Aberrant expression of HIF3A in plasma of patients with non‐small cell lung cancer and its clinical significance

BackgroundHypoxia‐inducible factors (HIFs) have been evaluated in various cancers and diseases. However, the specific role of hypoxia‐inducible factor 3 alpha (HIF3A) in non‐small cell lung cancer (NSCLC) remains controversial.Materials and MethodsWe investigated HIF3A mRNA expression in the plasma and tumor tissues of patients with NSCLC and explored its clinical significance. Plasma samples from 103 cases of lung adenocarcinoma (LUAD) and 96 cases of lung squamous cell carcinoma (LUSC), and tumor‐adjacent normal tissues from 58 LUAD and 62 LUSC cases were retrospectively evaluated at the No.8 People''s Hospital of Qing Dao. HIF3A expression was explored using RT‐qPCR. The clinical significance of HIF3A was evaluated in the plasma and tumor tissues using the receiver operating curve (ROC) and the area under the curve (AUC).ResultsHypoxia‐inducible factor 3 alpha expression was notably downregulated in the plasma or tumor tissues of patients with LUAD and LUSC, compared with the healthy control group or adjacent normal tissues. Furthermore, HIF3A expression had a significant positive correlation in the plasma and tumor tissues of LUAD and LUSC patients. Meanwhile, the ROC‐AUCs achieved a significantly higher range, from 0.84 to 0.93, with the plasma or tumor tissues of NSCLC patients. Thus, HIF3A expression was not only correlated with plasma and tumor tissues, but also showed potential significance in NSCLC.ConclusionHypoxia‐inducible factor 3 alpha is aberrantly detectable in NSCLC patients in the plasma and tumor tissues. HIF3A may be involved in hypoxic responses during the development and occurrence of NSCLC.     

Bioinformatics analysis of differentially expressed miRNAs in non‐small cell lung cancer

ObjectiveNon‐small cell lung cancer (NSCLC) contains 85% of lung cancer. Lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) are the largest NSCLC subgroups. The aim of the study was to investigate the underlying mechanism in developing more effective subtype‐specific molecular therapeutic procedures.MethodsA total of 876 specimens were used in this study: 494 LUAD tissues (ie, 449 LUAD tissues and 45 matched normal tissues) and 382 LUSC tissues (ie, 337 LUSC tissues and 45 matched normal tissues). The miRNA sequencing data were processed using R. The differential expressed miRNAs between lung cancer and normal tissues were analyzed using the limma package in R. Gene expression, Western blotting, hematoxylin and eosin staining, and luciferase assay were used to test LUAD and LUSC.ResultsLUAD and LUSC appear sharply distinct at molecular and pathological level. Let‐7a‐5p, miR‐338, miR‐375, miR‐217, miR‐627, miR‐140, miR‐147b, miR‐138‐2, miR‐584, and miR‐197 are top 10 relevant miRNAs and CLDN3, DSG3, KRT17, TMEM125, KRT5, NKX2‐1, KRT7, ABCC5, KRAS, and PLCG2 are top 10 relevant genes in NSCLC. At the same time, the miRNAs expression levels were also quite different between the two groups. Among the differential expressed miRNAs, let‐7a‐5p was significantly down‐regulated in LUAD while miR‐338 was markedly down‐regulated in LUSC. Bioinformatics analyses appeared that let‐7a‐5p directly targets high–molecular weight keratin 5 (KRT5) which were shown to be a strong risk factor for LUAD. And NK2 homeobox 1(NKX2‐1) which was associated with tumor progression in LUSC was identified as a target gene of miR‐338.ConclusionsDistinct profile of miRNAs can take a part in the development of LUAD and LUSC and thus could serve as a subtype‐specific molecular therapeutic target to protect against LUAD and LUSC.     

LncRNA PITPNA‐AS1/miR‐223‐3p/PTN axis regulates malignant progression and stemness in lung squamous cell carcinoma

