| Background Lung cancer,originating from bronchial epithelial cells or alveolar epithelial cells,is histologically divided into small cell lung cancer and non-small cell lung cancer(NSCLC),the latter accounting for about 85%of lung cancer.Lung adenocarcinoma(LUAD)and lung squamous cell carcinoma(LUSC)are the main histological subtypes of NSCLC.The latest global cancer statistics show that lung cancer is one of the most common malignancies in the world,and is the leading cause of cancer-related deaths worldwide.Metastasis is the main cause of high mortality of lung cancer,because many patients have local invasion or distant metastasis at the time of diagnosis,which makes the survival rate of lung cancer decline sharply.Although targeted drugs have been applied in patients with metastatic lung cancer,there are still many patients who cannot benefit from the treatment.In order to identify new diagnostic and prognostic markers for lung cancer and reveal the molecular mechanism of lung cancer metastasis,we analyzed the lung cancer transcriptome data from The Cancer Genome Atlas(TCGA)database,and found that glutathione peroxidase 2(GPX2)expression in NSCLC tissues was significantly higher than that in normal lung tissues,and the prognosis of NSCLC patients with high GPX2 expression was poor,which suggested that GPX2 may affect the progression and prognosis of NSCLC patients.GPX2 is a glutathione peroxidase,which is mainly distributed in the whole gastrointestinal tract,including esophagus and liver,and regulates intracellular reactive oxygen species.It participates in anti-inflammatory,prevents intestinal mucosal inflammation,and protects epithelial cells from damage caused by lipid peroxides intake.GPX2 participates in antioxidant,and inhibits the occurrence of inflammation-related tumors.The results of bioinformatics analysis based on TCGA data are shown that GPX2 is up-regulated in some cancers,including LUAD and LUSC.However,the mechanism of GPX2 in NSCLC is still unclear.In this study,we investigated the GPX2expression and its clinical significance in NSCLC,as well as the function and possible molecular mechanism of GPX2 in NSCLC cells,hoping to provide a new prospects for clinical diagnosis and treatment of NSCLC.MethodsⅠ.Expression and clinical significance of GPX2 in NSCLC1.The pan-cancer transcriptome data from TCGA database were downloaded to analyze the expression of GPX2 mRNA in various cancers including LUAD and LUSC.2.The expression of GPX2 mRNA in 10 pairs of fresh NSCLC tissues(cancer and adjacent normal lung tissues)was detected by RT-PCR and agarose gel electrophoresis.3.The expression of GPX2 mRNA in 46 pairs of fresh NSCLC tissues(cancer and adjacent normal lung tissues)was detected by RT-qPCR.4.The expression of GPX2 protein in 252 pairs of NSCLC paraffin-embedded tissues(cancer and adjacent normal lung tissues)was detected by immunohistochemistry staining.The expression level of GPX2 protein was evaluated by immunohistochemical score-based semi-quantitative method.The clinicopathological data of the patients were collected,and the survival status and survival time of the patients were investigated through telephone follow-up.5.X~2test was used to analyze the relationship between GPX2 protein expression level and clinicopathological factors in 252 NSCLC patients.Kaplan-Meier method and Log-rank test were used to analyze the relationship between GPX2 protein expression level and overall survival(OS)in all NSCLC patients and the patients with the same pathological factors.Cox regression was used to analyze the prognostic factors of NSCLC patients.6.Immunohistochemistry was used to detect the expression of GPX2 protein in tissues(cancer tissues,adjacent normal lung tissues and positive/negative lymph nodes)of 31 NSCLC patients with lymph node metastasis.Immunohistochemical score was performed by semi-quantitative method.Spearman correlation coefficient and X~2test were used to analyze the correlation between GPX2 protein expression in primary cancer tissues and that in lymph node metastases of NSCLC.7.The diagnostic value of GPX2 in NSCLC,LUAD and LUSC was analyzed by receiver operating characteristic(ROC)curve.Ⅱ.Function and mechanism of GPX2 in NSCLC cells(Ⅰ)Effects of GPX2 on EMT,migration,invasion and metastasis of NSCLC cells1.Expression of GPX2 mRNA in 7 NSCLC cell lines was detected by RT-qPCR,with human bronchial epithelial cell line(HBE)and human lung fibroblast cell line(HLF)used as control.Western blotting and immunocytochemistry were used to detect the expression of GPX2 protein in 7 NSCLC cell lines.2.NSCLC cell lines with high endogenous GPX2 expression were selected to construct stable GPX2 knockdown cell lines through viral transfection.NSCLC cell lines with low endogenous GPX2 expression were selected to construct stable GPX2overexpression cell lines by virus transfection.Knockdown efficiency and overexpression efficiency of GPX2-stable knockdown or overexpression NSCLC cell lines