| Background:The incidence and mortality of lung cancer ranks first among all cancers.Most lung cancers(~80%)are Non-small cell lung cancer(NSCLC),causing at least 1.5 million deaths each year.The high incidence,high mortality,high metastasis,severe complications,short life cycle and physical and psychological damage caused by NSCLC are urgently urging people to explore the molecular mechanism of NSCLC development.Establish effective targets for new therapeutic strategies.CD300 a is a type I transmembrane protein expressed in the bone marrow and lymphoid lineage and regulates the immune response by its inhibitory capacity.CD300 a has an immunoglobulin(Ig)V-like extracellular domain,a transmembrane domain,and a cytoplasmic tail.Interestingly,the CD300 a gene locus is one of the few sites in the genome that is under strong positive evolutionary selection and may indicate the importance of this receptor family in immune regulation.Currently,it has been confirmed that CD300 a functions as an inhibitory receptor in various immune cell types.Crosslinking of human CD300 a with m Ab inhibits Ca2+ mobilization in response to stimulation mediated by B-cell receptor(BCR),T-cell receptor(TCR),Fce RI and Fcg RIIa.It also inhibits NK cell-mediated cytotoxicity,B cell proliferation,Ig E-dependent mediator release from mast cells,stem cell factor(SCF)-mediated mast cell activation,differentiation and survival,and basophils Ig E-mediated CD63 expression,eosinophil chemokine-induced eosinophil migration and IL-5 and GM-CSF-induced eosinophil survival,Fcg RIIa-mediated neutrophil-producing reactive oxygen species Substance,LPS and Cp G oligodeoxynucleotides(Cp G)-induced myelomonocytic THP-1and U937 cells secrete IL-8.CD300 a is also involved in the mechanisms by which viruses are used to develop immune evasion strategies and to infect host cells.The involvement of CD300 a in viral infections includes lipid-dependent and non-dependent mechanisms.Specifically,the virus covers Phosphatidylserine(PS)and Phosphatidy-lethanolamine(PE)in various ways.Simulate apoptotic cells and debris.It is well known that exogenous PS is essential for apoptotic cell clearance of professional(eg macrophages)and non-professional phagocytic cells.It has been reported that CD300 a can be involved in the process of virus entry into host cells as a PS-binding protein.Similarly,CD300 a binds to apoptotic cells via PS and PE and transmits an inhibitory signal that prevents monocyte-derived macrophages(MDM)from phagocytizing dead cells.Furthermore,blocking the receptor with an antibody(Abs)in MDM that naturally expresses CD300 a results in a decrease in the number of infected cells.In addition to viral particle binding associated with PSand PE,CD300 a has also been shown to bind and promote viral entry in a PSand PE-independent manner.In large-scale protein interaction screening,the interaction of several human adenovirus E3 proteins with host cells was identified and validated.Among them,the human adenovirus-D47 E3/49 K protein binds to CD300 a and CD300 c pairing receptors.Their extracellular domains share extensive homology,but intracellular domains determine their inhibitory and activating functions.CD300 a is also involved in viral escape mechanisms,such as inhibition of NK cell-mediated cytotoxicity against infected cells.Or participate in T cell failure during chronic HIV-1 infection.In the past few years,there has been increasing evidence that the receptor family in which CD300 a is located is involved in the pathogenesis of many diseases.For example,expression of CD300 a on mast cells,eosinophils,and basophils is involved in allergic reactions that initiate and regulate these three important cell types,leading to the design of bispecific antibody fragments targeting CD300 a and other receptors.Body,the purpose is to downgrade these functions.In addition to its role in allergic processes,mast cells are also known to play an important role in mouse cecal ligation and puncture peritonitis models,with a large number of cells undergoing apoptosis in the peritoneal cavity.After cecal ligation and puncture,CD300a-deficient peritoneal mast cells produce more chemoattractants,leading to neutrophil recruitment and better bacterial clearance.Therefore,CD300a2/2 mice showed prolonged survival.Theseresults indicate that CD300 a regulates the inflammatory response of mast cells to microbial infection.Combined with three other genes,CD300 a has been proposed as a blood-based biomarker that distinguishes between ulcerative colitis and Crohn's disease and non-inflammatory diarrhea.This may contribute to the serological detection of inflammatory bowel disease