High expression of ISG20L2 promotes proliferation and invasion of A549 cells and is associated with poor prognosis in lung adenocarcinoma

High expression of ISG20L2 promotes proliferation and invasion of A549 cells and is associated with poor prognosis in lung adenocarcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article High expression of ISG20L2 promotes proliferation and invasion of A549 cells and is associated with poor prognosis in lung adenocarcinoma Xinyu Zhang, Dan Yu, Ming Liu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3843095/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Interferon-stimulated 20kDa exonuclease-like 2 (ISG20L2) is a gene that exhibits differential expression in lung adenocarcinoma (LUAD). However, its expression and function in LUAD remain poorly understood. The aim of this study was to investigate the expression of ISG20L2 in LUAD and its correlation with prognosis, as well as to explore its impact on the biological behavior of LUAD. Methods The researchers analyzed the expression of ISG20L2 using both The Cancer Genome Atlas (TCGA) database and immunohistochemistry (IHC). Enrichment analysis was performed using the "GOplot" and "clusterprofile" R packages. The correlation between ISG20L2 expression and prognosis of LUAD patients was assessed through IHC and Kaplan-Meier survival analysis. Additionally, the diagnostic value of ISG20L2 in LUAD was evaluated using ROC curve analysis. The relationship between ISG20L2 expression and clinicopathological characteristics was examined through IHC. Overexpression and knockout experiments of ISG20L2 were conducted via transient transfection. The biological properties of ISG20L2 in A549 cells, including cell proliferation, apoptosis, migration, and invasion abilities, were investigated using assays such as cell counting kit-8 (CCK-8), flow cytometry, and Transwell assays. Results The findings indicated that ISG20L2 was highly expressed in LUAD, and its high expression was closely associated with poor prognosis. In vitro experiments further confirmed a positive correlation between ISG20L2 expression level and the proliferation, migration, and invasion abilities of LUAD cells, while no significant effect on apoptotic ability was observed. Conclusion Our study indicates that ISG20L2 promotes the proliferation, migration, and invasion of LUAD cells, and its high expression predicts a poorer prognosis for LUAD patients. This study suggests that ISG20L2 has the potential to serve as a molecular marker for the treatment and prognosis of LUAD. Lung adenocarcinoma ISG20L2 Proliferation Prognosis Targeted regulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Background Lung cancer is the leading cause of cancer-related deaths worldwide, with an incidence that increases yearly[ 1 , 2 ].It can be classified into two main types: non-small cell lung carcinoma (NSCLC) and small-cell lung carcinoma (SCLC). NSCLC accounts for approximately 85% of all lung cancers, with about 40% being lung adenocarcinoma (LUAD)[ 3 – 5 ]. Targeted therapies have revolutionized the treatment for oncogene-driven NSCLC[ 6 ]. A significant proportion, up to 69%, of patients with advanced NSCLC may have specific molecular targets that can be targeted by treatment[ 7 ]. The approach to treating advanced NSCLC has shifted from selecting treatments based on clinical and pathological features to utilizing biomarker-driven treatment algorithms that consider the patient's tumor molecular profile[ 8 ]. Molecularly targeted therapy involves using drugs or other substances that specifically target certain molecules (molecular targets) to inhibit the growth and spread of cancer cells[ 9 ]. This treatment approach offers advantages such as precise and efficient drug delivery, as well as reduced toxicity compared to traditional chemotherapy drugs, which improves the survival rate of many types of malignant tumors[ 10 ]. While significant progress has been made in the systemic treatment of advanced NSCLC through molecular targeted therapy and immunotherapy, the long-term survival rate for lung cancer patients remains low[ 11 – 13 ]. There is still a need to identify new biomarkers and prognostic genes, develop next-generation drugs with more specific targeting effects, and create drugs that can effectively counter specific drug-resistant mutations. These advancements aim to provide patients with more effective treatments and extend their survival time. Such efforts align with the research focus in the era of precision medicine. Interferon-stimulated 20-kDa exonuclease-like 2 (ISG20L2) is a member of the vertebrate exonuclease family that plays a role in ribosome biogenesis degradation. Research has demonstrated that ISG20L2 accumulates in the nucleoli of HeLa cells and exhibits exoribonucleolytic activity, degrading RNA from the 3'- to 5'-terminus. This activity has been linked to cellular proliferation and apoptosis[ 14 ]. High expression of ISG20L2 has been observed in various cancers, including lung cancer[ 15 ], liver cancer[ 16 , 17 ], breast cancer[ 18 ], etc. The latest research has found that ISG20L2 is an exonuclease with priority specificity for uridylated miRNA, which can regulate T cell activation[ 19 ]. Notably, in myeloma, ISG20L2 weakens the binding of bortezomib to the proteasome 20S subunit β5 (PSMB5), leading to reduced inhibition of proteasome activity and decreased efficacy of bortezomib treatment[ 20 ] .These findings highlight the close association between ISG20L2 expression levels and the effectiveness and prognosis of cancer treatment. However, the specific role of ISG20L2 LUAD remains unclear. This study aims to investigate the expression of ISG20L2 in lung cancer and evaluate its diagnostic value using bioinformatics analysis. Subsequently, the expression of ISG20L2 will be validated in tissue samples through immunohistochemistry. The relationship between ISG20L2 and clinicopathological features of LUAD will be explored, along with its prognostic value. Additionally, in vitro experiments will be conducted to examine the impact of ISG20L2 on the biological behavior of LUAD cells. 2. Materials and methods 2.1 Bioinformatics analysis The clinical data of ISG20L2 expression profile related LUAD were downloaded from the TCGA dataset. The adenocarcinoma microarray dataset GSE19804 was retrieved from the Gene Expression Omnibus (GEO) database[ 21 ], which is maintained by the National Center for Biotechnology Information (NCBI). To perform differential analysis, we employed the R package clusterProfiler. 2.2 Gene Ontology and Kyoto Encyclopedia of Genes and Genomes Enrichment Analyses The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed on the shared genes by the “GOplot” and the “clusterprofiler” R package. P < 0.05 was considered statistically significant. 2.3 Diagnostic Value Analysis The receiver operating characteristic (ROC) curve was used to assess the diagnostic value of ISG20L2 in LUAD. The closer the area under the curve (AUC) is (1), the better the diagnostic effect is. AUC in 0.5–0.7 has a low accuracy, AUC in 0.7–0.9 has a certain accuracy, and AUC above 0.9 has a high accuracy. 2.4 Immunohistochemical (IHC) staining We obtained a lung adenocarcinoma tissue chip (catalog number HLugA180Su08) from Shanghai Outdo Biotech Co., Ltd., which consisted of 95 LUAD and 80 paracancerous specimens. The tissue chip was initially baked at 63°C for 1 hour. Subsequently, we removed the tissue chip using xylene treatment (twice, for 15 minutes each) and performed gradient alcohol dehydration (100% twice every 7 minutes, 90% once every 5 minutes, 80% once every 5 minutes, and 70% once every 5 minutes). Antigen retrieval was then carried out using the Dako fully automated immunohistochemical pretreatment system (Dako North America, Inc). Furthermore, the tissue chip was incubated overnight at 4°C with the ISG20L2 antibody (rabbit polyclonal, dilution 1:50, catalog number NBP1-82287, Novus Biologicals, USA). The following day, we treated the chip with anti-rabbit horseradish peroxidase IgG polymer (Abcam, Cambridge, UK) as a secondary antibody for 30 minutes at room temperature. The chip was stained with DAB, counterstained with hematoxylin, and finally sealed with a neutral resin. For scoring, staining intensity was categorized as follows: 0 (negative), 1 (1+), 2 (2+), and 3 (3+). The proportion of positive staining ranged from 0–100%. The overall score was calculated by multiplying the staining intensity score by the proportion of staining results (0-300%). In the survival analysis group, a total score of less than 27.5 indicated the ISG20L2 low expression group, while a score of 27.5 or higher indicated the ISG20L2 high expression group. 2.5 Cell Culture and transfection We purchased A549 cells from Procell Life Science & Technology Co., Ltd. (Wuhan, China) and cultured them in Ham's F-12K medium supplemented with 10% fetal bovine serum and 1% double antibody (100%) × penicillin-streptomycin solution. The cells were incubated in a humidified incubator containing 5% CO2 at 37°C. The pcDNA empty vector, pcDNA-ISG20L2, negative control, and siRNA-ISG20L2 were designed and provided by GenePharma Co., Ltd (Suzhou, China). A549 cells were inoculated on 6-well cell culture plates at a density of 1×10 5 cells/well. Lipofectamine3000 (Invitrogen, Thermo Fisher Scientific, USA) was added to the plasmids or oligonucleotides mentioned above when the cells reached 70%∼80% confluence. Cells were harvested and extracted for further study 48 hours after transfection. 2.6 Western blot analysis Protein samples were extracted from A549 cells using RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with protease and phosphatase inhibitors (Beyotime). The protein concentration was measured using a BCA protein detection kit (Beyotime). Subsequently, the protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were then blocked with 5% skim milk at room temperature for 1.5 hours. Primary antibodies against ISG20L2 (catalog number NBP2-83084, Novus Biologicals, USA) and GAPDH (Abcam, Cambridge, UK) were incubated with the membranes overnight at 4°C. After three washes, the membranes were incubated with a secondary antibody, goat anti-rabbit IgG (Abcam, Cambridge, UK), for 1.5 hours. Proteins were visualized using an ECL luminescent kit (Tanon, Shanghai, China). GAPDH was used as an internal control, and the relative protein expression levels were quantified using Image J software (NIH, Bethesda) by calculating the grayscale value of the bands. All experiments were performed in triplicate. 2.7 Cell proliferation assay To assess the proliferation of tumor cells, we utilized the Cell Counting Kit-8 (CCK-8, Dojindo, Tokyo, Japan). A549 cells were seeded in 96-well plates at a density of 2×10 3 cells per well. Following incubation for 0, 12, 24, 48, and 72 hours, 10 µL of the CCK-8 reagent was added to each well and incubated at 37°C for 1 hour. The absorbance at 450 nm in each well was subsequently measured using a microplate reader (Thermo, Waltham, MA, USA). All experiments were conducted in triplicate. 2.8 Flow cytometry To detect early and late apoptotic cells, we employed flow cytometry in conjunction with the Annexin V-FITC/PI Apoptosis Detection kit (KeyGEN BioTECH, Nanjing, China). Cells were harvested using EDTA-free Trypsin, washed twice with chilled PBS, and centrifuged at 1,000 g for 5 minutes. A 500 µL binding buffer was added to the cell suspension, followed by the addition of 5 µL Annexin V-FITC and 5 µL Propidium Iodide. The mixture was then incubated at room temperature in the dark for 10 minutes. Finally, an optical instrument known as a flow cytometer (Becton Dickinson and Co., Franklin Lakes, NJ, USA) was employed to detect the apoptotic cells. All experiments were conducted in triplicate. 