were detected by RT-qPCR and Western blotting.3.Morphological changes of NSCLC cells with GPX2 stable overexpression were observed by light microscope.4.Western blotting was used to detect the expression of epithelial-mesenchymal transition(EMT)-related molecular markers in GPX2-stable knockdown or overexpression NSCLC cell lines.5.Wound healing assay was used to evaluate the migration abilitity of GPX2-stable knockdown or overexpression NSCLC cell lines.6.Transwell assays(without or with matrigel)were performed to evaluate the migration and invasion ability of GPX2-stable knockdown or overexpression NSCLC cell lines.7.The lung metastasis model with tumor cells injected into the tail vein of nude mice was established,and the metastasis ability of GPX2-stable knockdown NSCLC cells in vivo were evaluated by the changes in number and size of lung metastasis nodules.Immunohistochemical staining was used to detect the expression of GPX2 and EMT-related markers in pulmonary metastatic nodules.(Ⅱ)Molecular mechanism of GPX21.After DCFH-DA probe treatment,fluorescence microscopy and flow cytometry were used to detect the level of reactive oxygen species(ROS)levels in GPX2-stable knockdown or overexpression NSCLC cell lines.2.RNA-sequencing(RNA-seq)was performed in GPX2 knockdown or control NSCLC cell lines.Transcriptome data were analyzed and differentially expressed genes(DEGs)were screened.3.Gene Ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis were performed for the above DEGs.4.The expression of the related-proteins in downstream signaling pathway regulated by GPX2 was validated by western blotting.In GPX2-stable overexpression or knockdown NSCLC cells.ResultsⅠ.GPX2 was overexpressed in NSCLC and was associated with poor prognosis1.A pan-cancer analysis based on TCGA database showed that GPX2 mRNA was differentially expressed in multiple human tumors.The expression of GPX2 mRNA was significantly higher in both LUAD and LUSC tissues than their adjacent normal lung tissues.2.In 10 pairs of fresh NSCLC tissues,GPX2 mRNA was not detected in adjacent normal lung tissues,but it was detected in 5 cancer tissues with a positive rate of 50%.3.In 46 pairs of fresh NSCLC tissues,the mRNA level of GPX2 in cancer tissues was significantly higher than that in adjacent normal lung tissues.4.In 252 pairs of paraffin-embedded tissues,GPX2 protein was expressed in both NSCLC,LUAD and LUSC,but almost not expressed in adjacent normal lung tissues.GPX2 protein was expressed in 166 NSCLC samples(86 samples with high GPX2expression and 80 samples with low GPX2 expression),while it GPX2 protein was not expressed in 86 NSCLC samples.5.In 252 patients with NSCLC,GPX2 protein expression was correlated with gender(P<0.001),histological type(P<0.001),lymph node metastasis(P<0.001),tumor size(P=0.017),and TNM stage(P=0.022).6.NSCLC,LUAD,or LUSC patients with high GPX2 expression had a shorter overall survival than those with low GPX2 expression(P<0.001 for NSCLC and LUAD patients;P=0.017 for LUSC patients).7.Univariate Cox model analysis showed that GPX2 expression,lymph node metastasis,tumor size,and TNM stage were all significant prognostic factors for NSCLC patients(all of them,P<0.001).Multivariate Cox model analysis showed that GPX2,as well as tumor size and TNM stage,was identified as an independent prognostic factor for NSCLC patients(P=0.013,hazard ratio HR=1.600).8.By stratified patients with clinicopathological factors,we found that high GPX2expression could also predict poor overall survival in patient subgroup with tumor size>3cm(P=0.001),positive lymph node metastasis(P=0.038),TNM stage I(P=0.010),or TNM stage III(P<0.001),but not in patients with tumor size≤3cm,negative lymph node metastasis,or TNM stage II(P>0.05).9.In samples of 31 NSCLC patients with positive lymph node metastasis,GPX2was expressed in the metastatic lung cancer cells of lymph node,but not in lymphocyte of lymph nodes.The expression of GPX2 protein was higher in primary cancer tissues and lymph node metastases than that in adjacent normal lung tissues(P<0.001).A strong correlation between GPX2 expression in lung cancer tissues and that in lymph node metastases was observed(Spearman r=0.834,P<0.001).These results suggest that GPX2 is closely related to lymph node metastasis of NSCLC.10.The area under the curve(AUC)of GPX2 in NSCLC,LUAD,and LUSC was0.793,0.767,and 0.835,respectively.Meanwhile,based on the analysis of NSCLC data in TCGA database,we found the result showed that the AUC of GPX2 mRNA in NSCLC,LUAD,and LUSC was also more than 0.7.These results suggest that GPX2has high sensitivity for NSCLC,LUAD and LUSC diagnosis.Ⅱ.The function and possible molecular mechanism of GPX2 in NSCLC(Ⅰ)GPX2 induced EMT in NSCLC cells and promoted migration and invasion of NSCLC cells,while knockdown of GPX2 inhibited the metastasis of NSCLC cells in nude mice.1.The expression of GPX2 mRNA in A549 and H520 cells was higher than that in other lung cancer cell lines,as well as HBE and HLF cells.The expression of GPX2protein in A549 and H520 cells was higher than that in other lung cancer cell lines.2.Compared with control cells,the expression levels of both GPX2 mRNA and protein were up-regulated in GPX2-stable overexpression Anip973 cell lines,and were down-regulated in GPX2-stable knockdown A549 and H520 cell lines(sh1 and sh2).These results indicate that GPX2-stable overexpression or knockdown NSCLC cell lines were successfully constructed.3.Anip973 cell lines with GPX2-stable overexpression showed a trend of mesenchymal-like morphological feature compared with control cell lines.4.Compared with control cells,GPX2 overexpression led to downregulated E-cadherin but upregulated Vimentin and Snail in Anip973 cells,while GPX2knockdown had the opposite effects in A549 or H520 cells.These results suggeste that GPX2 may promote EMT by up-regulating Snail protein.5.Wound healing assay showed that overexpression of GPX2 prominently promoted the scratch healing capacity of Anip973 cells,while knockdown of GPX2suppressed this capacity in A549 or H520 cells.6.In Transwell assay without or with matrigel,overexpression of GPX2 enhanced the migration and invasion capacities of Anip973 cells,while knockdown of GPX2suppressed the migration and invasion capacities in A549 or H520 cells.7.In the experiment of lung metastasis by injection of tumor cells into the tail vein of nude mice,the number and size of lung metastatic nodules in the experimental group(the mice injected with GPX2-stable knockdown A549 cells)were less than those in the control group(the mice injected with control A549 cells).The result was further confirmed by H&E staining.Meanwhile,immunohistochemical staining results showed that knockdown of GPX2 upregulated E-cadherin expression,but decreased Vimentin expression.These findings suggest that knockdown of GPX2 can inhibit the metastasis ability of NSCLC cells in vivo.(Ⅱ)GPX2 downregulates ROS levels of NSCLC cell lines and activates PI3K/AKT/mTOR signaling pathway1.The results of fluorescence microscopy and flow cytometry showed that overexpression of GPX2 reduced the fluorescence intensity of probes in Anip973 cells,while knockdown of GPX2 enhanced the fluorescence intensity of probes in A549 or H520 cells.These results suggest that GPX2 can downregulate ROS levels of NSCLC cell lines.2.The result of RNA-seq showed that 296 DEG_Swith 249 upregulated and 47downregulated genes were identified,when GPX2 was knocked down by sh1,while150 DEGs with 102 upregulated and 48 downregulated genes were screened out,when GPX2 was knocked down by sh2.3.GO enrichment analysis of these DEGs showed that in sh1 vs shNC(Control)dataset,the functions of DEGs were mainly enriched in cell-cell adhesion via plasma-membrane adhesion,extracellular matrix,plasma membrane protein complex,extracellular structure organization,proteinaceous extracellular matrix and so on.Meanwhile,in sh2 vs shNC dataset,the functions of DEGs were mainly enriched in inflammatory response,adherens junction,extracellular matrix,proteinaceous extracellular matrix,membrane region and so on.4.KEGG analysis of these DEGs showed that in sh1 vs shNC dataset,the main enrichment pathways were PI3K-AKT signaling pathway,cell adhesion molecules(CAMs),Ras signaling pathway and so on.In sh2 vs shNC dataset,the main enrichment pathways were PI3K-AKT signaling pathway,Ras signaling pathway,Relaxin signaling pathway and so on.Collectively,this result indicates that PI3K/AKT signaling pathway is the important downstream pathway regulated by GPX2.5.Compared with control cells,GPX2 overexpression increased the PI3K and AKT phosphorylation level but not total PI3K and AKT in Anip973 cells.In contrast,GPX2 knockdown decreased the PI3K and AKT phosphorylation levels,but not total PI3K and AKT in A549 or H520 cells.6.To further confirm the effect of GPX2 on PI3K/AKT pathway,we evaluated the phosphorylation level of mTOR,which is the direct downstream target of PI3K/AKT pathway.The result showed that compared with control cells,overexpression of GPX2increased the mTOR phosphorylation(p-mTOR)level but not total mTOR in Anip973cells,while knockdown of GPX2 led to decreased p-mTOR level in A549 or H520cells.Conclusion1.GPX2 was highly exprssed in NSCLC tissues,GPX2 was almost not expressed in adjacent normal lung tissues.2.High GPX2 protein expression was associated with poor prognosis of NSCLC patients.3.GPX2 protein expression was correlated with lymph node metastasis,tumor size,and TNM stage of NSCLC patients.4.There was a strong correlation between GPX2 protein expression in lung cancer tissues and that in lymph node metastases of NSCLC patients.5.GPX2 prometed EMT,migration and invasion of NSCLC cells in vitro,while knockdown of GPX2 inhibited the metastasis of NSCLC cells in nude mice.6.GPX2 downregulated ROS levels of NSCLC cell lines and activated PI3K/AKT/mTOR signaling pathway. |
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