currently based on reactivity to bacterial antigens.Other genetic correlation studies have shown that CD300 a may be involved in the etiology of Alzheimer's disease.Currently,to the best of our knowledge,four studies have reported the role of CD300 a in tumors.Jiang et al found a negative correlation between CD300 A m RNA levels and overall survival in patients with diffuse large B-cell lymphoma(DLBCL).Decreased levels of CD300 a in OCI-Ly01,Farage and SUDHL-4 cells significantly inhibited cell proliferation but did not affect VAL,OCI-Ly10,or SUDHL-8 cell proliferation.In addition,xenograft animal models have also shown that a decrease in CD300 a levels in OCI-Ly01 and Farage cells significantly inhibits tumor formation in vivo.Molecularly,it was found that decreased expression of CD300 a resulted in a significant decrease in AKT phosphorylation,a key molecular event in tumorigenesis in OCI-Ly01,Farage and SUDHL-4 cells.In order to determine the new markers for the detection of Minimal residual disease(MRD)in acute lymphoblastic leukemia(ALL),Smith et al.compared the genome-wide expression and normalization of lymphocytes in 270 newly diagnosed children with ALL.Whole genomeexpression of CD19+CD10+ B cell progenitor cells,and differential expression of CD300 a and other markers in ALL was identified.In 16 differentially expressed markers including CD300 a,minimal residual disease detection,including antibodies against CD19,CD10 and CD34,and one of the new markers was verified by 4-color flow cytometry analysis.The latest study found that CD300 a is highly expressed in acute myeloid leukemia(AML)and is associated with prognosis.Decreased expression of CD300 a promotes apoptosis and inhibits proliferation and migration of the AML cell line U937,as well as promotes activation of the AKT/m TOR pathway.These results demonstrate that CD300 A acts as a tumor promoter in AML cells.In this study,we first discovered the role of CD300 a in the pathogenesis and development of NSCLC beyond viral infection and cellular immunity.These results suggest that CD300 a still has many very critical functions that we don't know,and it is worthwhile for us to continue our research.Methods:CD300a m RNA was analyzed using RNA data from 483 lung adenocarcinoma(LUAD)samples and 347 paracancerous tissues in GEPIA,as well as RNA data from 486 lung squamous cell carcinoma(LUSC)samples and 338 adjacent tissues.Expression changes in NSCLC.Subsequently,we collected 23 clinical samples of tumors from NSCLC patients and corresponding adjacent normal tissues and their serum samples,and recruited23 healthy volunteers to also collect their serum samples.Using q RT-PCR technology,we examined the m RNA expression levels of CD300 a in these tissues and serum samples.We used GEPIA to analyze the survival of patients with high and low expression of CD300 a.The 23 patients with NSCLC were also divided into two groups according to the expression of CD300a: low expression of CD300a(12 cases)and high expression of CD300a(11 cases).),and the correlation analysis between CD300 a expression level and overall survival was performed on 23 patients.Subsequently,at the cellular level,NSCLC cell lines A549 and H1650 were used as cell models in vitro,and CD300 a knockdown(KD)and CD300 a overexpressing(OV)cell lines were constructed by transfecting si RNA-CD300 a and pc DNA3.1-CD300 a,respectively.The effects of CD300 a overexpression or knockdown on cell proliferation and growth were examined using CCK8 assays and colony formation assays.Transwell experiments were used to investigate the effect of upregulation or downregulation of CD300 a expression on cell migration.The effect of CD300 a overexpression or knockdown on apoptosis was detected by flow cytometry,and the expression of apoptosis-related proteins was detected by weatern blot.Finally,an in-depth look at the signaling pathways involved in the role of CD300 a in NSCLC cells.In vivo animal model experiments,nude mice xenograft models of CD300 a knockdown(KD),CD300 a overexpression(OV)and their corresponding negative controls(NC-KD and NC-OV)wereconstructed,respectively.Every 3 days,the volume of the transplanted tumor was measured and recorded,and then the tumor growth curve was drawn.After18 days,the nude mice were sacrificed,the transplanted tumor was taken out,and the apoptosis of the tumor tissue was detected by TUNEL staining.Results:GEPIA analysis showed that CD300 a m RNA expression was significantly down-regulated in NSCLC(P < 0.05).The clinical sample tests we collected showed that the m RNA expression of CD300 a in NSCLC tumor tissues was significantly down-regulated compared to adjacent normal