2.9 Transwell migration and invasion assays To assess the migration and invasion of cancer cells, we utilized Transwell chambers (8 mm pore size, Corning Inc, NY, USA) and 24-well plates (Corning Inc, NY, USA). For the transwell migration and invasion assays, 200 µL of serum-free medium containing 1 × 10 5 cells was seeded into the upper chamber of a transwell. In the case of the transwell invasion assay, the upper chamber was pre-coated with matrigel (Corning Inc, NY, USA). The lower chamber was filled with 700 µL of Ham's F-12k supplemented with 10% FBS. After incubating for 24 hours, any remaining tumor cells in the upper chamber were removed using cotton swabs. The invaded cells were fixed with 4% paraformaldehyde for 20 minutes and stained with crystal violet for 15 minutes at room temperature. Subsequently, the number of invading cells was counted in 5 randomly selected fields under an inverted microscope (Olympus, Tokyo, Japan). All experiments were conducted in triplicate. 2.10 Statistical analysis We employed R software v4.3.0 for statistical analysis. The Wilcoxon test was utilized to perform differential analysis of ISG20L2 expression in cancer and paracancerous tissues. The Fisher test was employed to analyze the relationship between ISG20L2 expression and clinicopathological variables. For survival univariate analysis, we utilized Kaplan-Meier survival analysis and log-rank statistical test. Additionally, we used the Cox proportional risk regression model for univariate and multivariate analysis of total survival time. A statistically significant difference was considered when P < 0.05. 3. Results 3.1 ISG20L2 is highly expressed in LUAD and High ISG20L2 expression is associated with poor LUAD prognosis To investigate the role of ISG20L2 in LUAD, we initially analyzed its differential expression in LUAD mRNA using The Cancer Genome Atlas (TCGA) database[22] (https://genome-cancer.ucsc.edu/). Our analysis revealed that ISG20L2 expression was significantly upregulated in LUAD compared to corresponding healthy tissues (Fig. 1A; P<0.001). To confirm this finding, we performed immunohistochemical (IHC) staining on a chip containing 95 LUAD and 80 paracancerous specimens. The results demonstrated that ISG20L2 protein was highly expressed in lung adenocarcinoma tissues but exhibited lower expression in paracancerous tissues (Fig. 1B; P<0.001). These findings collectively indicated the increased expression of ISG20L2 in LUAD, suggesting its involvement in LUAD pathogenesis. Furthermore, we conducted ROC curve analysis to assess the diagnostic value of ISG20L2 in LUAD. The results indicated that ISG20L2 exhibited a certain level of accuracy (AUC＝0.829) in predicting LUAD (Fig. 1C). Additionally, we examined the associations between ISG20L2 expression and various clinical characteristics in LUAD patients. However, no significant correlations were observed between ISG20L2 expression and factors such as gender, age, tumor size, T stage, N stage, M stage, pathological grade, and TNM stage (Table 1). To evaluate the prognostic significance of ISG20L2 expression at the protein level, we analyzed the relationship between ISG20L2 expression and patient prognosis using IHC staining results. Our analysis revealed that low ISG20L2 expression was associated with a better prognosis (Fig. 1D; P<0.05). Moreover, we employed the Cox regression model to assess the link between ISG20L2 expression and patient survival (Table 2). Univariate analysis demonstrated that ISG20L2 expression, age, N stage, and TNM stage were factors influencing the overall survival of lung adenocarcinoma patients. However, multivariate analysis indicated that these factors were not independent risk factors for the overall survival of lung adenocarcinoma patients. Overall, our findings suggest that ISG20L2 is upregulated in LUAD and may play a role in its pathogenesis. Furthermore, ISG20L2 expression shows diagnostic potential and is associated with patient prognosis in LUAD. Table 1．The relationship between the protein expression of ISG20L2 and clinicopathological characteristics in patients with lung adenocarcinoma variables ISG20L2 expression total p value r value low high sex 0.143 0.162 Male 21 18 39 Female 21 35 56 age 1 -0.009 ≤60 21 27 48 >60 21 26 47 Tumor_size 0.512 0.071 ≤4cm 29 32 61 >4cm 12 18 30 T 0.124 0.184 Ⅰ 12 8 20 Ⅱ-Ⅳ 27 44 71 N 0.061 0.21 N0 25 20 45 N1/N2/N3 17 32 49 M 1 0.092 M0 42 52 94 M1 0 1 1 grade 0.569 0.071 Ⅰ-Ⅱ 37 44 81 Ⅲ 5 9 14 TNM 0.086 0.193 Ⅰ 20 15 35 Ⅱ-Ⅳ 22 37 59 r：Correlation Coefficient, P < 0.05 was considered statistically significant. Table 2. Univariate and multivariate analyses of the factors correlated with Overall survival of lung cancer patients variables Univariate analysis Multivariate analysis HR 95%CI p value HR 95%CI p value Lower limit Upper limit Upper limit expression 1.706 1.005 2.895 0.0477 1.35 0.78 2.32 0.282 sex 0.991 0.593 1.656 0.971 age 1.953 1.161 3.284 0.0117 1.47 0.86 2.53 0.16 Tumor_size 1.166 0.673 2.019 0.585 T 1.838 0.927 3.645 0.0814 N 3.369 1.929 5.884 0.0000197 2.68 0.95 7.55 0.0617 M 1.343 0.185 9.734 0.77 grade 1.331 0.654 2.706 0.43 TNM 2.979 1.639 5.412 0.000341 1.11 0.37 3.38 0.853 HR: Hazard Ratio, CI: Confidence Interval. P < 0.05 was considered statistically significant. 3.2 GO and KEGG Enrichment Analyses To gain a better understanding of the biological processes and pathways involved in lung cancer, we performed GO functional enrichment analysis and KEGG pathway analysis. The GO functional enrichment analysis revealed that the primary biological processes (BP) involved extracellular matrix organization, extracellular structure organization, and external encapsulating. The cellular component (CC) was primarily enriched in collagen-containing extracellular matrix, membrane raft, and membrane microdomain. The molecular function (MF) was mainly involved in glycosaminoglycan binding, extracellular matrix structural constituent, and heparin binding (Fig. 2A). The KEGG pathway enrichment analysis identified several pathways related to lung cancer, including cytokine-cytokine receptor interaction, protein digestion and absorption, IL-17 signaling pathway, and lipid and atherosclerosis (Fig. 2B). These findings suggest that alterations in extracellular matrix organization and cytokine signaling may play a critical role in the development and progression of lung cancer. These results provide valuable insights into the biological processes and pathways associated with lung cancer, which may aid in the development of more effective diagnostic and therapeutic strategies for this disease. 3.3 ISG20L2 overexpression promotes proliferation, migration, and invasion of A549 cells but without affecting apoptosis To assess the efficiency of ISG20L2 overexpression, Western blot analysis was performed in both the pcDNA3.1 and pcDNA3.1-ISG20L2 groups. The results showed a significant increase in ISG20L2 expression in the pcDNA3.1-ISG20L2 group compared to the pcDNA3.1 group (Fig. 3A; P<0.001). To further investigate the impact of ISG20L2 on the biological function of A549 cells, a CCK-8 assay was conducted to measure cell proliferation. The results demonstrated a significant increase in cell proliferation in the pcDNA3.1-ISG20L2 group after 48 hours compared to the pcDNA3.1 group (Fig. 3B; P 0.05). These findings suggest that ISG20L2 overexpression enhances the growth rate of A549 cells in vitro but does not affect cell apoptosis. To evaluate the migratory and invasive abilities of A549 cells, transwell assays were performed. The results indicated that cell migration and invasion were significantly increased in the pcDNA3.1-ISG20L2 group compared to the pcDNA3.1 group (Fig. 4A; Fig. 4B; P<0.05). These findings suggest that ISG20L2 overexpression promotes the migration and invasion of A549 cells. Collectively, our results demonstrate that ISG20L2 overexpression enhances the proliferative capacity, migration, and invasion of A549 cells, indicating its potential role in promoting the progression of lung adenocarcinoma. 3.4 ISG20L2 knockdown decreases proliferation, migration, and invasion of A549 cells but without affecting apoptosis To further investigate the effects of ISG20L2 on LUAD cells, ISG20L2 knockdown was performed, and the transfection efficiency was measured using Western blot analysis. The results revealed a reduction in ISG20L2 expression in the si - ISG20L2 group compared to the NC group (Fig. 3A; P<0.001). Subsequently, a CCK-8 assay was conducted to evaluate the cell proliferative capacity. The results indicated a significant decrease in cell proliferation in the si - ISG20L2 group compared to the NC group (Fig. 5A; P 0.05). These findings suggest that ISG20L2 knockdown reduces the proliferative ability of A549 cells, while similar to the results observed with ISG20L2 overexpression, ISG20L2 knockout does not affect apoptosis. Furthermore, transwell assays were performed to assess cell migration and invasion. The si - ISG20L2 group exhibited significantly decreased migration and invasion abilities compared to the NC group (Fig. 5C; Fig. 5D; P<0.05). In summary, the aforementioned experiments demonstrate that ISG20L2 knockdown decreases the migration and invasion of A549 cells. Taken together, these findings suggest that ISG20L2 plays a crucial role in promoting the proliferative capacity, migration, and invasion of LUAD cells, highlighting its potential as a target for therapeutic interventions in lung adenocarcinoma. 4. Discussion Lung adenocarcinoma is a prevalent form of cancer worldwide, but its precise underlying mechanisms of occurrence and development remain incompletely understood. Investigating the differential genes associated with lung adenocarcinoma not only contributes to unraveling its molecular mechanisms but also holds significant potential for identifying diagnostic and prognostic markers for this disease. Limited literature has explored the relationship between ISG20L2 and cancer, primarily employing bioinformatics approaches. Consequently, its precise cellular function remains largely unknown. Notably, bioinformatics analysis has revealed that ISG20L2 exhibits elevated expression in hepatocellular carcinoma, correlating with reduced patient survival. Consequently, it holds promise as a potential molecular target for immunotherapy in this cancer type[ 17 ]. In breast cancer, ISG20L2 has been associated with the expression of the tumor cell proliferation marker MKI67 (Ki-67) and the prognosis of breast cancer patients[ 18 ]. In the context of LUAD, our study employed bioinformatics analysis and immunohistochemistry tests to identify elevated mRNA and protein levels of ISG20L2. Survival analysis demonstrated that ISG20L2 serves as a high-risk gene, with increased expression correlating with poor prognosis in LUAD patients. ROC analysis indicated that ISG20L2 holds diagnostic potential for lung adenocarcinoma. These findings suggest that ISG20L2 may play a crucial role in improving the prognosis of LUAD patients by aiding in diagnosis and accurate prognostic evaluation. In terms of GO analysis, ISG20L2 enrichment is associated with proliferation and energy metabolism, including extracellular matrix tissue and extracellular structural tissue. Cellular component terms indicate involvement in membrane microdomains and membrane rafts. Bioenergetic stress from abnormal energy metabolism can lead to signaling in tumor cells[ 23 ]. Notably, membrane microdomains play