Expression Of BTLA And Its Role In Promoting Invasion And Migration In NSCLC

Objective: The incidence of lung cancer ranks the first in the world and is one of the main causes of malignant tumor death.NSCLC accounts for about 80% to 85% of lung cancers,adenocarcinoma(ADC,about 40% to 50%)and squamous cell carcinoma(SCC,about 20% to 30%)are the main histological subtypes of NSCLC.Only a small proportion of patients with NSCLC are diagnosed at an early stage(stage I or II),when the tumor can be treated by surgical excision.More than 70% of patients with NSCLC have locally advanced or metastatic disease(stage III or IV)at the time of diagnosis and have lost the opportunity of complete surgical resection.At present,the main treatment methods of advanced NSCLC include chemotherapy,radiotherapy,targeted drugs and TCM treatment.In the past decade,significant breakthroughs have been made in targeted drug therapy,which has become the standard treatment method and significantly improved the survival prognosis of lung adenocarcinoma with driver gene mutation.However,for non-adenocarcinoma patients lacking effective therapeutic targets,the overall survival benefit is limited.Moreover,targeted drugs are still unable to avoid the progression of the disease,and the emerging problem of drug resistance urgently requires new treatment methods to improve the prognosis.Immunotherapy for tumors develops a new way of treatments,which is different from traditional ones.Immunotherapy is not limited to specific pathological types and tumors with target gene mutations.It is an anticancer treatment aimed at activating the human immune system and killing cancer cells and tumor tissues by autoimmune function.Checkpoint inhibitors are one of the important means of immunotherapy,which activate cytotoxic T lymphocytes through blocking the immune checkpoint,reverse the immune escape of tumor cells,and then exclude the tumor.Checkpoint inhibitors have been approved for clinical treatment of malignant melanoma,non-small cell lung cancer,kidney cancer and other malignant tumors.Despite the breakthrough in checkpoint inhibitor therapy,only a small number of patients(about 20%)benefited from monoclonal antibody therapy and showed resistance after treatment for some time,which illustrates the complexity of checkpoint effect in tumor microenvironment.Recent literature has shown that multiple immune checkpoints are often co-expressed on tumor-depleted T cells,suggesting that immune suppression of tumor microenvironment may be co-regulated by multiple groups of synergistic inhibitory molecules.Combined use of checkpoint inhibitors is a feasible strategy for treatment,so it is urgent to find new therapeutic targets.BTLA(B and T lymphocyte attenuator,CD272)was an immunosuppressive receptor newly discovered in CD28 superfamily,which was an important potential target of immunotherapy.Previous studies have found that the high expression of BTLA in melanoma limits the expression and function of CD8+T cells.HSP70 vaccine down-regulated the expression of BTLA in mouse cervical cancer model to improve its anti-tumor effect.Studies on single nucleotide polymorphism of BTLA have shown that BTLA polymorphism is related to the occurrence and prognosis of breast cancer.BTLA is more highly expressed in gastric cancer tissues than in adjacent normal tissues,and is associated with clinical characteristics such as lymph node metastasis,tumor invasion depth,and high-level differentiation,etc.,and it is an influential factor for poor prognosis.So far,little is known about the expression and function of BTLA protein in NSCLC.Therefore,this study aims to investigate the expression of BTLA in NSCLC and its correlation with immune checkpoint PD-1/ PD-L1,tumor infiltrating lymphocyte(TILs),clinicopathological characteristics and survival prognosis,as well as further explore the regulatory effect of BTLA on the cell function of NSCLC and the possible signal transduction mechanism.Research methods: 1.Expression of BTLA,PD-1,PD-L1 and TILs in non-small cell lung cancer and their related survival and prognosis analysis.This study collected 87 cases of Shengjing hospital of China medical university with a diagnosis of NSCLC(I-III)in patients with paraffin embedding tissue section and the corresponding pathological data and total survival time,through immunohistochemical method to detect BTLA,PD-1,PD-L1 expression in cancerous tissue,then through a chi-square test to evaluate BTLA expression and clinical pathological characteristics of correlation,the Spearman method evaluating BTLA with PD-1,PD-L1,the correlation of TILs,Kaplan-meier method was used to analyze the relationship between the expression of immune checkpoints and the survival time of patients.2.Expression of BTLA in four NSCLC cell linesThe expression of BTLA protein in four NSCLC cell lines(A549,H226,H1650 and H460)was detected by immunofluorescence assay,and the expression of cell line protein in the four NSCLC cell lines was determined by Wbstem blot.Two NSCLC cell lines with high BTLA protein expression were selected for subsequent experiments.3.Effect of BTLA protein silencing on the biological behavior of NSCLC cell linesIn this study,BTLA sh RNA recombinant plasmid sh-BTLA was used to construct btla-silenced A549 and H226 non-small cell lung cancer cell lines,and transfected negative control sh RNA recombinant plasmid(sh-NC)and non-transfected blank cells were used as control.Real-time fluorescence quantitative PCR(quantitative PCR)and Wbstem blot were used to determine the expression of BTLA at 48 h after transfection.Cell Counting kit-8(cck-8),Annexin v-fitc /PI double