The Role And Mechanism Of ADAMTSL4 And PZP In Regulating The Development Of Lung Adenocarcinoma

Part 1,The mechanism of ADAMTSL4 in inhibiting the development of lung adenocarcinoma by regulating GPX2 expression Background and Aims: Lung cancer is a malignant tumor with high morbidity and mortality in the world,and it ranks first in China.Lung adenocarcinoma(LUAD)is main subtype,and despite the availability of various treatments such as radiotherapy,chemotherapy,targeted therapy,and immunotherapy,the 5-year survival rate of patients is still less than20%,mainly due to the following reasons: LUAD is prone to local invasion and distant metastasis,as well as the lack of diagnostic and prognostic indicators with high sensitivity and specificity.ADAMTSL4 is a secreted glycoprotein widely expressed in tissues such as eye,brain and lung,and studies in some tumors such as nasopharyngeal carcinoma and glioblastoma have shown its involvement in tumor development.However,its role and mechanism in LUAD have not been reported.The aim of this study was to detect the expression of ADAMTSL4 in LUAD,analyze the relationship between its expression level and patient prognosis,explore the function of ADAMTSL4 in LUAD,and then explore its mechanism in the development of LUAD.The study will provide new ideas and methods for the clinical diagnosis and treatment of LUAD.Methods:(1)We analysis of m RNA expression levels of ADAMTSL4 in pancancer including LUAD using bioinformatic methods.And IHC was used to detect the expression of ADAMTSL4 protein in 110 LUAD tissues and paracancerous tissues,and WB was used to detect the basal expression level of ADAMTSL4 protein in lung cancer cell lines and normal cell lines.The relationship between ADAMTSL4 protein expression levels and clinicopathological factors in 110 LUAD patients was analyzed by the chi-square test,the relationship between high and low ADAMTSL4 protein expression levels and overall survival(OS)of patients was analyzed by the Kaplan-Meier method,and the prognostic factors affecting LUAD patients were analyzed by Cox regression.The diagnostic value of ADAMTSL4 in patients with LUAD was evaluated by receiver operating characteristic(ROC).(2)The LUAD cell lines A549 and PC-9 with low endogenous ADAMTSL4 expression were used to construct stable transgenic cell lines with ADAMTSL4 overexpression and knockdown by lentiviral transfection,then we detect the overexpression and knockdown efficiency by WB.CCK-8 and clone formation assays were used to detect the proliferation ability of ADAMTSL4 overexpression and knockdown LUAD cells.Wound healing and transwell assays were used to detect the cell migration and invasion ability.Flow cytometry(FCM)was used to detect the cell cycle and apoptosis rate.In addition,a subcutaneous tumor formation model was established in nude mice to observe the growth volume and plot the growth curve,the mice were executed and weighed after 35 days to observe the effect of ADAMTSL4 on the proliferation ability of tumor cells in vivo.(3)Transcriptomics and proteomics were used to analyze the m RNA and protein expression profiles of ADAMTSL4 overexpressing LUAD cells,compared with the control cell line,the differentially expressed genes and proteins were screened,and cluster analysis and KEGG signaling pathway enrichment analysis were performed to screen out the significantly down-regulated molecule glutathione peroxidase GPX2.Further,the m RNA expression levels of GPX2 in different cancer species were first analyzed using bioinformatics methods,and KM online data was used to analyze the effect of GPX2 expression on patients’ OS.Next,GPX2 was transfected into LUAD cells stably overexpressing ADAMTSL4 by plasmid transfection technique.q RT-PCR and WB were applied to detect the m RNA and protein expression levels of ADAMTSL4 and GPX2 in each group of cells.CCK-8 and EDU were used to detect cell proliferation ability,wound healing and transwell assays were used to detect cell migration and invasion ability,FCM were used to detect cell cycle and apoptosis rate.Fluorescence microscopy was used to observe the cell positive rate of ROS occurrence in cells,and FCM was used to detect the mean fluorescence intensity(MFI)of ROS occurrence in cells.Results:(1)Bioinformatics analysis suggested that ADAMTSL4 m RNA was expressed at lower levels in a variety of tumor tissues than in normal tissues(both paired and unpaired),with ADAMTSL4 m RNA expression significantly downregulated in LUAD tissues compared to normal lung tissues.In 110 paired clinical samples of LUAD,IHC assays revealed significantly lower expression of ADAMTSL4 in tumor tissues than in paracancerous tissues,WB results showed lower levels of ADAMTSL4 protein in lung cancer cell lines than in normal cell lines.ADAMTSL4 expression was correlated with clinicopathological stage in LUAD patients,but not with gender,age,or smoking,and univariate and multivariate COX regression analyses showed that ADAMTSL4 was an independent risk factor,and OS was significantly lower in patients with low ADAMTSL4 expression than in patients with high expression,and the ROC curve showed that ADAMTSL4 had an area under curve(AUC)value of 0.753(95% CI: 0.688-0.817)in LUAD patients,suggesting that ADAMTSL4 has some diagnostic value in patients with LUAD.