a role in immune receptor signaling[ 24 , 25 ]. Previous studies have reported ISG20L2's involvement in immune cell invasion in hepatocellular carcinoma[ 17 ]. Therefore, it is necessary to further investigate whether ISG20L2 is involved in immune cell invasion in LUAD and elucidate how ISG20L2 mediates the relationship between immune receptor signaling and immune cell infiltration. Regarding the KEGG analysis, ISG20L2 is implicated in several pathways closely related to the inflammatory response, such as the cytokine-cytokine receptor interaction pathway and the IL17 signaling pathway[ 26 – 28 ].These findings suggest that ISG20L2 may participate in immune-related processes within the context of LUAD. Further research is warranted to comprehensively understand the precise mechanisms by which ISG20L2 influences immune responses and its potential impact on immune cell infiltration in lung adenocarcinoma. Our study also investigated the relationship between the expression level of ISG20L2 and clinical pathological parameters in LUAD patients. We observed that the expression of ISG20L2 did not show significant correlations with gender, age, tumor size, TNM staging, or pathological grading in LUAD patients. However, COX regression model analysis revealed an association between ISG20L2 expression and the survival rate of LUAD patients. To gain insights into the impact of ISG20L2 on the biological behavior of LUAD cells, we conducted various cell experiments. Our findings demonstrated a positive correlation between ISG20L2 expression and the proliferation, migration, and invasion abilities of LUAD cells. It is well known that abnormal proliferation is closely related to the development and progression of cancer and cancer invasion and metastasis accounts for the majority of cancer related mortality[ 29 , 30 ]. However, we did not observe any significant effect of ISG20L2 on the apoptosis ability of LUAD cells. It is important to note that our study has certain limitations. Accumulated evidence indicates that miR-139-3p was decreased in breast cancer[ 31 ] tissues and colon cancer[ 32 ] serum, and decreased miR-139-3p is associated with a poor prognosis in cancer patients. Although we acknowledge that ISG20L2 is a target gene for miR-139-3p [ 33 ], we have not conducted in-depth research on the specific mechanisms of action involved. Additionally, we have not investigated the regulatory effect of ISG20L2 on LUAD in vivo . 5. Conclusion In summary, our study indicates that ISG20L2 promotes the proliferation, migration, and invasion of LUAD cells, and its high expression predicts a poorer prognosis for LUAD patients. These findings suggest that ISG20L2 may serve as a potential therapeutic target and prognostic factor for LUAD. Further research is needed to elucidate the underlying mechanisms and validate these observations in vivo . Abbreviations NSCLC non-small cell lung carcinoma SCLC small-cell lung carcinoma LUAD lung adenocarcinoma ISG20L2 Interferon-stimulated 20-kDa exonuclease-like 2 PSMB5 proteasome 20S subunit β5 GEO Gene Expression Omnibus NCBI National Center for Biotechnology Information GO Gene Ontology KEGG Kyoto Encyclopedia of Genes and Genomes ROC receiver operating characteristic AUC area under the curve TNM Tumor Node Metastasis IHC immunohistochemical SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis PVDF polyvinylidene difluoride CCK-8 cell counting kit-8 EDTA ethylenediaminetetraacetic acid BP biological processes CC cellular component MF molecular function IL17 interleukin 17 Declarations Availability of data and materials The clinical data of ISG20L2 expression profile related LUAD were downloaded from the TCGA dataset (https://portal.gdc.com).The adenocarcinoma microarray dataset GSE19804 was retrieved from the Gene Expression Omnibus (GEO) database of the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/geo/). Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding This study was supported by grants from Top Talent Of Changzhou “The 14 th Five-Year Plan” High-Level Health Talents Training Project (2022CZBJ056) and Changzhou Science and Technology Project (ZD202337). Author contributions Xinyu Zhang and Ming Liu conceived and designed the research. Xinyu Zhang performed the experiments and analyzed the data. Dan Yu analyzed the data. Xinyu Zhang and Dan Yu wrote the manuscript, and Ming Liu made revisions to it . All authors read and approved the final manuscript. Acknowledgments The authors thank the efforts and contributions of all the staff in this study. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. References Oliver AL. Lung Cancer: Epidemiology and Screening. 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JCI Insight. 2022;7:e157081. doi: 10.1172/jci.insight.157081 Barrett T, Wilhite SE, Ledoux P, Evangelista C, Kim IF, Tomashevsky M, et al. NCBI GEO: archive for functional genomics data sets–update. Nucleic Acids Res. 2013;41:D991-995. doi: 10.1093/nar/gks1193 Tomczak K, Czerwińska P, Wiznerowicz M. The Cancer Genome Atlas (TCGA): an immeasurable source of knowledge. Contemp Oncol (Pozn). 2015;19:A68-77. doi: 10.5114/wo.2014.47136 Parlani M, Jorgez C, Friedl P. Plasticity of cancer invasion and energy metabolism. Trends Cell Biol. 2023;33:388–402. doi: 10.1016/j.tcb.2022.09.009 Horejsi V, Hrdinka M. Membrane microdomains in immunoreceptor signaling. FEBS Letters. 2014;588:2392–2397. doi: 10.1016/j.febslet.2014.05.047 Otáhal P, Angelisová P, Hrdinka M, Brdicka T, Novák P, Drbal K, et al. A new type of membrane raft-like microdomains and their possible involvement in TCR signaling. J Immunol. 2010;184:3689–3696. doi: 10.4049/jimmunol.0902075 Lu L, Liu LP, Gui R, Dong H, Su YR, Zhou XH, et al. Discovering common pathogenetic processes between COVID-19 and sepsis by bioinformatics and system biology approach. Front Immunol. 2022;13:975848. doi: 10.3389/fimmu.2022.975848 Amatya N, Garg AV, Gaffen SL. IL-17 Signaling: The Yin and the Yang. Trends Immunol. 2017;38:310–322. doi: 10.1016/j.it.2017.01.006 Qian Z, Zhang Z, Wang Y. T cell receptor signaling pathway and cytokine-cytokine receptor interaction affect the rehabilitation process after respiratory syncytial virus infection. PeerJ. 2019;7:e7089. doi: 10.7717/peerj.7089 Githaka JM, Pirayeshfard L, Goping IS. Cancer invasion and metastasis: Insights from murine pubertal mammary gland morphogenesis. Biochim Biophys Acta Gen Subj. 2023;1867:130375. doi: 10.1016/j.bbagen.2023.130375 Zhu X, Zhou L, Li R, Shen Q, Cheng H, Shen Z, et al. AGER promotes proliferation and migration in cervical cancer. Bioscience Reports. 2018;38:BSR20171329. doi: 10.1042/BSR20171329 Zhang W, Xu J, Wang K, Tang X, He J. miR–139–3p suppresses the invasion and migration properties of breast cancer cells by targeting RAB1A. Oncol Rep. 2019;42:1699–1708. doi: 10.3892/or.2019.7297 Fu G, Xu H, Zhou C. The Value of Serum miR-139-3p Expression Level in Predicting Postoperative Survival of Colon Cancer Patients. Int J Gen Med. 2022;15:1405–1412. doi: 10.2147/IJGM.S346674 Zhu Y, Zhou C, He Q. High miR-139-3p expression predicts a better prognosis for hepatocellular carcinoma: a pooled analysis. J Int Med Res. 2019;47:383–390. doi: 10.1177/0300060518802727 Additional Declarations No competing interests reported. Supplementary Files WBGAPDH.tif WBISG20L2.tif Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3843095","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":266193297,"identity":"94960bfa-8dc5-4597-adec-95ce63737771","order_by":0,"name":"Xinyu Zhang","email":"","orcid":"","institution":"Dalian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xinyu","middleName":"","lastName":"Zhang","suffix":""},{"id":266193298,"identity":"0d89f09b-61cd-43d8-8c55-ee9fb5ed29dd","order_by":1,"name":"Dan Yu","email":"","orcid":"","institution":"Department of Respiratory and Critical Care Medicine,The Affiliated Changzhou No. 2 People’s Hospital of Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Dan","middleName":"","lastName":"Yu","suffix":""},{"id":266193299,"identity":"195c72dc-92f0-41e1-9f2c-02e60f0462ae","order_by":2,"name":"Ming Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYBACgxs5Zsw8BgwM/MzMhx8QqyWNeQ5Qi2Q7W5oBcVrOnDnG/AfEOM+jIEGcluO9bcw5BXfsNh/mYTBgqLGJJqjF/jA/G3OOwbPkbYd5DzxgOJaW20DQFoiWw8lmh/kSDBgbDhOhBeQwHqAW42YeAwnitIC8D9RiZ8BMtBZQIAO1JEgcBgZyAlF+uZFj/pnnz2F7/v7Dhx98qLEhrAUGEsEqE4hVDgL2pCgeBaNgFIyCEQYAih1CD1kwqusAAAAASUVORK5CYII=","orcid":"","institution":"Department of Respiratory and Critical Care Medicine,The Affiliated Changzhou No. 2 People’s Hospital of Nanjing Medical University","correspondingAuthor":true,"prefix":"","firstName":"Ming","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2024-01-07 17:14:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3843095/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3843095/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49491795,"identity":"58dce5c9-ca8d-4ae9-a6f6-b2e709e408fa","added_by":"auto","created_at":"2024-01-11 18:23:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":375406,"visible":true,"origin":"","legend":"\u003cp\u003eISG20L2 is highly expressed in lung adenocarcinoma and is associated with poor prognosis. (A)Differential expression analysis of ISG20L2 in lung adenocarcinoma in TCGA database.(B) ISG20L2 expression difference in lung adenocarcinoma and paracancerous tissue. (C) ROC curve analysis of the diagnostic value of ISG20L2 in lung adenocarcinoma.(D) Prognostic correlation analysis between ISG20L2 and lung adenocarcinoma patients. *** P\u0026lt;0.001\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3843095/v1/8f5ef9b3dd76174b5cf7a6ce.png"},{"id":49491801,"identity":"cc825735-f3d3-4294-9aa2-3f4105500d52","added_by":"auto","created_at":"2024-01-11 18:23:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":9194249,"visible":true,"origin":"","legend":"\u003cp\u003eGO and KEGG Enrichment Analyses. (A) GO terms of biological ,cellular component and molecular function .(B) KEGG pathways terms.