staining apoptosis assay,Cell scratch assay,and Transwell invasion assay were used to detect the proliferation,apoptosis,migration,and invasion abilities of A549 and H226 NSCLC Cell lines transfected with sh-BTLA and sh-NC as well as the blank control group,respectively.4.Effects of BTLA silencing on EMT and NF-κB signaling pathwaysWestern stem blot was used to determine the expression levels of certain EMT and NF-κB signaling pathways in A549 and H226 NSCLC cell lines transfected with sh-BTLA,the constructed silent BTLA group,the negative control group transfected with sh-NC,and the blank control group.The expression levels and distribution of p65 phosphorylated in the NF-κB signaling pathway were detected by immunofluo-rescence in two cell lines(A549 and H226)transfected with sh-BTLA,negative control group with sh-NC,and blank control group without transfection.Results: 1.BTLA and PD-L1 are mainly expressed in the cytoplasm and cell membrane of tumor tissues in NSCLC,and are occasionally expressed in lymphocytes in tumor interstitium.PD-1 protein is mainly expressed in lymphocytes in tumor interstitium.The expression of BTLA in the group with lymph node metastasis was higher than that in the group without lymph node metastasis(P=0.045),and the expression in the tumor stage III group was higher than that in the I-II group(P=0.034).The expression of PD-L1 was closely related to smoking(P=0.011)and lymph node metastasis(P=0.019),among which the expression of PD-L1 in smokers was higher than that in non-smokers(44.8% vs.17.2%),and the expression of PD-L1 in the lymph node positive group was higher than that in the negative group(50.0% vs.22.5%).The expression of PD-1 was not significantly correlated with the clinical and pathological characteristics.2.The high expression of BTLA was positively correlated with the high expression of PD-L1(P=0.011),but not with the expression of PD-1 and TILs.3.The RFS of patients with positive BTLA expression were shorter than those with negative BTLA expression(P=0.029).RFS and OS of patients with positive expression of PD-L1 were shorter than those with negative expression of PD-L1(P=0.016;P = 0.034).4.Patients with negative BTLA and negative PD-L1 had longer RFS and OS than patients with positive BTLA or positive PD-L1 or positive at both checkpoints(P=0.012;P = 0.031).5.The expression of BTLA protein in NSCLC cell lines(A549,H226,H1650,H460)was detected by cell immunofluorescence method,and the expression of BTLA protein in four NSCLC cell lines was determined by Wbstem blot.The A549 and H226 cell lines with high expression were screened for subsequent experiments.6.Both quantitative PCR and Westem blot results showed that the expression levels of BTLA in A549 and H226 cells transfected with sh-BTLA were significantly lower than those transfected with sh-NC and blank cells(P<0.05).7.The results of cck-8 assay and Annexin v-alexa Flour 647/PI kit double staining assay showed no statistical difference in cell proliferation and apoptosis between the experimental group transfected with sh-BTLA and the negative control group transfected with sh-NC and the blank control group.8.Cell scratch experiments were conducted at 0h,24 h and 48 h after sh-BTLA transfection.The results showed that the cell migration ability of the experimental group transfected with sh-BTLA was weaker than that of the negative control group transfected with sh-NC and the blank control group(P<0.05).9.Transwell assay was performed 48 h after plasmid transfection,and the results showed that the cell invasion ability of the experimental group transfected with sh-BTLA was weaker than that of the negative control group transfected with sh-NC and the blank control group(P<0.05).10.Proteins were extracted from cells treated with transfection plasmid for 48 h,and Western Blot results showed that e-cadherin protein was up-regulated,n-cadherin protein was down-regulated,Vimentin protein was down-regulated,Snail protein was down-regulated,and Slug protein was down-regulated in the btla-silenced group(P<0.05).11.Adding the NF-κB inhibitor bay11-7082 with a concentration of 5μM in the medium for 72 hours,the protein was collected for Western blot detection,which significantly inhibited the phosphorylation of NF-κB p65,decreased the expression of Snail protein,and increased the expression of e-cadherin protein(P<0.05).12.Proteins were extracted from cells treated with transfected plasmids for 48 h.Western Blot results showed that the expression of NF-κB phosphorylated p65 protein was down-regulated and the expression of phosphorylated IKb-αprotein was down-regulated in the btla-silenced group.Cell immunofluorescence assay showed that NF-κB p65 nuclear transfer was down-regulated.Conclusion: 1.BTLA and PD-L1 are expressed in non-small cell lung cancer(NSCLC),higher than normal tissue around.BTLA is higher expressed in the lymph node positive group than the negative group,and higher in the stage III tumor than in the stage I-II patients.The expression of PD-L1 in smokers was higher than that in non-smokers,and the expression in the lymph node positive group was higher than that in the negative group.2.The expression of BTLA in non-small cell lung cancer is positively correlated with the expression of PD-L1.3.Patients with high expression of BTLA have shorter RFS,while patients with high expression of PD-L1 have shorter RFS and OS.4.Patients with both high expression of BTLA and PD-L1 had worse prognosis.5.BTLA silencing may inhibit the epithelial mesenchymal transtion of NSCLC cells by inhibiting the NF-κB/Snail signaling pathway,thereby inhibiting the migration and invasion of NSCLC cells.