Objective·To investigate the expression of protein tyrosine phosphatase receptor type N(PTPRN)in lung adenocarcinoma and its potential molecular mechanisms in promoting lung adenocarcinoma metastasis.Methods·A highly bone-metastatic A549-BM cell line was established through multiple rounds of intracardiac injection.RNA sequencing(RNA-seq),combined with Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses,was performed to identify PTPRN as a key metastasis-related gene.Subsequently,The Cancer Genome Atlas(TCGA)database was utilized to evaluate PTPRN expression in patients with lung adenocarcinoma and its correlation with clinical prognosis.Co-expression analysis based on TCGA data was conducted to identify and analyze key genes co-expressed with PTPRN.Small interfering RNA(siRNA)targeting PTPRN(siPTPRN)was transfected into A549-BM cells,and Transwell assays were performed to assess its effects on cell migration and invasion.Western blotting was used to detect the expression of epithelial-mesenchymal transition(EMT)-related proteins and the activation of the PI3K-AKT signaling pathway.siPTPRN-transfected A549-BM cells were injected into a mouse model via intracardiac injection,and in vivo metastasis was assessed.Additionally,multiple database analyses were integrated to predict BCL6 as an upstream transcription factor of PTPRN,and siBCL6 transfection experiments were performed to validate the regulatory effect of BCL6 on PTPRN expression.Results·RNA-seq and GO/KEGG enrichment analyses demonstrated that PTPRN was significantly upregulated in highly metastatic A549-BM cells and enriched in metastasis-associated pathways,including the PI3K-AKT signaling pathway and extracellular matrix(ECM)-receptor interactions.Analysis of the TCGA database further confirmed that PTPRN was highly expressed in lung adenocarcinoma patients and significantly associated with poor prognosis.Co-expression analysis based on TCGA data,combined with GO/KEGG enrichment analyses,revealed that PTPRN-associated genes were mainly enriched in biological processes such as neural signaling,endocrine regulation,cell communication,and ECM-receptor interactions.In vitro experiments demonstrated that siPTPRN transfection significantly inhibited the migration and invasion of A549-BM cells,accompanied by downregulation of EMT-related proteins and reduced activation of the PI3K-AKT signaling pathway.In vivo experiments further showed that PTPRN knockdown markedly suppressed the metastatic potential of A549-BM cells,confirming its pro-metastatic role.Additionally,siBCL6 transfection experiments demonstrated that BCL6 knockdown upregulated PTPRN expression.Conclusion·PTPRN is highly expressed in lung adenocarcinoma tissues and promotes tumor cell migration and metastasis by enhancing EMT and activating the PI3K-AKT signaling pathway.High PTPRN expression is significantly correlated with poor prognosis in lung adenocarcinoma patients,while PTPRN enhances lung adenocarcinoma cell invasiveness and metastatic potential.BCL6 may act as an upstream transcriptional regulator of PTPRN,influencing its expression levels.