\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3843095/v1/d8fbea0ed201ee3a2cb070f5.png"},{"id":49491797,"identity":"001c72a2-8c26-4ef9-bc2e-e74ee7223313","added_by":"auto","created_at":"2024-01-11 18:23:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4073735,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of ISG20L2 overexpression on the proliferation and apoptosis of A549 cells. (A)The transient transfection efficiency was tested by western-blot. (B) Proliferation of the pc-DNA3.1 and pc-DNA3.1-ISG20L2 groups was assessed using the CCK8 assay. (C) The apoptotic rate in the pc-DNA and pc-DNA-ISG20L2 groups was detected using flow cytometry. \u0026nbsp;ns: no significant (P \u0026gt; 0.05),**P\u0026lt;0.01, *** P\u0026lt;0.001\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3843095/v1/fef48ca2c928be35cf19bc4f.png"},{"id":49491798,"identity":"b5ffdc79-2c28-4bef-851c-46457a732c25","added_by":"auto","created_at":"2024-01-11 18:23:17","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5742620,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of ISG20L2 overexpression on the migration and invasion of A549 cells. (A) \u0026nbsp;Migratory \u0026nbsp;ability of pc-DNA3.1 and pc-DNA3.1-ISG20L2 groups was detected by the Transwell migration assay (magnification, ×200) . (B) Invasive ability of the pc-DNA and pc-DNA-ISG20L2 groups was detected by the Transwell invasion assay (magnification, ×200). *P\u0026lt;0.05 ,**P\u0026lt;0.01\u003c/p\u003e","description":"","filename":"figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3843095/v1/2863ec5babcb04820e69168f.jpg"},{"id":49492803,"identity":"d0e1920d-aed9-4535-9aa9-080d789dc360","added_by":"auto","created_at":"2024-01-11 18:31:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2735475,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of ISG20L2 knockdown on the biological behavior of A549 cells. (A) Proliferation of the NC and si\u003cem\u003e-\u003c/em\u003eISG20L2 groups was assessed using the CCK8 assay. (B) The apoptotic rate in the NC and si\u003cem\u003e-\u003c/em\u003eISG20L2 groups was detected using flow cytometry. (C) Migratory ability of NC and si\u003cem\u003e-\u003c/em\u003eISG20L2 groups was detected by the Transwell migration assay (magnification, ×200) . (D) Invasive ability of the NC and si-ISG20L2 groups was detected by the Transwell invasion assay. **P\u0026lt;0.01, *** P\u0026lt;0.001\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3843095/v1/95311629ad824e2383428853.png"},{"id":49556607,"identity":"6bcb7b01-dc00-46ad-9e07-655fa1762351","added_by":"auto","created_at":"2024-01-13 04:22:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1674603,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3843095/v1/70043af7-3f96-4aba-b4a4-bafdfb701e77.pdf"},{"id":49491799,"identity":"ad1c1c95-50a4-4505-852e-a366ee955420","added_by":"auto","created_at":"2024-01-11 18:23:17","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1491600,"visible":true,"origin":"","legend":"","description":"","filename":"WBGAPDH.tif","url":"https://assets-eu.researchsquare.com/files/rs-3843095/v1/47a587d1a943052abce5eb8b.tif"},{"id":49491796,"identity":"4d0eb6e0-9363-4f66-abc8-04c69d22c2a5","added_by":"auto","created_at":"2024-01-11 18:23:17","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1494388,"visible":true,"origin":"","legend":"","description":"","filename":"WBISG20L2.tif","url":"https://assets-eu.researchsquare.com/files/rs-3843095/v1/4ae73e6ada1299ef3f2a2218.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"High expression of ISG20L2 promotes proliferation and invasion of A549 cells and is associated with poor prognosis in lung adenocarcinoma","fulltext":[{"header":"1. Background","content":"\u003cp\u003eLung cancer is the leading cause of cancer-related deaths worldwide, with an incidence that increases yearly[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].It can be classified into two main types: non-small cell lung carcinoma (NSCLC) and small-cell lung carcinoma (SCLC). NSCLC accounts for approximately 85% of all lung cancers, with about 40% being lung adenocarcinoma (LUAD)[\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Targeted therapies have revolutionized the treatment for oncogene-driven NSCLC[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. A significant proportion, up to 69%, of patients with advanced NSCLC may have specific molecular targets that can be targeted by treatment[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The approach to treating advanced NSCLC has shifted from selecting treatments based on clinical and pathological features to utilizing biomarker-driven treatment algorithms that consider the patient's tumor molecular profile[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Molecularly targeted therapy involves using drugs or other substances that specifically target certain molecules (molecular targets) to inhibit the growth and spread of cancer cells[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This treatment approach offers advantages such as precise and efficient drug delivery, as well as reduced toxicity compared to traditional chemotherapy drugs, which improves the survival rate of many types of malignant tumors[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. While significant progress has been made in the systemic treatment of advanced NSCLC through molecular targeted therapy and immunotherapy, the long-term survival rate for lung cancer patients remains low[\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. There is still a need to identify new biomarkers and prognostic genes, develop next-generation drugs with more specific targeting effects, and create drugs that can effectively counter specific drug-resistant mutations. These advancements aim to provide patients with more effective treatments and extend their survival time. Such efforts align with the research focus in the era of precision medicine.\u003c/p\u003e \u003cp\u003eInterferon-stimulated 20-kDa exonuclease-like 2 (ISG20L2) is a member of the vertebrate exonuclease family that plays a role in ribosome biogenesis degradation. Research has demonstrated that ISG20L2 accumulates in the nucleoli of HeLa cells and exhibits exoribonucleolytic activity, degrading RNA from the 3'- to 5'-terminus. This activity has been linked to cellular proliferation and apoptosis[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. High expression of ISG20L2 has been observed in various cancers, including lung cancer[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], liver cancer[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], breast cancer[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], etc. The latest research has found that ISG20L2 is an exonuclease with priority specificity for uridylated miRNA, which can regulate T cell activation[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Notably, in myeloma, ISG20L2 weakens the binding of bortezomib to the proteasome 20S subunit β5 (PSMB5), leading to reduced inhibition of proteasome activity and decreased efficacy of bortezomib treatment[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] .These findings highlight the close association between ISG20L2 expression levels and the effectiveness and prognosis of cancer treatment. However, the specific role of ISG20L2 LUAD remains unclear.\u003c/p\u003e \u003cp\u003eThis study aims to investigate the expression of ISG20L2 in lung cancer and evaluate its diagnostic value using bioinformatics analysis. Subsequently, the expression of ISG20L2 will be validated in tissue samples through immunohistochemistry. The relationship between ISG20L2 and clinicopathological features of LUAD will be explored, along with its prognostic value. Additionally, in vitro experiments will be conducted to examine the impact of ISG20L2 on the biological behavior of LUAD cells.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Bioinformatics analysis\u003c/h2\u003e \u003cp\u003eThe clinical data of ISG20L2 expression profile related LUAD were downloaded from the TCGA dataset. The adenocarcinoma microarray dataset GSE19804 was retrieved from the Gene Expression Omnibus (GEO) database[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], which is maintained by the National Center for Biotechnology Information (NCBI). To perform differential analysis, we employed the R package clusterProfiler.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Gene Ontology and Kyoto Encyclopedia of Genes and Genomes Enrichment Analyses\u003c/h2\u003e \u003cp\u003eThe Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed on the shared genes by the \u0026ldquo;GOplot\u0026rdquo; and the \u0026ldquo;clusterprofiler\u0026rdquo; R package. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Diagnostic Value Analysis\u003c/h2\u003e \u003cp\u003eThe receiver operating characteristic (ROC) curve was used to assess the diagnostic value of ISG20L2 in LUAD. The closer the area under the curve (AUC) is (1), the better the diagnostic effect is. AUC in 0.5\u0026ndash;0.7 has a low accuracy, AUC in 0.7\u0026ndash;0.9 has a certain accuracy, and AUC above 0.9 has a high accuracy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Immunohistochemical (IHC) staining\u003c/h2\u003e \u003cp\u003eWe obtained a lung adenocarcinoma tissue chip (catalog number HLugA180Su08) from Shanghai Outdo Biotech Co., Ltd., which consisted of 95 LUAD and 80 paracancerous specimens. The tissue chip was initially baked at 63\u0026deg;C for 1 hour. Subsequently, we removed the tissue chip using xylene treatment (twice, for 15 minutes each) and performed gradient alcohol dehydration (100% twice every 7 minutes, 90% once every 5 minutes, 80% once every 5 minutes, and 70% once every 5 minutes). Antigen retrieval was then carried out using the Dako fully automated immunohistochemical pretreatment system (Dako North America, Inc). Furthermore, the tissue chip was incubated overnight at 4\u0026deg;C with the ISG20L2 antibody (rabbit polyclonal, dilution 1:50, catalog number NBP1-82287, Novus Biologicals, USA). The following day, we treated the chip with anti-rabbit horseradish peroxidase IgG polymer (Abcam, Cambridge, UK) as a secondary antibody for 30 minutes at room temperature. The chip was stained with DAB, counterstained with hematoxylin, and finally sealed with a neutral resin.\u003c/p\u003e \u003cp\u003eFor scoring, staining intensity was categorized as follows: 0 (negative), 1 (1+), 2 (2+), and 3 (3+). The proportion of positive staining ranged from 0\u0026ndash;100%. The overall score was calculated by multiplying the staining intensity score by the proportion of staining results (0-300%). In the survival analysis group, a total score of less than 27.5 indicated the ISG20L2 low expression group, while a score of 27.5 or higher indicated the ISG20L2 high expression group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Cell Culture and transfection\u003c/h2\u003e \u003cp\u003eWe purchased A549 cells from Procell Life Science \u0026amp; Technology Co., Ltd. (Wuhan, China) and cultured them in Ham's F-12K medium supplemented with 10% fetal bovine serum and 1% double antibody (100%) \u0026times; penicillin-streptomycin solution. The cells were incubated in a humidified incubator containing 5% CO2 at 37\u0026deg;C. The pcDNA empty vector, pcDNA-ISG20L2, negative control, and siRNA-ISG20L2 were designed and provided by GenePharma Co., Ltd (Suzhou, China). A549 cells were inoculated on 6-well cell culture plates at a density of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well. Lipofectamine3000 (Invitrogen, Thermo Fisher Scientific, USA) was added to the plasmids or oligonucleotides mentioned above when the cells reached 70%\u0026sim;80% confluence. Cells were harvested and extracted for further study 48 hours after transfection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Western blot analysis\u003c/h2\u003e \u003cp\u003eProtein samples were extracted from A549 cells using RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with protease and phosphatase inhibitors (Beyotime). The protein concentration was measured using a BCA protein detection kit (Beyotime). Subsequently, the protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes.\u003c/p\u003e \u003cp\u003eThe PVDF membranes were then blocked with 5% skim milk at room temperature for 1.5 hours. Primary antibodies against ISG20L2 (catalog number NBP2-83084, Novus Biologicals, USA) and GAPDH (Abcam, Cambridge, UK) were incubated with the membranes overnight at 4\u0026deg;C. After three washes, the membranes were incubated with a secondary antibody, goat anti-rabbit IgG (Abcam, Cambridge, UK), for 1.5 hours. Proteins were visualized using an ECL luminescent kit (Tanon, Shanghai, China). GAPDH was used as an internal control, and the relative protein expression levels were quantified using Image J software (NIH, Bethesda) by calculating the grayscale value of the bands. All experiments were performed in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Cell proliferation assay\u003c/h2\u003e \u003cp\u003eTo assess the proliferation of tumor cells, we utilized the Cell Counting Kit-8 (CCK-8, Dojindo, Tokyo, Japan). A549 cells were seeded in 96-well plates at a density of 2\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells per well. Following incubation for 0, 12, 24, 48, and 72 hours, 10 \u0026micro;L of the CCK-8 reagent was added to each well and incubated at 37\u0026deg;C for 1 hour. The absorbance at 450 nm in each well was subsequently measured using a microplate reader (Thermo, Waltham, MA, USA). All experiments were conducted in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Flow cytometry\u003c/h2\u003e \u003cp\u003eTo detect early and late apoptotic cells, we employed flow cytometry in conjunction with the Annexin V-FITC/PI Apoptosis Detection kit (KeyGEN BioTECH, Nanjing, China). Cells were harvested using EDTA-free Trypsin, washed twice with chilled PBS, and centrifuged at 1,000 g for 5 minutes. A 500 \u0026micro;L binding buffer was added to the cell suspension, followed by the addition of 5 \u0026micro;L Annexin V-FITC and 5 \u0026micro;L Propidium Iodide. The mixture was then incubated at room temperature in the dark for 10 minutes. Finally, an optical instrument known as a flow cytometer (Becton Dickinson and Co., Franklin Lakes, NJ, USA) was employed to detect the apoptotic cells. All experiments were conducted in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Transwell migration and invasion assays\u003c/h2\u003e \u003cp\u003eTo assess the migration and invasion of cancer cells, we utilized Transwell chambers (8 mm pore size, Corning Inc, NY, USA) and 24-well plates (Corning Inc, NY, USA). For the transwell migration and invasion assays, 200 \u0026micro;L of serum-free medium containing 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells was seeded into the upper chamber of a transwell. In the case of the transwell invasion assay, the upper chamber was pre-coated with matrigel (Corning Inc, NY, USA). The lower chamber was filled with 700 \u0026micro;L of Ham's F-12k supplemented with 10% FBS. After incubating for 24 hours, any remaining tumor cells in the upper chamber were removed using cotton swabs. The invaded cells were fixed with 4% paraformaldehyde for 20 minutes and stained with crystal violet for 15 minutes at room temperature. Subsequently, the number of invading cells was counted in 5 randomly selected fields under an inverted microscope (Olympus, Tokyo, Japan). All experiments were conducted in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Statistical analysis\u003c/h2\u003e \u003cp\u003eWe employed R software v4.3.0 for statistical analysis. The Wilcoxon test was utilized to perform differential analysis of ISG20L2 expression in cancer and paracancerous tissues. The Fisher test was employed to analyze the relationship between ISG20L2 expression and clinicopathological variables. For survival univariate analysis, we utilized Kaplan-Meier survival analysis and log-rank statistical test. Additionally, we used the Cox proportional risk regression model for univariate and multivariate analysis of total survival time. A statistically significant difference was considered when P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 ISG20L2 is highly expressed in LUAD and High ISG20L2 expression is associated with poor LUAD prognosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the role of ISG20L2 in LUAD, we initially analyzed its differential expression in LUAD mRNA using The Cancer Genome Atlas (TCGA) database[22]\u0026nbsp;(https://genome-cancer.ucsc.edu/). Our analysis revealed that ISG20L2 expression was significantly upregulated in LUAD compared to corresponding healthy tissues (Fig. 1A; P\u0026lt;0.001). To confirm this finding, we performed immunohistochemical (IHC) staining on a chip containing 95 LUAD and 80 paracancerous specimens. The results demonstrated that ISG20L2 protein was highly expressed in lung adenocarcinoma tissues but exhibited lower expression in paracancerous tissues (Fig. 1B; P\u0026lt;0.001). These findings collectively indicated the increased expression of ISG20L2 in LUAD, suggesting its involvement in LUAD pathogenesis.\u003c/p\u003e\n\u003cp\u003eFurthermore, we conducted ROC curve analysis to assess the diagnostic value of ISG20L2 in LUAD. The results indicated that ISG20L2 exhibited a certain level of accuracy (AUC＝0.829) in predicting LUAD (Fig. 1C). Additionally, we examined the associations between ISG20L2 expression and various clinical characteristics in LUAD patients. However, no significant correlations were observed between ISG20L2 expression and factors such as gender, age, tumor size, T stage, N stage, M stage, pathological grade, and TNM stage (Table 1).\u003c/p\u003e\n\u003cp\u003eTo evaluate the prognostic significance of ISG20L2 expression at the protein level, we analyzed the relationship between ISG20L2 expression and patient prognosis using IHC staining results. Our analysis revealed that low ISG20L2 expression was associated with a better prognosis (Fig. 1D; P\u0026lt;0.05). Moreover, we employed the Cox regression model to assess the link between ISG20L2 expression and patient survival (Table 2). Univariate analysis demonstrated that ISG20L2 expression, age, N stage, and TNM stage were factors influencing the overall survival of lung adenocarcinoma patients. However, multivariate analysis indicated that these factors were not independent risk factors for the overall survival of lung adenocarcinoma patients.\u003c/p\u003e\n\u003cp\u003eOverall, our findings suggest that ISG20L2 is upregulated in LUAD and may play a role in its pathogenesis. Furthermore, ISG20L2 expression shows diagnostic potential and is associated with patient prognosis in LUAD.\u003c/p\u003e\n\u003cp\u003eTable 1．The relationship between the protein expression of ISG20L2 and clinicopathological characteristics in patients with lung adenocarcinoma\u0026nbsp;\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"536\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.553072625698324%\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"15.828677839851025%\" rowspan=\"2\"\u003evariables\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"26.443202979515828%\" colspan=\"2\"\u003eISG20L2 \u0026nbsp; \u0026nbsp; expression\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.359404096834265%\" rowspan=\"2\"\u003etotal\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.221601489757914%\" rowspan=\"2\"\u003ep value\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.594040968342645%\" rowspan=\"2\"\u003er value\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.68292682926829%\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"29.26829268292683%\"\u003elow\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"28.048780487804876%\"\u003ehigh\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.58955223880597%\" valign=\"top\"\u003esex\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"15.85820895522388%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.432835820895523%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"12.873134328358208%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.380597014925373%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.246268656716419%\" valign=\"top\"\u003e0.143\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.619402985074627%\" valign=\"top\"\u003e0.162\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.58955223880597%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"15.85820895522388%\" valign=\"top\"\u003eMale\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.432835820895523%\" valign=\"top\"\u003e21\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"12.873134328358208%\" valign=\"top\"\u003e18\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.380597014925373%\" valign=\"top\"\u003e39\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.246268656716419%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.619402985074627%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.58955223880597%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"15.85820895522388%\" valign=\"top\"\u003eFemale\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.432835820895523%\" valign=\"top\"\u003e21\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"12.873134328358208%\" valign=\"top\"\u003e35\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.380597014925373%\" valign=\"top\"\u003e56\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.246268656716419%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.619402985074627%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.58955223880597%\" valign=\"top\"\u003eage\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"15.85820895522388%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.432835820895523%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"12.873134328358208%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.380597014925373%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.246268656716419%\" valign=\"top\"\u003e1\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.619402985074627%\" valign=\"top\"\u003e-0.009\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.58955223880597%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"15.85820895522388%\" valign=\"top\"\u003e\u0026le;60\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.432835820895523%\" valign=\"top\"\u003e21\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"12.873134328358208%\" valign=\"top\"\u003e27\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.380597014925373%\" valign=\"top\"\u003e48\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.246268656716419%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.619402985074627%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.58955223880597%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"15.85820895522388%\" valign=\"top\"\u003e\u0026gt;60\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.432835820895523%\" valign=\"top\"\u003e21\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"12.873134328358208%\" valign=\"top\"\u003e26\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.380597014925373%\" valign=\"top\"\u003e47\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.246268656716419%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.619402985074627%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.58955223880597%\" valign=\"top\"\u003eTumor_size\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"15.85820895522388%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.432835820895523%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"12.873134328358208%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.380597014925373%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.246268656716419%\" valign=\"top\"\u003e0.512\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.619402985074627%\" valign=\"top\"\u003e0.071\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.58955223880597%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"15.85820895522388%\" valign=\"top\"\u003e\u0026le;4cm\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.432835820895523%\" valign=\"top\"\u003e29\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"12.873134328358208%\" valign=\"top\"\u003e32\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.380597014925373%\" valign=\"top\"\u003e61\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.246268656716419%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.619402985074627%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.58955223880597%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"15.85820895522388%\" valign=\"top\"\u003e\u0026gt;4cm\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.432835820895523%\" valign=\"top\"\u003e12\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"12.873134328358208%\" valign=\"top\"\u003e18\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.380597014925373%\" valign=\"top\"\u003e30\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.246268656716419%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.619402985074627%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.58955223880597%\" valign=\"top\"\u003eT\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"15.85820895522388%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.432835820895523%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"12.873134328358208%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.380597014925373%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.246268656716419%\" valign=\"top\"\u003e0.124\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.619402985074627%\" valign=\"top\"\u003e0.184\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.58955223880597%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"15.85820895522388%\" valign=\"top\"\u003eⅠ\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.432835820895523%\" valign=\"top\"\u003e12\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"12.873134328358208%\" valign=\"top\"\u003e8\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.380597014925373%\" valign=\"top\"\u003e20\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.246268656716419%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.619402985074627%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.58955223880597%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"15.85820895522388%\" valign=\"top\"\u003eⅡ-Ⅳ\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.432835820895523%\" valign=\"top\"\u003e27\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"12.873134328358208%\" valign=\"top\"\u003e44\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.380597014925373%\" valign=\"top\"\u003e71\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.246268656716419%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.619402985074627%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.58955223880597%\" valign=\"top\"\u003eN\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"15.85820895522388%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.432835820895523%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"12.873134328358208%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.380597014925373%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.246268656716419%\" valign=\"top\"\u003e0.061\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.619402985074627%\" valign=\"top\"\u003e0.21\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.58955223880597%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"15.85820895522388%\" valign=\"top\"\u003eN0\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.432835820895523%\" valign=\"top\"\u003e25\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"12.873134328358208%\" valign=\"top\"\u003e20\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.380597014925373%\" valign=\"top\"\u003e45\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.246268656716419%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.619402985074627%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.58955223880597%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"15.85820895522388%\" valign=\"top\"\u003eN1/N2/N3\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.432835820895523%\" valign=\"top\"\u003e17\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"12.873134328358208%\" valign=\"top\"\u003e32\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.380597014925373%\" valign=\"top\"\u003e49\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.246268656716419%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.619402985074627%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.58955223880597%\" valign=\"top\"\u003eM\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"15.85820895522388%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.432835820895523%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"12.873134328358208%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.380597014925373%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.246268656716419%\" valign=\"top\"\u003e1\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.619402985074627%\" valign=\"top\"\u003e0.092\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.58955223880597%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"15.85820895522388%\" valign=\"top\"\u003eM0\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.432835820895523%\" valign=\"top\"\u003e42\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"12.873134328358208%\" valign=\"top\"\u003e52\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.380597014925373%\" valign=\"top\"\u003e94\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.246268656716419%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.619402985074627%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.58955223880597%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"15.85820895522388%\" valign=\"top\"\u003eM1\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.432835820895523%\" valign=\"top\"\u003e0\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"12.873134328358208%\" valign=\"top\"\u003e1\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.380597014925373%\" valign=\"top\"\u003e1\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.246268656716419%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.619402985074627%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.58955223880597%\" valign=\"top\"\u003egrade\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"15.85820895522388%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.432835820895523%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"12.873134328358208%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.380597014925373%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.246268656716419%\" valign=\"top\"\u003e0.569\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.619402985074627%\" valign=\"top\"\u003e0.071\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.58955223880597%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"15.85820895522388%\" valign=\"top\"\u003eⅠ-Ⅱ\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.432835820895523%\" valign=\"top\"\u003e37\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"12.873134328358208%\" valign=\"top\"\u003e44\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.380597014925373%\" valign=\"top\"\u003e81\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.246268656716419%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.619402985074627%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.58955223880597%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"15.85820895522388%\" valign=\"top\"\u003eⅢ\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.432835820895523%\" valign=\"top\"\u003e5\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"12.873134328358208%\" valign=\"top\"\u003e9\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.380597014925373%\" valign=\"top\"\u003e14\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.246268656716419%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.619402985074627%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.58955223880597%\" valign=\"top\"\u003eTNM\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"15.85820895522388%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.432835820895523%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"12.873134328358208%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.380597014925373%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.246268656716419%\" valign=\"top\"\u003e0.086\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.619402985074627%\" valign=\"top\"\u003e0.193\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.58955223880597%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"15.85820895522388%\" valign=\"top\"\u003eⅠ\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.432835820895523%\" valign=\"top\"\u003e20\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"12.873134328358208%\" valign=\"top\"\u003e15\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.380597014925373%\" valign=\"top\"\u003e35\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.246268656716419%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.619402985074627%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.58955223880597%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"15.85820895522388%\" valign=\"top\"\u003eⅡ-Ⅳ\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.432835820895523%\" valign=\"top\"\u003e22\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"12.873134328358208%\" valign=\"top\"\u003e37\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.380597014925373%\" valign=\"top\"\u003e59\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.246268656716419%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.619402985074627%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;r：Correlation Coefficient, P \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e\n\u003cp\u003eTable 2. Univariate and multivariate analyses of the factors correlated with Overall survival of lung cancer patients\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"94%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.19191919191919%\"\u003evariables\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"44.44444444444444%\" colspan=\"4\"\u003eUnivariate analysis\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"36.36363636363637%\" colspan=\"4\"\u003eMultivariate analysis\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.791666666666668%\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.333333333333334%\"\u003eHR\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"20.833333333333332%\" colspan=\"2\"\u003e95%CI\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"14.583333333333334%\"\u003ep value\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.291666666666667%\"\u003eHR\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"18.75%\" colspan=\"2\"\u003e95%CI\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\"\u003ep value\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20%\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.421052631578947%\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\"\u003eLower limit\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\"\u003eUpper limit\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"14.736842105263158%\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.368421052631579%\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.368421052631579%\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\"\u003eUpper