(2)We successfully constructed ADAMTSL4 overexpressing and knockdown LUAD cell lines A549 and PC-9.CCK-8 assay showed that the OD value of cells in the ADAMTSL4 overexpression group was lower than that of the control group,while the OD value of cells in the knockdown group was higher than that of the control group.Clone formation assay showed that the number of clone formation was significantly reduced after ADAMTSL4 overexpression,while that of cells in the knockdown group was significantly increased,indicating that ADAMTSL4 could inhibit the proliferation ability of LUAD cells.The wound healing assay showed that the migration ability of cells was reduced after ADAMTSL4 overexpression and enhanced in the knockdown group.Transwell assay showed that the number of cells in the overexpression group penetrating the vesicles was less than that in the control group,while the number of cells in the knockdown group was more than that in the control group,indicating that ADAMTSL4 could inhibit the migration and invasion ability of LUAD cells.The FCM results showed that the percentage of apoptosis was higher in the overexpression group and lower in the knockdown group than in the control group,while the percentage of G0/G1 phase was increased in the overexpression group and decreased in the knockdown group,indicating that ADAMTSL4 could block the cell cycle in(G0/G1)phase,reduce DNA synthesis and thus inhibit cell division.Subcutaneous tumorigenesis experiments in nude mice revealed that the ADAMTSL4 overexpression group had slower subcutaneous tumor growth and significantly smaller tumor volume and weight than the control group,indicating that ADAMTSL4 could inhibit tumor cell proliferation in vivo.(3)Transcriptomic analysis screened a total of 2995 differential genes,including 1688 up-regulated genes and 1307 down-regulated genes.Proteomic analysis screened 372 differential proteins,including 228 upregulated proteins and 144 down-regulated proteins.Pathway enrichment analysis of differential genes and proteins revealed significant enrichment of ROS and glutathione metabolic pathways,among which,glutathione peroxidase GPX2 was significantly down-regulated,suggesting that ADAMTSL4 may be involved in the development of LUAD through regulating GPX2 expression.Further studies showed that GPX2 m RNA was expressed at elevated levels in most tumors,including LUAD,and analysis of KM online data showed that patients with high GPX2 expression in LUAD had a poor prognosis.Simultaneous overexpression of GPX2 in ADAMTSL4 overexpressing cell lines,and CCK-8 and EDU assays together showed that GPX2 was able to reverse the ability of ADAMTSL4 to inhibit the proliferation of LUAD cells.Wound healing assay and transwell assay showing that GPX2 reverses the ability of ADAMTSL4 to inhibit the migration and invasion of LUAD cells.FCM showing that GPX2 reverses ADAMTSL4-induced apoptosis and cycle arrest in LUAD cells.Meanwhile,ADAMTSL4 could elevate intracellular ROS levels in LUAD cells and could be reversed by GPX2,indicating that ADAMTSL4 could regulate intracellular ROS levels through GPX2.Conclusions: ADAMTSL4 may serve as a new prognostic biomarker in LUAD.ADAMTSL4 inhibits the development of LUAD through the GPX2/ROS pathway,and targeting the ADAMTSL4/GPX2 pathway may be a new strategy for the treatment of LUAD.Part 2,The study of PZP in affecting the immune microenvironment of lung adenocarcinoma by regulating M1 polarization of macrophage Background and Aims: Tumor development is a complex process,which is regulated by multiple mechanisms.In recent years,a large number of studies have suggested the interaction between tumor immune microenvironment and tumor cells.PZP is an immune-related protein widely expressed in liver,uterus,lung and other tissues,and plays an important role in pregnancy immunity,liver cancer immunity,and heart transplantation immunity,but its role in LUAD is unknown.The purpose of this study was to investigate the prognostic value of PZP in LUAD and its role in the immune microenvironment.Methods:(1)The m RNA expression level of PZP in pan-cancer was analyzed by bioinformatics.IHC was used to detect the expression of PZP protein in110 LUAD tissues and corresponding paracancerous tissues.q RT-PCR and WB were used to detect the expression levels of PZP m RNA and protein in LUAD cell lines and normal lung epithelial cell lines,and ELISA was used to detect the amount of PZP secreted protein in the supernatant.The chi-square test was used to analyze the relationship between PZP protein expression level and clinicopathological factors in110 patients with LUAD,Kaplan-Meier method was used to analyze the relationship between PZP expression level and OS of patients,Cox regression analysis was used to analyze the factors related to prognosis of LUAD patients,R packages rms was used to construct nomogram to predict prognostic information,consistency index and calibration plots were used to evaluate the nomogram,and ROC curves were drawn to evaluate the diagnostic value of PZP in LUAD patients.