Objective·To investigate the expression level of O-GlcNAc transferase(OGT)in non-small cell lung cancer(NSCLC)and its impact on lung cancer proliferation,as well as to explore the underlying mechanisms.Methods·The expression of OGT in NSCLC tumors and adjacent normal tissues was detected by immunohistochemistry(IHC).The dataset(GSE31210)from the GEO database was analyzed to assess the correlation between OGT expression and NSCLC patient prognosis.siRNA transfection was performed to knock down OGT expression in H460 and H1299 cells,followed by total RNA extraction and transcriptome sequencing.Pathway enrichment analysis was conducted on differentially downregulated genes in the knockdown group compared with the control group,and Western blotting was used to validate the enrichment results.The effects of OGT knockdown on cell proliferation and colony formation in H460 and H1299 cells were evaluated using the cell counting kit-8(CCK-8)assay and colony formation assay,respectively.The impact of overexpressing downstream genes was also examined.Stable OGT-knockdown cell lines were generated using shRNA and subcutaneously inoculated into nude mice to monitor tumor growth.Results·IHC revealed that OGT expression was significantly upregulated in NSCLC tumor tissues compared to adjacent normal tissues.Patients with high OGT expression exhibited shorter survival times and poorer prognoses than those with low expression.Transcriptome sequencing demonstrated that genes downregulated after OGT knockdown were primarily enriched in the mitogen-activated protein kinase(MAPK)signaling pathway.Western blotting showed that total extracellular regulated protein kinase 1/2(ERK1/2)levels remained unchanged in H460 and H1299 cells after OGT knockdown,while phosphorylated ERK1/2(p-ERK1/2)and its downstream proto-oncogene JUNB protein were markedly reduced.Suppression of OGT expression attenuated the proliferation rate and colony formation capacity of H460 and H1299 cells,whereas JUNB overexpression rescued the proliferation defects induced by OGT knockdown.Notably,H460 cells with stable OGT knockdown formed significantly smaller tumors in nude mice.Conclusion·OGT is highly expressed in NSCLC and correlates with poor prognosis.Knockdown of OGT inhibits NSCLC cell proliferation and clonogenicity in vitro,and tumor growth in vivo.Mechanistically,OGT appears to promote NSCLC progression by activating the ERK/JUNB signaling axis.

Objective·To investigate the expression of protein tyrosine phosphatase receptor type N(PTPRN)in lung adenocarcinoma and its potential molecular mechanisms in promoting lung adenocarcinoma metastasis.Methods·A highly bone-metastatic A549-BM cell line was established through multiple rounds of intracardiac injection.RNA sequencing(RNA-seq),combined with Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses,was performed to identify PTPRN as a key metastasis-related gene.Subsequently,The Cancer Genome Atlas(TCGA)database was utilized to evaluate PTPRN expression in patients with lung adenocarcinoma and its correlation with clinical prognosis.Co-expression analysis based on TCGA data was conducted to identify and analyze key genes co-expressed with PTPRN.Small interfering RNA(siRNA)targeting PTPRN(siPTPRN)was transfected into A549-BM cells,and Transwell assays were performed to assess its effects on cell migration and invasion.Western blotting was used to detect the expression of epithelial-mesenchymal transition(EMT)-related proteins and the activation of the PI3K-AKT signaling pathway.siPTPRN-transfected A549-BM cells were injected into a mouse model via intracardiac injection,and in vivo metastasis was assessed.Additionally,multiple database analyses were integrated to predict BCL6 as an upstream transcription factor of PTPRN,and siBCL6 transfection experiments were performed to validate the regulatory effect of BCL6 on PTPRN expression.Results·RNA-seq and GO/KEGG enrichment analyses demonstrated that PTPRN was significantly upregulated in highly metastatic A549-BM cells and enriched in metastasis-associated pathways,including the PI3K-AKT signaling pathway and extracellular matrix(ECM)-receptor interactions.Analysis of the TCGA database further confirmed that PTPRN was highly expressed in lung adenocarcinoma patients and significantly associated with poor prognosis.Co-expression analysis based on TCGA data,combined with GO/KEGG enrichment analyses,revealed that PTPRN-associated genes were mainly enriched in biological processes such as neural signaling,endocrine regulation,cell communication,and ECM-receptor interactions.In vitro experiments demonstrated that siPTPRN transfection significantly inhibited the migration and invasion of A549-BM cells,accompanied by downregulation of EMT-related proteins and reduced activation of the PI3K-AKT signaling pathway.In vivo experiments further showed that PTPRN knockdown markedly suppressed the metastatic potential of A549-BM cells,confirming its pro-metastatic role.Additionally,siBCL6 transfection experiments demonstrated that BCL6 knockdown upregulated PTPRN expression.Conclusion·PTPRN is highly expressed in lung adenocarcinoma tissues and promotes tumor cell migration and metastasis by enhancing EMT and activating the PI3K-AKT signaling pathway.High PTPRN expression is significantly correlated with poor prognosis in lung adenocarcinoma patients,while PTPRN enhances lung adenocarcinoma cell invasiveness and metastatic potential.BCL6 may act as an upstream transcriptional regulator of PTPRN,influencing its expression levels.

