| BackgoundILT4(immunoglobulin-like transcript 4),also known as lymphocyte immunoglobulin-like receptor 2(LIR2),monocyte/macrophage immunoglobulin-like receptor 10(MIR-10),or CD85d,The protein was discovered in 1997 and belongs to the family of activated and inhibitory immUnder normal physiological conditions,ILT4 is mainly expressed in innate immune cells,such as monocytes,macrophages,dendritic cells(DC)and granulocytes,unoglobulin-like transcripts(ILTs),which regulate the activation of immune cells.When ILT4 on the surface of immune cells binds to ligands,SHP-1 or SHP-2 phosphatase containing SH-2 domain can be recruited to inhibit calcium mobilization and thus inhibit the activation signal of these cells.The expression of ILT4 in innate immune cells and endothelial cells can be significantly induced by some pathological factors,including inflammatory cytokines(IL-10,TGF-beta,IFN-alpha,IL-16 or thrombopoietin),interaction with regulatory T cells(Treg)and mesenchymal stem cells,and immunosuppressive drugs(dexamethasone,nifluoric acid,rapamycin).Vegetatin and resveratrol,pathogen infection and Toll-like receptor(TLR)signal transduction.It is reported that abnormal or ectopic expression of ILT4 mediated by these pathological factors can promote the progress of allograft rejection,autoimmune and infectious diseases.Although significant progress has been made in the study of the role of ILT4 in immune-related diseases,its role in tumorigenesis and progression has not been fully studied.Recent studies have shown that ILT4 is highly expressed in tumor cells and related stromal cells of some tumors,including leukemia,non-small cell lung cancer(NSCLC),breast cancer,esophageal cancer and pancreatic cancer.Studies have shown that ILT4 may directly regulate the biological behavior of these malignant tumor cells.However,the role of ILT4 in NSCLC and its related mechanisms are still unclear.MMPs(matrix metalloproteinases)secreted from connective tissue are a family of proteases that degrade extracellular matrix.There are high structural homologies among the members of the MMPs family.The MMPs protease family plays an important role in wound healing,extracellular matrix formation,and tumor invasion and metastasis.MMP-2 is an important member of the MMPs family.MMP-2 can not only degrade extracellular matrix,but also decompose collagen type IV(the main component of basement membrane).MMP-2 plays an important role in regulating cell migration,promoting tumor growth and angiogenesis.To create a more suitable microenvironment for invasion and metastasis of tumors.Relevant studies have shown that MMP-2 is overexpressed in many kinds of tumors.It is also confirmed that the expression of MMP-2 is closely related to the invasion,metastasis and prognosis of tumors,which is of great significance in predicting the risk of metastasis and recurrence of tumors.PI3K is an important member of the phospholipid kinase family.According to the functional and structural differences of PI3K,PI3K can be divided into three types:I,? and ?.So far,PI3K,which is relatively widely studied,is PI3K of type I,and PI3K of type I can be further divided into two subtypes,A and B,according to the difference between the binding subunits of PI3K and p110.IA-type P13K is composed of catalytic subunits,i.e.p110a,p110beta,p110delta and regulatory subunits(p85).Compared with IA-type PI3K,IB-type PI3K has p110gamma catalytic subunit.PI3K reacts with tyrosine residues(growth factor receptor,connexin,Ras protein)to activate PI3K.The activated PI3K induces the phosphorylation of phosphatidylinositol 4,5-bisphosphonate(PIP2)to phosphatidyl inositol 3,4,5-triphosphate(PIP3),while the PH domain in Akt can be combined with PIP3,making the catalytic domain of AKT1 thr308(Thr309 of AKT2 and Thr305 of AKT3)were phosphorylated by phosphatidyl inositol-dependent kinase 1(PDK1),Ser473 of C-terminal hydrophobic region of AKT1,Ser474 of AKT2 and Ser472 of AKT3 were phosphorylated by MTORC2 to activate AKT.P-AKT activates MTORC1,p70S6 kinase(p70s6 kinase,p70s6K)and eukaryotic initiating factor 4E binding protein 1(4E-binding protein 1,4E-BP1)by phosphorylating 1/2 of tuberous sclerosis complex(TSC)to convert Rheb GDP into Rheb GTP.Research purposeA large number of studies have shown that ILT4 is highly expressed in different types of malignant tumors,and the abnormal high expression of ILT4 is one of the important factors leading to the occurrence and development of tumors.However,the role of ILT4 in invasion and metastasis of non-small cell lung cancer(NSCLC)and its molecular mechanism are still unclear.Numerous studies have shown that MMP-2 and MMP-9 play an important role in the invasion and metastasis of various tumors,especially NSCLC.However,it is not clear whether ILT4 plays an important role in invasion and metastasis of NSCLC through MMP-2 or MMP-9.In view of this,this study first observed the expression of ILT4,MMP-2 and MMP-9 in NSCLC by immunohistochemistry and analyzed the correlation;then through the cell experiment in vitro,to explore the impact of ILT4 knockout on the invasion and metastasis of non-small cell lung cancer,and further explore the impact of ILT4 knockout on the relevant mechanism.Research Methods1.The expression of ILT4,MMP-2 and MMP-9 in non-small cell lung cancer tissues was verified by immunohistochemical method at protein level.The expression differences of ILT4,MMP-2 and MMP-9 in adjacent normal tissues and NSCLC cancer tissues were statistically