| Background and Objective:It has been confirmed that cancer metastasis-related molecules play an important role in the process of cancer metastasis and can be used to evaluate the risk of disease progression after surgery.Metastasis-associated protein 1(MTA1)plays an important role in the invasion and metastasis of various cancers,but its mechanism in the invasion and metastasis of non-small cell lung cancer(NSCLC)has not been fully elucidated.The proportion of multifocal non-small cell lung cancer(MNSCLC)in NSCLC is increasing year by year,and the formulation of postoperative management strategies for ≤3 cm MNSCLC is based on its correct classification.For MNSCLC with the same histological type,there is still a lack of clinically operable classification criteria,which makes it more difficult to formulate postoperative management strategies and seriously affects the prognosis of patients.This study aimed to reveal the mechanism of MTA1 in the invasion and metastasis of NSCLC,and to clarify the clinical application of MTA1 protein expression in risk stratification of disease progression in ≤3 cm MNSCLC.Methods:This study was conducted in two parts:1.The mechanism of MTA1 in the invasion and metastasis of NSCLC1.1 The role of MTA1 in the adhesion,invasion and migration of NSCLC:The experimental cells were selected according to the basal level of MTA1 protein expression in the cells and cell migration ability.MTA1 knockout H1299 cells were constructed by CRISPR/Cas9 technology,and MTA1 overexpressed A549 cells were constructed by lentiviral transfection technology.The effects of MTA1 on cell proliferation,clone formation,invasion,migration and adhesion were evaluated from the cellular level.The effects of MTA1 on H1299 cells in the formation of subcutaneous xenograft tumors and lung metastases in nude mice were evaluated from the animal level.1.2 MTA1 affected the adhesion,invasion and migration of NSCLC cells by inhibiting the expression of tight junction protein 1(TJP1)and regulating tight junctions between cells:The proteins interacting with MTA1 in H1299 cells were identified by immunoprecipitation combined with mass spectrometry.TJP1,a MTA1 interacting protein involved in tumor cell adhesion was screened by bioinformatics.The expression of TJP1 in tumor tissues was evaluated by immunohistochemistry.The direct interaction between MTA1 and TJP1 was clarified from the aspects of immunofluorescence co-localization and reverse immunoprecipitation.The regulatory effect of MTA1 on TJP1 was revealed from the cellular and nude mouse tissue levels.The regression experiment was used to prove that MTA1 regulated the adhesion,invasion and migration of NSCLC cells by inhibiting TJP1 expression.2.Clinical application of MTA1 protein expression in risk stratification of disease progression in ≤3 cm MNSCLCPatients with ≤3cm MNSCLC who were pathologically diagnosed after surgery in the Third Affiliated Hospital of Kunming Medical University were selected according to the inclusion and exclusion criteria,the tissue sections and clinical data of the patients were collected.Immunohistochemical techniques and semi-quantitative immune response scoring methods were used to evaluate the level of MTA1 protein expression in tumor tissue.Repeated measure analysis of variance and other methods were used to analyze the correlation between MTA1 protein expression and clinical characteristics of patients with ≤3 cm MNSCLC.Cox proportional hazards regression model was used to analyze the clinical value of MTA1 protein expression in risk stratification of disease progression in MNSCLC ≤3 cm.Results:1.The mechanism of MTA1 in the invasion and metastasis of NSCLC1.1 The role of MTA1 in the adhesion,invasion and migration of NSCLC:1)H1299 cells with strong migration ability and the highest level of MTA1 protein expression and A549 cells with strong migration ability and low level of MTA1 protein expression were selected as experimental cells.2)MTA1 knockout H1299 cells and MTA1 overexpression A549 cells were successfully constructed.3)Knockout of MTA1 significantly inhibited the migration and invasion ability of H1299 cells,but enhanced the adhesion ability,and had no significant effect on proliferation and clone formation.Overexpression of MTA1 significantly enhanced the migration and invasion ability of A549 cells,but inhibited adhesion ability,and had no significant effect on proliferation and clone formation.4)Knockout of MTA1 had no significant effect on the subcutaneous tumor formation of H1299 cells in nude mice,but inhibited H1299 cells from forming lung metastases in nude mice.1.2 MTA1 affected the adhesion,invasion and migration of NSCLC cells by inhibiting the