Objective·To investigate the expression level of O-GlcNAc transferase(OGT)in non-small cell lung cancer(NSCLC)and its impact on lung cancer proliferation,as well as to explore the underlying mechanisms.Methods·The expression of OGT in NSCLC tumors and adjacent normal tissues was detected by immunohistochemistry(IHC).The dataset(GSE31210)from the GEO database was analyzed to assess the correlation between OGT expression and NSCLC patient prognosis.siRNA transfection was performed to knock down OGT expression in H460 and H1299 cells,followed by total RNA extraction and transcriptome sequencing.Pathway enrichment analysis was conducted on differentially downregulated genes in the knockdown group compared with the control group,and Western blotting was used to validate the enrichment results.The effects of OGT knockdown on cell proliferation and colony formation in H460 and H1299 cells were evaluated using the cell counting kit-8(CCK-8)assay and colony formation assay,respectively.The impact of overexpressing downstream genes was also examined.Stable OGT-knockdown cell lines were generated using shRNA and subcutaneously inoculated into nude mice to monitor tumor growth.Results·IHC revealed that OGT expression was significantly upregulated in NSCLC tumor tissues compared to adjacent normal tissues.Patients with high OGT expression exhibited shorter survival times and poorer prognoses than those with low expression.Transcriptome sequencing demonstrated that genes downregulated after OGT knockdown were primarily enriched in the mitogen-activated protein kinase(MAPK)signaling pathway.Western blotting showed that total extracellular regulated protein kinase 1/2(ERK1/2)levels remained unchanged in H460 and H1299 cells after OGT knockdown,while phosphorylated ERK1/2(p-ERK1/2)and its downstream proto-oncogene JUNB protein were markedly reduced.Suppression of OGT expression attenuated the proliferation rate and colony formation capacity of H460 and H1299 cells,whereas JUNB overexpression rescued the proliferation defects induced by OGT knockdown.Notably,H460 cells with stable OGT knockdown formed significantly smaller tumors in nude mice.Conclusion·OGT is highly expressed in NSCLC and correlates with poor prognosis.Knockdown of OGT inhibits NSCLC cell proliferation and clonogenicity in vitro,and tumor growth in vivo.Mechanistically,OGT appears to promote NSCLC progression by activating the ERK/JUNB signaling axis.

Objective:To explore the diagnostic value of laboratory examination in bronchoalveolar lavage fluid(BALF) for interstitial lung disease (ILD) and its application value in assessing the degree of fibrosis in the disease.Methods:Retrospective analysis. The clinical data of ILD patients treated in Shanghai First People′s Hospital from January 1, 2021 to December 31, 2023[104 cases, male︰female=48︰56, (62.79±1.24) years] were collected. According to the imaging scores, they were divided into a mild fibrosis ILD group [53 cases, male︰female=26︰27, (61.32±1.71) years] and a moderate to severe ILD fibrosis group [51 cases, male︰female=22︰29, (64.31±1.88) years]. Patients with community-acquired pneumonia without fibrotic lesions by HRCTduring the same period were selected as the control group [49 cases, male︰female=25︰24, (65.37±1.65)years]. The clinical information of all study subjects, as well as BALF lymphocyte subset analysis, cytokine and cytology count detection results were collected. Furthermore, the Kruskal-Wallis H test was used to screen the differential indexes, and the receiver operating characteristic (ROC) curve was used to evaluate the differential indexes to assess the degree of ILD pulmonary fibrosis.Results:Compared with the non-fibrotic pneumonia group, IL-6, IL-8, CD4+CD45RO+cells and macrophages (M%) were significantly upregulated in the mild fibrosis ILD group (P<0.05), and significantly higher in the moderate to severe fibrosis ILD group ( P<0.05). Compared with the non-fibrotic pneumonia group, IL-1β and white blood cell (WBC) count were significantly upregulated only in the moderate to severe fibrotic ILD group ( P<0.05). The correction model was constructed by stepwise logistic regression analysis, and the differential indexes were combined, and the proportion of IL-1β+IL-6+IL-8+CD4+CD45RO+cells+macrophages was finally screened as the optimal combined diagnostic mode, with an area under the curve of 0.925, sensitivity of 92.3%, and specificity of 80.0%. Conclusion:Compared with the non-fibrotic pneumonia group, BALF-derived IL-1β, IL-6, IL-8, CD4+CD45RO+cells, WBC count and M% can be used as potential biomarkers to assess the degree of fibrosis, and the combination of IL-1β+IL-6+IL-8+CD4+CD45RO+cells+macrophages has a better diagnostic efficacy for moderate to severe fibrotic ILD.

