| Background: Lung cancer,especially non-small cell lung cancer(NSCLC),is currently the leading cause of cancer-related death worldwide.NSCLC accounts for approximately 85% of lung cancer,of which 80% patients are locally advanced or with distant metastasis(stage IIIB-IV)at the time of diagnosis.Radical concurrent chemoradiotherapy has been established as the standard treatment for unresectable locally advanced NSCLC yielding a local remission rate of 61%.However,once brain metastasis occurs,the natural survival of patients is only 2-4 months and the prognosis is extremely poor.The accuracy,sensitivity and specificity of common methods used to determine the mediastinal lymph node staging(MLNS)for unresectable LA-NSCLC needs to be improved urgently,otherwise the occurrence of radiation injury is inevitable.In addition,due to the existence of the blood-brain barrier,patients with brain metastasis lack effective treatment.Therefore,how to accurately delineate the mediastinal lymph node involvement field and prevent the occurrence of radiation lung injury and how to efficiently improve the control rate of brain metastases will provide a theoretical basis for improving the survival of patients with advanced NSCLC and achieving individualized treatment.Method: Ten frozen lung adenocarcinoma specimens with or without EGFR-sensitive mutations were analyzed by RNA-sequencing,bioinformatics analysis and q RT-PCR.The results showed that BCDIN3 D was abnormally overexpressed in lung cancer with EGFR-sensitive mutations in mediastinal lymph node metastasis.The migration ability(cell scratch test),metastasis ability(Transwell test without ECM),invasion ability(Transwell test with ECM)and EMT process(Western blot detection)of PC-9 cells were significantly inhibited after knocked down BCDIN3 D expression by sh RNA lentivirus infection.Using real-time quantitative PCR,Western blot and tissue microarray combined with immunohistochemistry,the efficacy causality,prognosis and clinical significance between BCDIN3 D.Transcriptome sequencing and bioinformatics analysis were performed on the tumor tissues of 10 patients(5 cases each with or without mediastinal lymph node metastasis)who were surgically resected and pathologically diagnosed as lung adenocarcinoma and confirmed as EGFR19 Del or L858 R mutations by EGFR sequencing.Differentially expressed genes(DEGs)between the two groups with and without mediastinal lymph node metastasis were identified and validated by q RT-PCR.The clinical data of 11 patients with lung primary lesions and brain metastases who were surgically resected and pathologically diagnosed as brain metastases of NSCLC in our hospital were collected.Tissue sections and preoperative peripheral venous blood were collected from these 11 patients.The expression levels of PD-L1 in primary lung and brain metastases were detected by immunohistochemistry.The preoperative NLR values were calculated.The ROC analysis was used to analyze the correlation of NLR with ORR,DCR,DFS and OS.Results:(1)RNA sequencing detected the whole transcriptome genes of two groups of NSCLC with/without EGFR-sensitive mutation of mediastinal lymph node metastasis,respectively.169 DEGs were selected by Cuffdiff software analysis,of which 68 DEGs were up-regulated and 101 DEGs were down-regulated in lung cancer samples with lymph node metastasis compared with those without lymph node metastasis.(2)We validated the expression of 68 DEGs in PC-9 cells by q RT-PCR,and screened target genes for the construction of lentivirus by RNAi interference experiment.The results showed that 49 genes highly expressed in PC-9 cells were candidate genes.(3)Interference targets were designed for each gene and corresponding plasmids were constructed.The results showed that when MOI = 1(cell infection has reached more than 60%),Namely,the virus with titer of 1×108 TU/m L was added with 0.1 ?L virus solution and Eni.S+Polybrene was added into the culture medium.Under this condition,the target cells were susceptible to infection and could enter the downstream experiment after lentivirus infection.(4)Three days after lentivirus infection,the expression of BCDIN3 D gene at the m RNA level was inhibited in PC-9 cells of the experimental group.(5)After 3 days of sh RNA lentivirus infection,cell scratch assay was performed,and Cellomics assay showed that the migration ability of PC-9cells in the experimental group was significantly inhibited.This result suggested that BCDIN3 D gene was significantly associated with the migration ability of PC-9 cells.(6)Three days after lentivirus infection,the metastatic ability of PC-9 cells in the experimental group was significantly inhibited.It is suggested that BCDIN3 D gene is significantly associated with the metastatic ability of PC-9 cells.(7)Three days after sh RNA lentivirus infection,the invasive ability of PC-9 cells in the experimental group was significantly inhibited.(8)Western blot showed that the expression of EMT markers of PC-9 cells in the experimental group was inhibited 5 days after sh RNA lentivirus infection.(9)The expression of BCDIN3 D m RNA was detected by q RT-PCR in 54 patients with lung adenocarcinoma who underwent surgical treatment.The results showed that BCDIN3 D was an independent predictor of mediastinal lymph node metastasis at the level of m RNA.(10)The expression of BCDIN3 D protein was detected by immunohistochemistry in primary lung lesions and mediastinal metastases of 54 patients with lung adenocarcinoma in our hospital and 78 patients with lung adenocarcinoma in other hospitals.The results indicated that BCDIN3 D at the protein level was an independent predictor of mediastinal lymph node metastasis in male and 19 Del patients.(11)Transcriptome sequencing was performed on tissue samples from 10 patients by 100 bp pair-end sequencing by using Illumina platform,and 100 genes with down-regulated expression and 69 genes with up-regulated expression were found.(12)Clustering analysis of expression patterns further suggested that EEF1A2 was significantly down-regulated in 19 Del patients and up-regulated in L858 R patients;ERN2 was significantly up-regulated in 19 Del patients and down-regulated in L858 R patients.(13)There were 4 cases with high expression of PD-L1 in lung primary lesions,3cases with high expression in brain metastases,and 5 cases with higher NLR cut-off value than 3.0553 in brain.(14)The results of univariate and multivariate analysis showed that the staining intensity of PD-L1 in brain metastases was an independent prognostic factor for OS(p=0.012).Moreover,the combined assessment of PD-L1 and NLR was significantly associated with OS(p=0.034).Conclusion:(1)Abnormally high expression of BCDIN3 D can predict mediastinal lymph node metastasis in NSCLC with EGFR-sensitive mutation,which could be able to reduce the severity of radiation lung injury by precisely delineating the mediastinal lymph node target of lung cancer in order to narrow the chest radiation field.(2)In the course of mediastinal lymph node metastasis in patients with EGFR19 Del and L858 R mutant lung adenocarcinoma,there are obvious differences in the expression patterns of multiple genes and related molecular mechanisms,suggesting that 19 Del and L858 R may be two relatively distinct diseases.(3)High expression of PD-L1 and high NLR value are associated with poor treatment response and poor prognosis.NLR may be a predictor of the efficacy of PD-L1 antibody. |
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