limit\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003eexpression\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.421052631578947%\" valign=\"top\"\u003e1.706\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e1.005\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e2.895\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"14.736842105263158%\" valign=\"top\"\u003e0.0477\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.368421052631579%\" valign=\"top\"\u003e1.35\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.368421052631579%\" valign=\"top\"\u003e0.78\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e2.32\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e0.282\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003esex\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.421052631578947%\" valign=\"top\"\u003e0.991\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e0.593\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e1.656\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"14.736842105263158%\" valign=\"top\"\u003e0.971\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.368421052631579%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.368421052631579%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003eage\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.421052631578947%\" valign=\"top\"\u003e1.953\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e1.161\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e3.284\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"14.736842105263158%\" valign=\"top\"\u003e0.0117\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.368421052631579%\" valign=\"top\"\u003e1.47\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.368421052631579%\" valign=\"top\"\u003e0.86\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e2.53\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e0.16\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003eTumor_size\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.421052631578947%\" valign=\"top\"\u003e1.166\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e0.673\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e2.019\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"14.736842105263158%\" valign=\"top\"\u003e0.585\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.368421052631579%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.368421052631579%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003eT\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.421052631578947%\" valign=\"top\"\u003e1.838\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e0.927\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e3.645\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"14.736842105263158%\" valign=\"top\"\u003e0.0814\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.368421052631579%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.368421052631579%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003eN\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.421052631578947%\" valign=\"top\"\u003e3.369\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e1.929\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e5.884\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"14.736842105263158%\" valign=\"top\"\u003e0.0000197\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.368421052631579%\" valign=\"top\"\u003e2.68\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.368421052631579%\" valign=\"top\"\u003e0.95\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e7.55\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e0.0617\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003eM\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.421052631578947%\" valign=\"top\"\u003e1.343\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e0.185\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e9.734\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"14.736842105263158%\" valign=\"top\"\u003e0.77\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.368421052631579%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.368421052631579%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003egrade\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.421052631578947%\" valign=\"top\"\u003e1.331\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e0.654\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e2.706\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"14.736842105263158%\" valign=\"top\"\u003e0.43\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.368421052631579%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.368421052631579%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003eTNM\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.421052631578947%\" valign=\"top\"\u003e2.979\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e1.639\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e5.412\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"14.736842105263158%\" valign=\"top\"\u003e0.000341\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.368421052631579%\" valign=\"top\"\u003e1.11\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.368421052631579%\" valign=\"top\"\u003e0.37\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e3.38\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.526315789473685%\" valign=\"top\"\u003e0.853\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eHR: Hazard Ratio, CI: Confidence Interval.\u0026nbsp;P \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 GO and KEGG Enrichment Analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo gain a better understanding of the biological processes and pathways involved in lung cancer, we performed GO functional enrichment analysis and KEGG pathway analysis. The GO functional enrichment analysis revealed that the primary biological processes (BP) involved extracellular matrix organization, extracellular structure organization, and external encapsulating. The cellular component (CC) was primarily enriched in collagen-containing extracellular matrix, membrane raft, and membrane microdomain. The molecular function (MF) was mainly involved in glycosaminoglycan binding, extracellular matrix structural constituent, and heparin binding (Fig. 2A).\u003c/p\u003e\n\u003cp\u003eThe KEGG pathway enrichment analysis identified several pathways related to lung cancer, including cytokine-cytokine receptor interaction, protein digestion and absorption, IL-17 signaling pathway, and lipid and atherosclerosis (Fig. 2B). These findings suggest that alterations in extracellular matrix organization and cytokine signaling may play a critical role in the development and progression of lung cancer.\u003c/p\u003e\n\u003cp\u003eThese results provide valuable insights into the biological processes and pathways associated with lung cancer, which may aid in the development of more effective diagnostic and therapeutic strategies for this disease.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 ISG20L2 overexpression promotes proliferation, migration, and invasion of A549 cells but without affecting apoptosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the efficiency of ISG20L2 overexpression, Western blot analysis was performed in both the pcDNA3.1 and pcDNA3.1-ISG20L2 groups. The results showed a significant increase in ISG20L2 expression in the pcDNA3.1-ISG20L2 group compared to the pcDNA3.1 group (Fig. 3A; P\u0026lt;0.001).\u003c/p\u003e\n\u003cp\u003eTo further investigate the impact of ISG20L2 on the biological function of A549 cells, a CCK-8 assay was conducted to measure cell proliferation. The results demonstrated a significant increase in cell proliferation in the pcDNA3.1-ISG20L2 group after 48 hours compared to the pcDNA3.1 group (Fig. 3B; P\u0026lt;0.01). However, no significant difference was observed in apoptosis between the two groups following ISG20L2 overexpression (Fig. 3C，P\u0026gt; 0.05). These findings suggest that ISG20L2 overexpression enhances the growth rate of A549 cells in vitro but does not affect cell apoptosis.\u003c/p\u003e\n\u003cp\u003eTo evaluate the migratory and invasive abilities of A549 cells, transwell assays were performed. The results indicated that cell migration and invasion were significantly increased in the pcDNA3.1-ISG20L2 group compared to the pcDNA3.1 group (Fig. 4A; Fig. 4B; P\u0026lt;0.05). These findings suggest that ISG20L2 overexpression promotes the migration and invasion of A549 cells.\u003c/p\u003e\n\u003cp\u003eCollectively, our results demonstrate that ISG20L2 overexpression enhances the proliferative capacity, migration, and invasion of A549 cells, indicating its potential role in promoting the progression of lung adenocarcinoma.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 ISG20L2 knockdown decreases proliferation, migration, and invasion of A549 cells but without affecting apoptosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate the effects of ISG20L2 on LUAD cells, ISG20L2 knockdown was performed, and the transfection efficiency was measured using Western blot analysis. The results revealed a reduction in ISG20L2 expression in the si\u003cem\u003e-\u003c/em\u003eISG20L2 group compared to the NC group (Fig. 3A; P\u0026lt;0.001).\u003c/p\u003e\n\u003cp\u003eSubsequently, a CCK-8 assay was conducted to evaluate the cell proliferative capacity. The results indicated a significant decrease in cell proliferation in the\u0026nbsp;si\u003cem\u003e-\u003c/em\u003eISG20L2 group compared to the NC group (Fig. 5A; P\u0026lt;0.01). Flow cytometry analysis demonstrated no difference in apoptosis rate between the NC group and the si\u003cem\u003e-\u003c/em\u003eISG20L2 group (Fig. 5B，P\u0026nbsp;\u0026gt; 0.05). These findings suggest that ISG20L2 knockdown reduces the proliferative ability of A549 cells, while similar to the results observed with ISG20L2 overexpression, ISG20L2 knockout does not affect apoptosis.\u003c/p\u003e\n\u003cp\u003eFurthermore, transwell assays were performed to assess cell migration and invasion. The\u0026nbsp;si\u003cem\u003e-\u003c/em\u003eISG20L2 group exhibited significantly decreased migration and invasion abilities compared to the NC group (Fig. 5C; Fig. 5D; P\u0026lt;0.05). In summary, the aforementioned experiments demonstrate that ISG20L2 knockdown decreases the migration and invasion of A549 cells.\u003c/p\u003e\n\u003cp\u003eTaken together, these findings suggest that ISG20L2 plays a crucial role in promoting the proliferative capacity, migration, and invasion of LUAD cells, highlighting its potential as a target for therapeutic interventions in lung adenocarcinoma.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eLung adenocarcinoma is a prevalent form of cancer worldwide, but its precise underlying mechanisms of occurrence and development remain incompletely understood. Investigating the differential genes associated with lung adenocarcinoma not only contributes to unraveling its molecular mechanisms but also holds significant potential for identifying diagnostic and prognostic markers for this disease.\u003c/p\u003e \u003cp\u003eLimited literature has explored the relationship between ISG20L2 and cancer, primarily employing bioinformatics approaches. Consequently, its precise cellular function remains largely unknown. Notably, bioinformatics analysis has revealed that ISG20L2 exhibits elevated expression in hepatocellular carcinoma, correlating with reduced patient survival. Consequently, it holds promise as a potential molecular target for immunotherapy in this cancer type[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In breast cancer, ISG20L2 has been associated with the expression of the tumor cell proliferation marker MKI67 (Ki-67) and the prognosis of breast cancer patients[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In the context of LUAD, our study employed bioinformatics analysis and immunohistochemistry tests to identify elevated mRNA and protein levels of ISG20L2. Survival analysis demonstrated that ISG20L2 serves as a high-risk gene, with increased expression correlating with poor prognosis in LUAD patients. ROC analysis indicated that ISG20L2 holds diagnostic potential for lung adenocarcinoma. These findings suggest that ISG20L2 may play a crucial role in improving the prognosis of LUAD patients by aiding in diagnosis and accurate prognostic evaluation.