Objective:To investigate the tissue methylation features of adenocarcinoma patients presenting as pulmonary nodules on CT scans.Methods:A retrospective analysis was conducted on 70 adenocarcinoma patients with pulmonary nodules diagnosed at the Shanghai General Hospital from June 1, 2022 to January 20, 2024. Participants were assigned to two groups using the random number table, with 40 in the discovery group and 30 in the validation group. In the discovery group, tissue samples were analyzed using reduced representation bisulfite sequencing (RRBS) technology to compare the average methylation levels between cancer tissues and paired adjacent non-cancerous tissues. Differentially methylated regions (DMRs) were screened for analysis of their distribution across various genomic functional elements, and hierarchical clustering was plotted. GO and KEGG pathway enrichment analyses were further conducted on the DMRs. Subsequently, candidate DMRs associated with lung adenocarcinoma were validated using TCGA lung adenocarcinoma cohort and targeted bisulfite sequencing technology in the validation group. The comparison of methylation levels between groups was conducted using t-tests or non-parametric tests, while rates and composition ratios were analyzed using chi-square tests or Fisher′s exact test.Results:In discovery cohort, the average methylation level in cancer tissues was lower compared to adjacent normal tissues [(42.369±4.627) vs (44.370±4.046), t=?2.059, P=0.043]. A total of 37 995 DMRs were identified, including 16 889 upregulated regions and 21 106 downregulated regions, predominantly locating in promoter regions (48.917%), introns (36.457%), and exons (10.812%). The DMR clustering heatmap revealed two distinct clusters corresponding to cancer tissues and adjacent non-cancerous tissues. GO analysis showed that DMRs associated genes were mainly located in the cell membrane and nuclear chromatin, and were primarily involved in RNA polymerase Ⅱ-related transcription and regulation. KEGG pathway enrichment analysis indicated that DMRs associated genes were mainly involved in neuroactive ligand-receptor interaction, cancer pathways, calcium signaling pathway, cAMP signaling pathway, and MAPK signaling pathway. Validation in the TCGA cohort confirmed 11 potential characteristic DMRs. In the validation group, TBS confirmed that the methylation levels of DMRs associated with MIR10B, DMRTA2, HOPX, TFAP2B and MARCH11 in cancer tissues were significantly higher than those in adjacent non-cancerous tissues [11.200(4.305, 27.088) vs 2.650(1.298, 4.645), Z=?4.539, P<0.05; 18.610(13.600, 33.025) vs 8.675(5.488, 13.085), Z=?4.554, P<0.05; 17.600(2.183, 76.015) vs 1.085(0.898, 1.835), Z=?5.131, P<0.05; 5.250(3.220, 7.693) vs 3.495(2.165, 4.383), Z=?2.861, P<0.05; 11.515(7.525, 21.033) vs 7.830(5.518, 11.488), Z=?2.440, P<0.05 ], and the differences were statistically significant. Conclusions:Lung adenocarcinoma tissue exhibits different methylation patterns compared with adjacent normal lung tissue. The identified DMRs are involved in the regulation of several key pathways. Results from the TCGA cohort and an independent validation group support the potential diagnostic value of DMRs such as MIR10B, DMRTA2, HOPX, TFAP2B, and MARCH11 in lung adenocarcinoma, though their clinical application requires further validation.