tissues(P < 0.001).This result is consistent with the results of the GEPIA analysis.Analysis of m RNA levels of CD300 a in serum samples showed that CD300 a m RNA levels were significantly down-regulated in NSCLC patients compared to healthy volunteers(P < 0.001).The m RNA expression profile of CD300 a in the serum of NSCLC patients is consistent with its expression trend in tumor tissues.It was also found that the expression level of CD300 a was significantly correlated with the prognosis of patients with NSCLC(P = 0.029).Compared with the cells in the CON group,the absorbance values of the A549 and H1650 cells in the OV group were not significantly changed after transfection of pc DNA3.1-CD300 a for 24 h and 48 h,but the absorbance at 72 h after transfection.The value was significantly reduced(P<0.05).Compared with the CON group,CD300 a knocked down had no significant effect on the absorbance value(P>0.05).The results of the clone formation experiment were consistent with the results of the CCK8 experiment.It was found that CD300 a overexpression significantly reduced the number of clones and the size of clones formed in A549 and H1650 cells compared with the CON group(P<0.01).However,CD300 a knockdown did not significantly affect the number and size of cell-forming clones(P>0.05).Compared with the control group,the number of migrated cells of CD30 A overexpressing A549 and H1650 cells decreased to about 1/2 and 2/3,respectively(P< 0.05);the number of migrated cells of CD50 A knockdown A549 and H1650 cells increased respectively.1.7 times and 1.3 times(P< 0.05).Compared with the control group,the apoptotic rate of CD300 a overexpressing A549 cells increased significantly from 8.78 ±0.95%to 27.45±4.63%(P<0.01);the apoptosis rate of CD300 a overexpressing H1650 cells was also 1.97 ± 1.45%.Significantly increased to19.13 3.56%(P < 0.01).In contrast,the apoptotic rates of CD300 a knockdown A549 and H1650 cells were reduced to 3.50 ±0.47% and 5.67 ± 0.75%,respectively(P<0.05).Compared with the control group,the expression of the key anti-apoptotic protein Bcl-2 in CD350 A overexpressing A549 and H1650 cells was significantly down-regulated,while the expression of Bcl-2 was up-regulated in CD300 a knockdown cells(P<0.05).At the same time,when CD300 a was up-regulated in A549 or H1650 cells,the expression of Bax and Caspase3 p17(key pro-apoptotic protein)was significantly increased;when CD300 a wasknocked down,the expression of Bax and Caspase3 p17 was decreased(P<0.05).We also observed a significant decrease in the expression of Wnt3 and?-catenin in the CD300 a overexpressing group compared with the control group(P< 0.05).When CD300 a was up-regulated in A549 and H1650 cells,the expression of downstream protein E-cad,a key cell adhesion-associated protein,increased accordingly(P< 0.05).In addition,knockdown of CD300 a resulted in a significant decrease in ?-catenin expression and a significant decrease in E-cad expression in A549 and H1650 cells,but no effect on Wnt3 expression.The experimental results on the xenograft model of nude mice are also basically consistent with the results of the cell experiments.Over time,the volume of subcutaneous xenograft tumors in the NC-OV group and the NC-KD group continued to increase from the initial 0.2 cm3 to 1.07 ± 0.14 cm3(day18),and there was no significant difference between the two groups.difference.After inoculation from xenograft tumors,the volume of subcutaneous xenograft tumors in nude mice of OV group was always smaller than that of NC-OV group;and there was a significant difference in volume after 18 days of inoculation(0.75 ± 0.08 cm3 vs.1.07 ± 0.14 cm3,P< 0.05).Unlike the effect of CD300 a overexpression on the growth of transplanted tumors,CD300 a knockdown did not affect the growth of transplanted tumors.This conclusion can be drawn from the fact that there is no significant difference between the subcutaneous xenograft volume in the KD group and the NC-KD group.Finally,the TUNEL staining results showed that compared with the NC-OV group,the apoptosis of the transplanted tumor tissues of the OV group was significantly increased.Compared with the NC-KD group,there was no significant change in the apoptosis of the transplanted tumor tissues of the KD group.Conclusion:This study found that CD300 a is abnormally low in NSCLC and can be used as a potential predictor.Low expression of CD300 a predicts a poor prognosis.In addition,we found that CD300 a may play a tumor suppressor role in the progression of NSCLC by inhibiting the Wnt/?-catenin pathway and regulate the biological function of NSCLC cells. |
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