BackgroundLong noncoding RNAs (lncRNAs) are a kind of molecule that cannot code proteins, and their expression is dysregulated in diversified cancers. LncRNA PITPNA‐AS1 has been shown to act as a tumor promoter in a variety of malignancies, but its function and regulatory mechanisms in lung squamous cell carcinoma (LUSC) are yet unknown.MethodsThe mRNA and protein expression of genes were examined by RT‐qPCR, western blot, and IHC assay. The cell proliferation, migration, invasion, and stemness were detected through CCK‐8, colony formation, Transwell and spheroid formation assays. The CD44⁺ and CD166⁺‐positive cells were detected through flow cytometry. The binding ability among genes through luciferase reporter and RNA pull‐down assays. The tumor growth was detected through in vivo nude mice assay.ResultsThe lncRNA PITPNA‐AS1 had increased expression in LUSC and was linked to a poor prognosis. In LUSC, PITPNA‐AS1 also enhanced cell proliferation, migration, invasion, and stemness. This mechanistic investigation showed that PITPNA‐AS1 absorbed miR‐223‐3p and that miR‐223‐3p targeted PTN. MiR‐223‐3p inhibition or PTN overexpression might reverse the inhibitory effects of PITPNA‐AS1 suppression on LUSC progression, as demonstrated by rescue experiments. In addition, the PITPNA‐AS1/miR‐223‐3p/PTN axis accelerated tumor development in vivo.ConclusionsIt is the first time we investigated the potential role and ceRNA regulatory mechanism of PITPNA‐AS1 in LUSC. The data disclosed that PITPNA‐AS1 upregulated PTN through sponging miR‐223‐3p to enhance the onset and progression of LUSC. These findings suggested the ceRNA axis may serve as a promising therapeutic biomarker for LUSC patients.     

Sperm-associated antigens as targets for cancer immunotherapy: expression pattern and humoral immune response in cancer patients

The identification of novel cancer-related and immunogenic proteins is still a challenge to be faced to improve antigen-specific tumor immunotherapy. The category of so-called cancer-testis (CT) antigens is one of the most perspective groups of proteins for anticancer immune response activation as normally they are expressed in immunoprivileged tissues and are immunogenic if aberrantly generated in tumors. The heterogeneous group of proteins called sperm-associated antigens (SPAG) might encompass novel CT antigens owing to their common expression in male germ cells, their ability to elicit immune response underlying infertility, and lately proposed oncogenic properties. We carried out a comprehensive analysis of the expression pattern in various normal and cancerous tissues and assessed the frequency of spontaneous humoral immune response against members of the SPAG group in cancer patients using phage-displayed antigen microarrays. Our results show that out of 15 analyzed SPAG genes only SPAG1, SPAG6, SPAG8, SPAG15, and SPAG17 are predominantly expressed in testis, whereas the others are ubiquitously expressed with only a testis-associated alternative splice variant of SPAG16. mRNA expression of SPAG1, SPAG6, and alternative splice variants of SPAG8, SPAG16, and SPAG17 was elevated in various tumors with frequencies ranging from approximately 10% to 70%. The upregulation of SPAG6 in lung and breast cancer was confirmed by immunohistochemical analysis of tumor and normal tissue microarrays. Cancer-associated spontaneous humoral immune response was detected against SPAG1, SPAG6, SPAG8, and a novel testis-specific splice variant of SPAG17 and ascribe these as novel CT antigens that potentially are applicable as immunotherapeutic targets and serologic biomarkers.     

Increased expression of core-fucosylated glycans in human lung squamous cell carcinoma

Lung cancer is the most frequent cancer and the leading cause of cancer around the world. As one of the major types of lung cancer, lung squamous cell carcinoma (LUSC) is closely associated with smoking and shows poor sensitivity to therapy and prognosis. Although alteration of glycopatterns are reliable indicators of cancer, little is known about the alterations of protein glycosylation related to LUSC. In this study, we compared the differential expression levels of glycopatterns in seven pairs of LUSC tissues and normal pericarcinomatous tissues (PCTs) using lectin microarrays. Fluorescence-based lectin histochemistry and lectin blotting were utilized to validate and assess the expression and distribution of certain glycans in LUSC tissues and PCTs. And we further analyzed their total N-linked glycans using MALDI-TOF/TOF-MS to provide more information about the aberrant glycopatterns. The results showed that the expression level of the core fucosylation recognized by Pisum sativum agglutinin (PSA) and Lens culinaris agglutinin (LCA) was significantly increased in LUSC tissues compared with PCTs. There were 10 and 15 fucosylated N-linked glycans that were detected in PCTs and LUSC tissues respectively, 10 fucosylated N-glycans were common, while five fucosylated N-glycans were unique to LUSC tissues. And the abundance of the fucosylated N-glycans was increased from 40.9% (PCTs) to 48.3% (LUSC). These finding is helpful to elucidate the molecular mechanisms underlying the lung diseases and develop new treatment strategies.

The expression level of fucosylated and core fucosylated N-linked glycans increased in lung squamous cell carcinoma tissues.     