Objective·To verify the performance and explore the clinical application value of a multi-modal pulmonary nodule diagnosis model combined with metabolic fingerprints,protein biomarker CEA and Image-AI via random forest(MPI-RF).Methods·This study enrolled 289 patients with pulmonary nodules who were admitted to the Shanghai Chest Hospital,Shanghai Jiao Tong University School of Medicine and were detected by low-dose helical computed tomography(LDCT).The patients were divided into malignant nodule group(n=197)and benign nodule group(n=92)based on postoperative pathological results,and the basic information of the two groups was collected and compared.Electrochemiluminescence was used to detect the preoperative serum CEA levels of the patients in the two groups,matrix-assisted laser desorption/ionization mass spectrometry(MALDI-MS)was used to detect the serum metabolic fingerprints,and the CT image artificial intelligence model Image-AI was used to calculate the image scores.CEA data,serum metabolic fingerprints data and image scores were integrated and input into MPI-RF to calculate the malignant probability score of each patient.The receiver operator characteristic curve(ROC curve)and area under the curve(AUC)were used to evaluate the performance of different models,and the DeLong test was used for comparative analysis,including the diagnostic performance of MPI-RF in different types(solid nodule,pure ground-glass nodule and part-solid nodule)and sizes(diameter<8 mm and diameter≥8 mm)of pulmonary nodules,the diagnostic performance comparison of MPI-RF with Mayo Clinic model,veterans administration(VA)model and Brock model,and the diagnostic performance comparison of MPI-RF with lung imaging reporting and data system(Lung-RADS)in benign and malignant nodules.Results·MPI-RF had good diagnostic performance in the differentiation of benign and malignant pulmonary nodules(AUC=0.887,95%CI 0.848?0.925,sensitivity 81.22%,specificity 83.70%).Among them,the AUC of MPI-RF for solid nodules was 0.877(95%CI 0.820?0.934),for part-solid nodules was 0.858(95%CI 0.771?0.946),and for pure ground-glass nodules was 0.978(95%CI 0.923?1.000).The AUC of MPI-RF was 0.840(95%CI 0.716?0.963)for nodules within 8 mm diameter and 0.891(95%CI 0.849?0.933)for nodules larger than 8 mm diameter.Compared with the existing models,the diagnostic performance of MPI-RF was better than that of Mayo Clinic model,VA model and Brock model(all P=0.000).Compared with Lung-RADS,MPI-RF had better diagnostic performance in the total samples and different types of nodules(all P=0.000).Conclusion·MPI-RF is a model for the differential diagnosis of benign and malignant pulmonary nodules with excellent performance,and has potential clinical application value.

[Abstract] Objective: To investigate the effect of miR-93/EphA4 (Eph receptor A4) axis on the proliferation and migration of nonsmall cell lung cancer (NSCLC) H460 and H1299 cells via regulating extracellular regulated protein kinases (ERK) pathway. Methods: The expression levels of miR-93 in H460 and H1299 cells was detected by qPCR. miR-93 mimics and EphA4 overexpression plasmids were transfected into H460 cells and miR-93 inhibitor was transfected into H1299 cells respectively, after which MTT assay and Transwell assay were used to detect the effects of miR-93 on proliferation and migration of transfected cells. The targeted regulatory relationship betweenmiR-93andEphA4wasverifiedbyDual-luciferasereportergeneassay.Theexpression levels of PCNA(proliferating cell nuclear antigen), EphA4, ERK and p-ERK were detected by Westernblotting.The effects of simultaneous overexpression of miR-93 and EphA4 on proliferation and migration of H460 cells were detected by MTT assay and Transwell assay. Results: The expression of miR-93 in H1299 cells was higher than that in H460 cells (P<0.01). Overexpression of miR-93 promoted proliferation and migration of H460 cells (all P<0.01), and knockdown of miR-93 inhibited proliferation and migration of H1299 cells (all P<0.01). The Dualluciferase reporter gene assay confirmed that miR-93 could target EphA4. Overexpression of miR-93 down-regulated the mRNA and protein expression levels of EphA4(allP<0.05), and promoted proliferation and migration of H460 cells through targeted regulation of EphA4 and activation of ERK pathway (all P<0.01). Conclusion: miR-93 promotes the proliferation and migration of NSCLC cells, and its mechanism may be related to the targeted regulation of EphA4 and activation of the ERK pathway.