(2)We used GSEA to analyze the signaling pathways associated with PZP expression in the TCGA-LUAD dataset.TIMER online database was used to analyze the correlation of PZP m RNA with 6 tumor infiltrating immune cells(TIICs),and CIBERSORT was used to analyze the correlation of PZP m RNA with 22 TIICs.IHC was used to verify the expression levels of PZP with CD4+ T cells and CD68+macrophages in clinical samples of LUAD patients,and Multiplex Immunohistochemistry(m IHC)staining was used to detect the expression levels of PZP with CD86+M1 macrophages and CD206+M2 macrophages.The PMAstimulated suspended THP-1 cells were used for the walled M0 macrophages,and the supernatant of PC-9 cell conditioned medium was used to culture M0 macrophages,while lipopolysaccharide(LPS)and interferon-γ(IFN-γ)were added to stimulate M1-type polarization,where the experimental group added PZP recombinant protein at a concentration of 100 ng/ml,and the effect of PZP on macrophage polarization was observed by detecting the proportion of CD86 fluorescent antibody by FCM after 24 h.Results:(1)Bioinformatic analysis suggested that PZP m RNA was expressed at lower levels in a variety of human tumor tissues,including LUAD,than in non-cancerous tissues(both paired and unpaired),and the m RNA expression level of PZP in LUAD was lower in stages III&IV than in stages I&II,and the ROC curve showed an AUC of 0.789(95% CI:0.759-0.819),indicating PZP has some accuracy in diagnosing LUAD.Survival analysis showed that OS was significantly prolonged in patients with high PZP expression compared with those with low expression,univariate and multivariate Cox regression analysis showed that PZP was an independent risk factor,and nomogram showed that the 1-,3-,and 5-year survival probabilities of LUAD patients were significantly associated with PZP expression.The calibration curve assessed the nomogram performance of PZP with a concordance index(C-index)of0.703(95% CI: 0.679-0.727)for OS,indicating that our nomogram is consistent with the actual results.The above bioinformatics analysis preliminarily revealed that PZP plays an anti-tumor role in the development of LUAD.(2)In 110 clinical samples collected from LUAD patients,PZP expression was significantly lower in tumor tissues than in paracancerous tissues by IHC.q RT-PCR and WB results showed that PZP expression levels in lung cancer cell lines were lower than in normal lung epithelial cell lines,and ELISA results showed that the amount of PZP protein secreted in the supernatant of LUAD cell lines was lower than in normal lung epithelial cell lines.PZP expression was correlated with clinicopathological stage in LUAD patients,but not with gender,age,or smoking,and univariate and multivariate COX regression analyses revealed that PZP was an independent risk factor,and OS was significantly lower in patients with low PZP expression than in patients with high PZP expression,and the ROC curve showed that the AUC of PZP in LUAD was 0.804(95% CI: 0.747-0.862),suggesting that PZP has a high diagnostic value in patients with LUAD.(3)GSEA analysis showed that high expression of PZP in LUAD was associated with multiple immune-related pathways or molecules.TIMER database shows that PZP expression is associated with B cells,CD4+ T cells,macrophages,neutrophils and dendritic cells in LUAD.CIBERSORT results showed that PZP expression was positively correlated with resting memory CD4+ T cells,M1 macrophages,resting mast cells,and activated mast cells,and negatively correlated with M0 macrophages.IHC showed that PZP expression was negatively correlated with the infiltration level of CD68+ M0 macrophages and positively correlated with the infiltration level of CD4+ T cells.In addition,m IHC showed that PZP expression led to an increase in CD86+ M1 macrophages and a decrease in CD206+ M2 macrophages.PZP recombinant protein promoted LPS and IFN-γ stimulation of THP-1-derived M0 macrophages to M1-type macrophage polarization in vitro,and these results suggest that PZP may regulate macrophage M1 polarization and affect the immune microenvironment of LUAD.Conclusions: PZP expression is reduced in LUAD and is associated with poor patient prognosis.In addition,PZP may be involved in the regulation of TIICs in the immune microenvironment of LUAD,especially in regulating macrophage polarization,and PZP may be a biomarker of LUAD prognosis and a potential immune-related therapeutic target.