A novel model based on liquid‐liquid phase separation–Related genes correlates immune microenvironment profiles and predicts prognosis of lung squamous cell carcinoma

ObjectiveThe aim of the study was to construct and validate a robust prognostic model based on liquid‐liquid phase separation (LLPS)–related genes in lung squamous cell carcinoma (LUSC).MethodsThe Cancer Genome Atlas dataset was used as the discovery set to identify the LLPS‐related differentially expressed genes (DEGs) between LUSC and normal tissue. These DEGs were screened by the LASSO Cox regression analysis to identify the genes with nonzero coefficient, which were next included in the multivariate Cox regression analysis to construct the prediction model. The dataset {"type":"entrez-geo","attrs":{"text":"GSE41271","term_id":"41271"}}GSE41271 was adopted as the validation set to verify the efficacy of the model. Enrichment analysis and the CIBERSORT were performed to illustrate potential immune mechanisms underlying the prediction model.ResultsA total of 48 LLPS‐related genes were aberrantly expressed in LUSC. Among them, 7 genes were selected by the LASSO Cox regression analysis to construct the prediction model. Risk index (RI) was calculated according to the model for each patient. The prognosis was significantly different between the patients with high and low RI in the discovery set and the validation set (p < 0.001 and p = 0.028, respectively). The multivariate survival analysis confirmed RI as an independent prognostic factor in LUSC (in the discovery set: p < 0.001, HR = 2.643, 95% CI = 1.986–3.518; in the validation set: p = 0.042, HR = 2.144, 95% CI = 1.026–4.480). A series of pathways involving immune cells were found to be related to RI. The distribution pattern of immune cells and chemokines varied according to the value of RI.ConclusionThe prediction model based on LLPS‐related genes was constructed and validated as a robust prognostic tool for LUSC using multiple datasets. LLPS might have an impact on LUSC through immune pathways.     

Low‐level gastrokine 2 promoted progress of NSCLC and as a potential biomarker

BackgroundGastrokine 2 (GKN2) is significantly downregulated in non‐small cell lung cancer (NSCLC) tissues than in normal tissues (NT), as assessed by mRNA microassay; however, the mechanism and clinical value of GKN2 is unknown in NSCLC.MethodsA total of 60 NSCLC samples and corresponding NT samples were prospectively collected GKN2 expression in NSCLC tissues was estimated. Also, the expression level of GKN2 promoter methylation and correlation with clinical data in NSCLC patients from public databases were analyzed. Cytology experiments were also carried out.ResultsThe GKN2 mRNA and protein expression level in NSCLC was significantly lower than that in the NT, and the GKN2 expression level in large tumors NSCLC was significantly lower than that in the small tumor group. Public data showed that expression of GKN2 in LUAD with P53 mutation group was lower than that of the P53 non‐mutation group, and GKN2 promoter methylation level of LUAD was significantly higher than its NT and close to age and clinical stage. Cell migration, invasion, and proliferation ability of GKN2 overexpressed were lower in A549 and PC9 groups than those in GKN2 overexpressed A549 and PC9 negative control groups, while the percentage of apoptotic cells increased in the GKN2 overexpressed A549 and PC9 groups. The DNMT3B mRNA expression levels were higher in PC9 and A549 cells than BEAS‐2B cells.ConclusionThe overexpression of GKN2 significantly inhibited cell proliferation, migration, and invasion and promoted apoptosis. Low‐level GKN2 promoted the progression of NSCLC via DNMT3B and is expected to be a biomarker for NSCLC.     

Expression of STK15 mRNA in hepatocellular carcinoma and its prognostic significance

ObjectivesTo investigate whether the STK15 mRNA expression correlates with clinicopathologic features and the prognosis of HCC patients.Design and methodsThree hepatoma cell lines, two normal liver epithelial cell lines, hepatoma tissues, adjacent tumor tissues and normal liver tissues were obtained from 46 HCC patients. Semi-quantitative RT-PCR assays were performed to detect the expression of STK15 mRNA in above cell lines and tissues. Moreover, the expression of STK15 protein in hepatoma tissues, adjacent tumor tissues and normal liver tissues was also examined by immunohistochemical staining. Finally, correlations between STK15 mRNA expression and the clinicopathological features and prognosis of HCC patients were evaluated.ResultsSTK15 mRNA showed higher levels in hepatoma cell lines than in normal liver epithelial cell lines. Moreover, the mean levels of STK15 mRNA and protein expression showed statistical difference between tumor tissues, tumor adjacent tissues and normal liver tissues (P < 0.01). By immunohistochemical analysis, we found that paraffin-embedded archival HCC tissues showed higher expression of STK15 than adjacent tumors and normal liver tissues. Furthermore, HCC patients with higher STK15 mRNA expression showed poorer prognosis than those with lower STK15 mRNA expression. The high level of STK15 mRNA expression was significantly correlated with tumor stage (P = 0.0081), more frequent lymph node (P = 0.0380) or hematogenous metastasis (P = 0.0066), and a higher incidence of cancer-related death (P = 0.0083). Furthermore, the disease-free survival (DFS) and overall survival (OS) rates of HCC patients with higher STK15 mRNA expression group (47.6% and 52.7%) were significantly lower than those of patients with low STK15 mRNA expression group (56.9% and 68.8%, P = 0.0018 and 0.0047).ConclusionsSTK15 mRNA might be a good marker for predicting the prognosis of HCC patients.     