\u003c/p\u003e \u003cp\u003eIn terms of GO analysis, ISG20L2 enrichment is associated with proliferation and energy metabolism, including extracellular matrix tissue and extracellular structural tissue. Cellular component terms indicate involvement in membrane microdomains and membrane rafts. Bioenergetic stress from abnormal energy metabolism can lead to signaling in tumor cells[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Notably, membrane microdomains play a role in immune receptor signaling[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Previous studies have reported ISG20L2's involvement in immune cell invasion in hepatocellular carcinoma[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore, it is necessary to further investigate whether ISG20L2 is involved in immune cell invasion in LUAD and elucidate how ISG20L2 mediates the relationship between immune receptor signaling and immune cell infiltration. Regarding the KEGG analysis, ISG20L2 is implicated in several pathways closely related to the inflammatory response, such as the cytokine-cytokine receptor interaction pathway and the IL17 signaling pathway[\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].These findings suggest that ISG20L2 may participate in immune-related processes within the context of LUAD. Further research is warranted to comprehensively understand the precise mechanisms by which ISG20L2 influences immune responses and its potential impact on immune cell infiltration in lung adenocarcinoma.\u003c/p\u003e \u003cp\u003eOur study also investigated the relationship between the expression level of ISG20L2 and clinical pathological parameters in LUAD patients. We observed that the expression of ISG20L2 did not show significant correlations with gender, age, tumor size, TNM staging, or pathological grading in LUAD patients. However, COX regression model analysis revealed an association between ISG20L2 expression and the survival rate of LUAD patients.\u003c/p\u003e \u003cp\u003eTo gain insights into the impact of ISG20L2 on the biological behavior of LUAD cells, we conducted various cell experiments. Our findings demonstrated a positive correlation between ISG20L2 expression and the proliferation, migration, and invasion abilities of LUAD cells. It is well known that abnormal proliferation is closely related to the development and progression of cancer and cancer invasion and metastasis accounts for the majority of cancer related mortality[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. However, we did not observe any significant effect of ISG20L2 on the apoptosis ability of LUAD cells. It is important to note that our study has certain limitations. Accumulated evidence indicates that miR-139-3p was decreased in breast cancer[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] tissues and colon cancer[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] serum, and decreased miR-139-3p is associated with a poor prognosis in cancer patients. Although we acknowledge that ISG20L2 is a target gene for miR-139-3p [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], we have not conducted in-depth research on the specific mechanisms of action involved. Additionally, we have not investigated the regulatory effect of ISG20L2 on LUAD in \u003cem\u003evivo\u003c/em\u003e.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn summary, our study indicates that ISG20L2 promotes the proliferation, migration, and invasion of LUAD cells, and its high expression predicts a poorer prognosis for LUAD patients. These findings suggest that ISG20L2 may serve as a potential therapeutic target and prognostic factor for LUAD. Further research is needed to elucidate the underlying mechanisms and validate these observations in \u003cem\u003evivo\u003c/em\u003e.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003eNSCLC\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003enon-small cell lung carcinoma\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003eSCLC\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003esmall-cell lung carcinoma\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003eLUAD\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003elung adenocarcinoma\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003eISG20L2\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003eInterferon-stimulated 20-kDa exonuclease-like 2\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003ePSMB5\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003eproteasome 20S subunit \u0026beta;5\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003eGEO\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003eGene Expression Omnibus\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003eNCBI\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003eNational Center for Biotechnology Information\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003eGO\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003eGene Ontology\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003eKEGG\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003eKyoto Encyclopedia of Genes and Genomes\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003eROC\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003ereceiver operating characteristic\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003eAUC\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003earea under the curve\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003eTNM\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003eTumor Node Metastasis\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003eIHC\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003eimmunohistochemical\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003eSDS-PAGE\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003esodium dodecyl sulfate-polyacrylamide gel electrophoresis\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003ePVDF\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003epolyvinylidene difluoride\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003eCCK-8\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003ecell counting kit-8\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003eEDTA\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003eethylenediaminetetraacetic acid\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003eBP\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003ebiological processes\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003eCC\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003ecellular component\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003eMF\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003emolecular function\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003eIL17\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003einterleukin 17\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eThe clinical data of ISG20L2 expression profile related LUAD were downloaded from the TCGA dataset (https://portal.gdc.com).The adenocarcinoma microarray dataset GSE19804 was retrieved from the Gene Expression Omnibus (GEO) database of the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/geo/).\u003c/p\u003e\n\u003cp\u003eConflict of Interest\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003eFunding\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study was supported by grants from\u0026nbsp;Top Talent Of Changzhou \u0026ldquo;The 14\u003csup\u003eth\u003c/sup\u003e Five-Year Plan\u0026rdquo; High-Level Health Talents Training Project (2022CZBJ056) and Changzhou Science and Technology Project (ZD202337).\u003c/p\u003e\n\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eXinyu Zhang and Ming Liu conceived and designed the research. Xinyu Zhang performed the experiments and analyzed the data.\u0026nbsp;Dan Yu analyzed the data. Xinyu Zhang\u0026nbsp;and\u0026nbsp;Dan Yu\u0026nbsp;wrote the manuscript, and Ming Liu made revisions to it . All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eThe authors thank the efforts and contributions of all the staff in this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOliver AL. Lung Cancer: Epidemiology and Screening. 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J Int Med Res. 2019;47:383\u0026ndash;390. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1177/0300060518802727\u003c/span\u003e\u003cspan address=\"10.1177/0300060518802727\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Lung adenocarcinoma, ISG20L2, Proliferation, Prognosis, Targeted regulation","lastPublishedDoi":"10.21203/rs.3.rs-3843095/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3843095/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eInterferon-stimulated 20kDa exonuclease-like 2 (ISG20L2) is a gene that exhibits differential expression in lung adenocarcinoma (LUAD). However, its expression and function in LUAD remain poorly understood. The aim of this study was to investigate the expression of ISG20L2 in LUAD and its correlation with prognosis, as well as to explore its impact on the biological behavior of LUAD.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe researchers analyzed the expression of ISG20L2 using both The Cancer Genome Atlas (TCGA) database and immunohistochemistry (IHC). Enrichment analysis was performed using the \"GOplot\" and \"clusterprofile\" R packages. The correlation between ISG20L2 expression and prognosis of LUAD patients was assessed through IHC and Kaplan-Meier survival analysis. Additionally, the diagnostic value of ISG20L2 in LUAD was evaluated using ROC curve analysis. The relationship between ISG20L2 expression and clinicopathological characteristics was examined through IHC. Overexpression and knockout experiments of ISG20L2 were conducted via transient transfection. The biological properties of ISG20L2 in A549 cells, including cell proliferation, apoptosis, migration, and invasion abilities, were investigated using assays such as cell counting kit-8 (CCK-8), flow cytometry, and Transwell assays.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe findings indicated that ISG20L2 was highly expressed in LUAD, and its high expression was closely associated with poor prognosis. In vitro experiments further confirmed a positive correlation between ISG20L2 expression level and the proliferation, migration, and invasion abilities of LUAD cells, while no significant effect on apoptotic ability was observed.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur study indicates that ISG20L2 promotes the proliferation, migration, and invasion of LUAD cells, and its high expression predicts a poorer prognosis for LUAD patients. This study suggests that ISG20L2 has the potential to serve as a molecular marker for the treatment and prognosis of LUAD.\u003c/p\u003e","manuscriptTitle":"High expression of ISG20L2 promotes proliferation and invasion of A549 cells and is associated with poor prognosis in lung adenocarcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-11 18:23:12","doi":"10.21203/rs.3.rs-3843095/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"67d7c1c2-a850-4b02-b89f-6fb994f327f7","owner":[],"postedDate":"January 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-13T04:14:12+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-11 18:23:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3843095","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3843095","identity":"rs-3843095","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}