analyzed,and the correlation between ILT4 and MMP-2 and MMP-9 was analyzed.2.The follow-up data of patients with non-small cell lung cancer were collected,survival curve was drawn by Kaplan-Meier method,and the relationship between ILT4 expression and survival time was analyzed by log-rank test.3.Silicon-ILT4 was transfected into A549 and/or H1299 cells by Lipofectaemine 2000 reagent,and ILT4 gene was knocked out in A549 and/or H1299 cells.4.Quantitative RT-PCR was used to detect the difference of MMP-2 and MMP-9 levels in A549 and H1299 cells after ILT4 knockout.5.By transfecting siRNA,the expression of ILT-4 in NSCLC cell lines A549 and H1299 was inhibited in vitro.Negative control group(transfected empty carrier)and normal control group without any treatment were set up to observe the migration ability,invasion ability and the level of related protein expression.6.Through cell migration test,we analyzed the effect of IL T-4 knockout on the migration ability of two kinds of non-small cell lung cancer cells in vitro.7.Transwell test was used to analyze the effect of ILT-4 knockout on the invasive ability of two kinds of non-small cell lung cancer cells in vitro.8.Werstern Blot method was used to detect the related proteins in each group of cells,and the effects of IL T4 knockdown on the related signaling pathways were analyzed.9.Silicon-ILT4 was transfected into NSCLC cells.Silicon-ILT4,which was used to knock out ILT4,was transfected into A549 cells by Lipofectaemine 2000,and the effect on cell morphology was observed.10.To verify the effect of ILT4 on the proliferation of A549 cells in NSCLC cell line,MTT method was used to detect the proliferation rates of NC,si-NC and si-ILT4 groups11.In order to investigate the effect of IL T4 knockout on apoptosis of A549 cells,flow cytometry was used to detect apoptosis of A549 cells treated with IL T4 knockout.At the same time,the cell cycle distribution of A549 cells in each group was detected.12.The effects of ILT4 on PI3K-AKT signaling pathway-related proteins and downstream P53 and MMP-2 proteins in A549 cells were detected by Western Blot.Research Results1.Immunohistochemical results showed that the expression levels of ILT4,MMP-2 and MMP-9 in cancer tissues of patients with stage ?-? and ?-? NSCLC were significantly higher than those in normal tissues adjacent to cancer(P<0.05).The expression of ILT-4 and MMP-2 was significantly higher in stage ?-? NSCLC than in stage ?-? NSCLC(P<0.05).However,there was no significant difference in MMP-9 expression between NSCLC patients in stage ?-? and ?-?(P>0.05).2.Survival analysis showed that the overall prognostic survival rate of ILT4 positive patients was significantly lower than that of ILT4 negative patients.3.Cell migration test showed that silencing ILT4 gene in A549 and H1299 cells significantly inhibited the migration ability of A549 and H1299 cells(P<0.05).The results showed that the expression of ILT4 in NSCLC cell lines A549 and H1299 was increased,and the migration ability of A549 and H1299 cells was significantly inhibited after silencing ILT4.4.Transwell invasion test showed that silencing ILT4 gene in A549 and H1299 cells significantly inhibited the invasion ability of A549 and H1299 cells(P<0.05).5.The results of qRT-PCR showed that the gene levels of ILT4 and MMP-2 in A549 and H1299 cells silenced ILT4 gene were significantly lower than those in control cells(P<0.05),but there was no significant difference in the gene level of MMP-9(P>0.05).6.Immunoblotting assay showed that the expression of ILT4 and MMP-2 in A549 and H1299 cells silenced ILT4 gene was significantly lower than that in control cells(P<0.05),but there was no significant difference in the expression of MMP-9(P>0.05).7.After ILT4 was silenced in A549 cells,the proliferation rate of A549 cells was significantly inhibited,while the apoptotic rate of A549 cells increased significantly with a large number of A549 cells remaining in G1 phase.8.In A549 cells,PI3K-AKT signaling pathway was significantly inhibited with the decrease of biological activity(proliferation,invasion and migration)of A549 cells after ILT4 silencing.Meanwhile,the expression of P53 downstream protein was significantly increased while the expression of MMP-2 protein was significantly decreased.Conclusion1.ILT4 is one of the main factors inducing metastasis and invasion of non-small cell lung cancer.The induction of ILT-4 may be achieved by up-regulating the expression of MMP-2,which has no significant correlation with the expression of MMP-9.ILT-4 is a potential diagnostic marker for the prognosis of non-small cell lung cancer(NSCLC)patients.2.ILT4 is highly expressed in NSCLC tissues,and the expression of ILT4 is closely related to the clinical stage of NSCLC,and there is a positive correlation between the expression of ILT4 and the clinical stage of NSCLC.ILT4 can be used as a diagnostic biomarker and therapeutic target for patients with advanced or metastatic non-small cell lung cancer.3.After ILT4 silencing,the invasion and migration ability of non-small cell lung cancer cell lines A549 and H1299 were significantly inhibited.4.The anti-cancer effect of ILT4 silencing may be achieved by inhibiting the activity of MMP-2 protein.5.ILT4 silencing can significantly inhibit the biological activity of non-small cell lung cancer,and its mechanism may be related to the positive regulation of PI3K-AKT signaling pathway by ILT4. |
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