Inverse correlation of ribosomal protein S27A and multifunctional protein YB-1 in hepatocellular carcinoma

ObjectiveThe detection of possible correlation between ribosomal protein RPS27A and multifunctional YB-1 expression in hepatocellular carcinoma (HCC).Design and methodsTissue microarray slides containing totally 80 cores with 19 tissues of HCC, 1 tissue of hepatocholangiocarcinoma, 10 tissues of liver cirrhosis and 10 normal liver tissues in duplicates were analyzed for expression of RPS27A and YB-1 by immunohistochemistry.ResultsAmong each of 10 LC and normal liver tissues all (100%) showed RPS27A positive expression but only 11 out of 19 HCC tissues (57.89%) showed RPS27A positive expression with significant difference compared (P < 0.05) with both LC and normal tissues. We found positive expression of YB-1 in 17 tissues out of 19 HCC tissues (89.47%) but only 4 tissues out of each 10 LC as well as normal liver tissues showed positive expression with significant (P < 0.01) difference compared to HCC tissues. A statistically significant inverse weak correlation (rho = − 0.293) between YB-1 expression and RPS27A expression was found.ConclusionThe present investigation concludes that the ribosomal protein RPS27A was down-regulated in viral induced HCC patients. RPS27A expression was found to have a weak inverse correlation with overexpression of multifunctional protein YB-1 in HCC tissues. This study opens up a new window for YB-1–RPS27A axis in HCC.     

IL-34在表皮生长因子受体突变肺腺癌中的表达及其预后评估价值

目的检测白介素34(interleukin-34,IL-34)在表皮生长因子受体（epidermal growth factor receptor,EGFR）突变的肺腺癌患者中的表达水平，探讨其对该类肺腺癌患者预后的评估价值。方法 收集2015年7月至2017年12月在复旦大学附属中山医院诊治的144例术前未接受抗肿瘤治疗的EGFR突变肺腺癌患者的手术切除标本及其临床资料。通过免疫组织化学染色，检测IL-34在肺癌组织及正常肺组织中的表达情况。用Kaplan-Meier生存曲线及Cox比例风险模型分析IL-34与患者预后的关系。结果 IL-34在EGFR突变的肺腺癌患者的肿瘤组织中相对高表达，在正常肺组织中低表达。随访64(42, 73)个月，IL-34高表达组患者（n=93）总生存率低于低表达组（n=51,P=0.006），累积复发率高于低表达组（P=0.011）。Cox比例风险模型示，IL-34表达为患者总生存（HR=2.218, P=0.015）及复发的独立预测因素（HR=2.486, P=0.018）。结论 IL-34升高可能提示EGFR突变肺腺癌患者术后预后较差。     

Correlation of ARNTL2 with Immune Infiltration and Its Role as a Potential Prognostic Biomarker in Lung Adenocarcinoma

BackgroundARNTL2 is a core component of the circadian clock genes and plays regulatory roles in the cell cycle and immune infiltration, but its mechanism in lung cancer (LC) remains unclear.ObjectiveTo investigate the clinical and therapeutic value of ARNTL2 in LC.MethodsThe Oncomine and Cancer Cell Line Encyclopedia (CCLE) databases were adopted for assessing the ARNTL2 expression, after which the Kaplan-Meier plotter and Gene Expression Profiling Interactive Analysis 2 (GEPIA2) databases were used to assess the correlation of ARNTL2 with prognosis. The univariate and multivariate Cox regression analyses were utilized to identify independent prognostic factors. Also, we explored how ARNTL2 expression is related to immune infiltration, and immunomodulators in non-small lung cancer (NSCLC) using the Tumor Immune Estimation Resource (TIMER) database, TISIDB database and Gene Set Enrichment Analysis (GSEA). Finally, coexpression of ARNTL2 and PD-L1 in lung adenocarcinoma (LUAD) was verified via immunofluorescence staining and COXPRESdb v7 database.ResultsOur study demonstrated a remarkable upregulated expression of ARNTL2 in multiple cell lines and cancers, including NSCLC. Prognostic analysis displayed a remarkable correlation between high ARNTL2 expression and unfavorable overall survival (OS) and first progressive (FP) survival among patients ailing from LUAD, and ARNTL2 was an independent predictor of prognosis for LUAD patients. GSEA analysis showed that overexpression of ARNTL2 was significantly linked with cell cycle and immunity. Furthermore, we reported a correlation of ARNTL2 expression with immunomodulators and lymphocytes. Immunofluorescence staining revealed that ARNTL2 and PD-L1 were elevated relative to normal tissue for LUAD, and colocalization of them was observed.ConclusionElevated ARNTL2 expression in LUAD revealed the prognostic values and its prospective role as a target for cell cycle and immune therapy.