Objective To estimate the diagnostic value of circulating tumor cell detection for non-small cell lung cancer.Methods A Non-intervention clinical study was conducted in this research.From October 2014 to April 2015, totally 162 NSCLC who presented at Thoracic Surgery Department, 119 benign pulmonary disease and 52 healthy individuals were collected from Shanghai Chest Hospital.Folate receptor ( FR) based polymerase chain reaction ( PCR) method was used to detect the circulating tumor cell ( CTC) level, CEA and CYFRA21-1 was detected by the flowcytometry fluorescence luminance method, SCC was detected with Chemiluminescent microparticle immunoassay.The differences among groups were analyzed by the Kruskal-Wallis test( multi group comparison) and the Mann-Whitney U test( two group comparison) , and the chi-square test was used in the positive rate comparison;the Receiver Operating Characteristics ( ROC) curve was established.Results The median level of CTC in NSCLC patients was 11.90 Units/3 ml, which was significantly higher than those of benign pulmonary disease ( 6.72 CTC Units/3 ml ) and healthy individuals (5.82 CTC Units/3 ml,χ2 =125.990, P<0.01).Areas Under Curve ( AUCs) of ROC curve for NSCLC was 0.853 2(95% CI: 0.809 5,0.896 9).The cut-off value for discriminating NSCLC with benign pulmonary disease/healthy people was 8.74 CTC Units/3 ml with sensitivity being 77.16% and specificity being 90.06%.The positive rate of CTC in Stage I NSCLC patients was 68.7%, which was much higher than that of the combination of tumor markers(χ2 =32.98,P<0.01).Conclusion With relatively high sensitivity and specificity, the detection of circulating tumor cell may has a clinical value of application and extension.

Objective To evaluate the application value of circulating tumor cell ( CTC ) in the differential diagnosis of solitary pulmonary nodule ( SPN ) . Methods Peripheral blood samples were collected from 134 patients with solitary pulmonary nodule in Shanghai Chest Hospital from September 2013 to January 2015, including 80 patients with malignant nodule and 54 with benign nodule.CTC levels of the above subjects were detected by ligand-targeted polymerase chain reaction ( LT-PCR ) assay, and serum carcinoembryonic antigen ( CEA ) and cytokeratin 19 fragment ( CYFRA21-1 ) were detected by flow fluorescence assay.Results By Mann-Whitney U Test, the CTC levels of malignant SPN patients [11.06 (8.77-14.41)units/3 ml] were significantly higher than those of benign SPN patients [6.65(4.49 -7.84)units/3 ml] (Z=-6.217,P<0.001).The sensitivity and specificity of differential diagnosis of SPN for CTC were 80%(64/80) and 85%(46/54) respectively.According to the diameter of SPN, the patients were divided into three groups to evaluate the diagnostic value of CTC in SPN with different size .For SPN with diameter less than 8 mm, the sensitivity and specificity of CTC were 6/9 and 4/5 respectively .For SPN with diameter between 8 mm and 20 mm, the sensitivity and specificity of CTC were 83%(35/42) and 85%(29/34).For SPN with diameter greater than 20 mm, the sensitivity and specificity of CTC were 79%(23/29) and 13/15.Conclusion Comparing with the traditional tumor markers, CTC could provide more clinical value in the differential diagnosis of solitary pulmonary nodule .