-2,cyclin D1 and cyclin E,as well as inhibiting the PI3K/AKT pathway.2.Id4 inhibit the cell invasion and metastasis of HCT116 by down-regulating MMP2 and MMP7,and up-regulating TIMP1 and TIMP2.Id4 inversed the EMT of HCT116 cells by up-regulation of E–cadherin,and down-regulation of ?-catenin,twist,slug and snail.3.The interaction of Id4 and keratin K18 might decrease the level of K18 or the phosphorylation,and result in the inhibition of invasion and metastasis of HCT116 cells.