Regulatory Role And Mechanism Of IFITM1 On Cell Cycle Of Human Non-small Cell Lung Carcinoma

The interferon-inducible transmembrane protein（IFITM）genes are a class of interferon-stimulated genes,all of which have a highly conserved CD225 domain.The human IFITM genes containing five members are located on chromosome 11.Among them,IFITM1,IFITM2 and IFITM3 have a broad-spectrum antiviral role.The mechanism of immune-related IFITM proteins against enveloped virus entry involves in inhibiting organelle acidification and augmenting membrane rigidity.It was first discovered that the function of IFITMs is related to tumors,which means IFITMs play a critical role in the development of cancers.The high expression of IFITMs is obviously related to the poor prognosis of many cancers,but they exert tumor suppressing effect in some cases,which shows the complexity of IFITMs in the oncogenesis.Although the research of IFITMs has focused on antiviral funtion in the past decade,their role and mechanism in tumorigenesis are worth exploring.IFITM1,also known as 9-27 or Leu13,is a cell surface molecule.It is a component of a multimeric complex involved in the cell adhesion,cell invasion,cell cycle and antiviral process.Due to the deficiency of N-terminal amino acid residues,IFITM1 is mainly located on the cell membrane rather than endocytosomes and lysosomes.Lung cancer is one of the most common cancer with high mortality in the world.Clinically,lung cancer is divided into small cell lung cancer and non-small cell lung cancer,and non-small cell lung cancer（NSCLC）accounts for 75-80%of all lung carcinoma.Our research will focus on the cell cycle regulation about IFITM1 of non-small cell lung cancer cells.Firstly,IFITM1 has been revealed to promote the occurrence and development of non-small cell lung adenocarcinoma at the cellular level.Through analysis of TCGA datebase,we obtained that the transcription level of IFITM1 is closely related to non-small cell lung adenocarcinoma.Thus,we detected the effects of IFITM1 on the proliferation and migration of human non-small cell lung cancer cell line A549.The results indicated that the over-expression of IFITM1 significantly promoted cell proliferation,migration and cell cycle.Meanwhile,A549 cells were deleted IFITM1gene by CRISPR/Cas9 technique,and then we found that the proliferation rate of the IFITM1-knockout A549 cells was reduced about 30%and the related migration rate was also significantly surpressed.Moreover,compared with the wide type A549 cells,G2/M-transition of IFITM1^-/-A549 cells was also obviously delayed through flow cytometric analysis.All these results indicate that the high expression of IFITM1accelerates cell cycle,promotes the proliferation and migration of non-small cell lung adenocarcinoma cells.Subsequently,we preliminarily explored the regulatory role of IFITM1 on the cell cycle of human non-small cell lung cancer A549 cells.Immunoblotting experiments showed that the overexpression of IFITM1 can activate the phosphorylation of AKT1 Ser473 in A549 cells.Meanwhile,the expression of p21was significantly decreased.In contrast,the phosphorylation level of AKT1 Ser473 in IFITM1^-/-A549 cells was significantly inhibited,while the downstream p21expression level of AKT1 was significantly increased.Therefore,we speculated that IFITM1 activates AKT1 by phosphorylating Ser473,and then the activated AKT1induces phosphorylation of p21 Thr145,causing p21 arrested in the cytoplasm.Such p21 plays an anti-apoptotic and proliferative role.IFITM1 may regulate the G2/M phase of A549 cells through the AKT1-p21 signal axis.Finally,we found that 14-3-3 protein,not CDK1,may interact with IFITM1.The proteins interacting with IFITM1 in HEK293 cells were pooled and identified by immunoaffinity and mass spectrometry methods.Baesed on the mass spectrometry data,the protein 14-3-3γwas screened and identified to possibly interact with IFITM1.In order to verify this interaction,vectors expressing CDK1 or 14-3-3 proteins have been constructed,and then Co-IP experiments confirmed that IFITM1 interacted with14-3-3γindependent of its conserved motif directly acting the phosphorylated substrates identified previously.Some studies have reported that 14-3-3γcan directly or indirectly regulate CDK1 activity,and this protein involved in G2/M-transition by disrupting the degradation of p21 protein.Furthermore,the biological significance of this interaction between IFITM1 and 14-3-3γremains to be explored.In summary,IFITM1 promotes the ability of proliferation,migration and cell cycle in A549 cells.IFITM1 may regulate G2/M-transition by activating AKT1-p21signal pathway and the interaction with 14-3-3γ.These results reveal the regulatory role and mechanism of IFITM1 on the cell cycle in human non-small cell lung adenocarcinoma,and provide a new perspective for understanding the biological function of IFITM1.