expression of TJP1 and regulating tight junctions between cells:1)951 proteins were identified by mass spectrometry from immunoprecipitation products,and cell adhesion related proteins were significantly enriched in biological processes,cell component and molecular functions by enrichment analysis.TJP1,the MTA1 interacting protein involved in tumor cell adhesion,was screened out by bioinformatics analysis and scoring value of protein identified by mass spectrometry.2)The expression levels of MTA1 protein and TJP1 protein in NSCLC cells had a negative correlation trend,and the expression level of TJP1 protein in NSCLC cancer tissue was lower than that in normal lung tissue.3)MTA1 protein co-located with TJP1 protein in the cytoplasm and membrane of NSCLC cells.Direct interaction between MTA1 protein and TJP1 protein was confirmed by reverse immunoprecipitation.4)MTA1 inhibited the expression of TJP1 protein in H1299 cells,and had the same effect in nude mice subcutaneously transplanted tumors and lung metastases.MTA1 regulated the intercellular tight junction of H1299 cells.5)The inhibitory effect of MTA1 on H1299 cell adhesion could be partially recovered by TJP1,and the promoting effect of MTA1 on H1299 cell migration and invasion could be partially recovered by TJP1.2.Clinical application of MTA1 protein expression in risk stratification of disease progression in ≤3 cm MNSCLC1)Among the 119 patients who met the inclusion and exclusion criteria,52 were multiple primary lung cancer(MPLC),23 were intrapulmonary metastasis(IPM),and 44 were classified as uncertain.There were 25 patients with mediastinal lymph node metastasis(MLNM).Among the 259 lesions,244 were adenocarcinoma,7 were squamous cell carcinoma,8 were other types.The 156 adenocarcinoma lesions were divided into low-risk(n=62),moderate-risk(n=73)and high-risk(n=21)according to the invasiveness of pathological subtypes.The follow-up time was 4.27-76.53(months),and the median follow-up time was 25.97(months).2)The expression of MTA1 protein in the adjacent lung tissue was negative or weakly positive,the expression in lung cancer tissue was positive to varying degrees and the expression in cancer metastatic lymph nodes was strongly positive.MTA1 protein was expressed in the nucleus and cytoplasm of cancer tissue,it was mainly expressed in the cytoplasm of lung mucinous adenocarcinoma.The median value of immune response score of MTA1 protein expression in lung cancer tissue was 5.6,79 patients had high expression of MTA1 protein,and 40 patients had low expression of MTA1 protein.3)The level of MTA1 protein expression was significantly correlated with nodule density,MLNM and lesion classification.The rate of strongly positive MTA1 protein expression in lung cancer tissue of MLNM patients was significantly higher than that of lung cancer tissue of patients without MLNM.The level of MTA1 protein expression in IPM cancer tissue was higher than MPLC.The level of MTA1 protein expression in in highly invasive lesions of lung adenocarcinoma is higher than that in moderate and low invasive lesions.The disease-free survival of patients with high MTA1 protein expression was significantly worse than that of patients with low MTA1 protein expression.4)The results of Cox proportional hazards model univariate and multivariate analysis showed that high MTA1 protein expression,non-Xuanwei area,non-ipsilateral lung disease and MLNM were predictors of worsening disease-free survival in MNSCLC patients<3 cm.Patients with response score>5.6 have an increased risk of postoperative recurrence,and MTA1 protein can be used to assess the risk of disease progression in ≤3 cm MNSCLC patients.Conclusion:1.MTA1 inhibits NSCLC cell adhesion,promotes cell migration and invasion,and promotes NSCLC cells to form lung metastases in nude mice,suggesting that MTA1 may be a potential important molecular marker reflecting NSCLC invasion and metastasis.2.MTA1 can inhibit the expression of TJP1 protein co-located in the cytoplasm and cell membrane,weaken the tight junctions between cells,and change the ability of cell adhesion,migration and invasion,which may be the mechanism of MTA1 promoting the invasion and metastasis of NSCLC.3.MTA1 protein expression level is significantly correlated with MLNM,intrapulmonary metastasis and lung adenocarcinoma pathological subtype aggressiveness in patients with ≤3 cm MNSCLC.MTA1 protein can be used for risk stratification of disease progression in patients with ≤3 cm MNSCLC,screening out high-risk patients with postoperative disease progression,and providing reference for the selection of postoperative management strategies for such patients. |
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