Serum Of Non-small Cell Lung Cancer Patients Research Articles

NSCLC extracellular vesicles containing miR-374a-5p promote leptomeningeal metastasis by influencing blood‒brain barrier permeability.

Leptomeningeal metastasis (LM) is a devastating complication of advanced non-small cell lung cancer (NSCLC). Diagnosis and monitoring of LM can be challenging. Extracellular vesicles (EVs) microRNAs (miRNAs) have become a new noninvasive diagnostic biomarker. The purpose of this study was to examine the clinical value and role of EVs miRNAs in NSCLC-LM. According to next-generation sequencing (NGS), miRNAs with differential expression of EVs in serum of NSCLC patients with LM and non-LM were detected to find biological markers for the diagnosis of LM. Cellular and in vivo experiments were conducted to explore the pathogenesis of EVs miRNA promoting LM in NSCLC. In the present study, we first demonstrated the serum level of EV-associated miR-374a-5p in patients with LM of lung cancer was much higher than that in patients without LM and was correlated with the survival time of patients with LM. Further studies showed that EVs miR-374a-5p efficiently destroys tight junctions and the integrity of the cerebral microvascular endothelial cell barrier, resulting in increased blood-brain barrier (BBB) permeability. Mechanistically, miR-374a-5p regulates the distribution of ZO-1 and occludin in endothelial cells by targeting ADD3, increasing vascular permeability and promoting LM. Implications: These results suggest that serum NSCLC-derived EVs miR-374a-5p is involved in premetastatic niche formation by regulating the permeability of BBB to promote NSCLC-LM, and can be used as a blood biomarker for the diagnosis and prognosis of NSCLC-LM.

A novel molecular mechanism mediated by circCCDC134 regulates non-small cell lung cancer progression.

Circular RNA (circRNA) plays a crucial role in non-small cell lung cancer (NSCLC) progression. However, the role of circCCDC134 in NSCLC is still largely unknown. Quantitative real-time PCR was utilized for measuring circCCDC134, microRNA (miR)-625-5p and nuclear factor of activated T cell 5 (NFAT5) expression. Cell functions were evaluated by colony formation, EdU, transwell, and wound healing assays and flow cytometry. Glucose consumption, lactate production, and ATP level were determined to analyze cell glycolysis. Western blot analysis was used to detect protein expression. Animal experiments were performed to assess the effect of circCCDC134 on NSCLC tumor growth. RNA interaction was evaluated by dual-luciferase reporter assay and RIP assay. Exosomes were isolated from the serum of NSCLC patients and healthy normal controls. Highly expressed circCCDC134 was found in NSCLC tissues and cells, as well as in the serum exosomes of NSCLC patients. Downregulated circCCDC134 restrained NSCLC cell growth, metastasis and glycolysis. CircCCDC134 sponged miR-625-5p to regulate NFAT5. MiR-625-5p inhibitor abolished the regulation of circCCDC134 knockdown on NSCLC progression, and NFAT5 overexpression eliminated the effects of miR-625-5p on NSCLC cell behaviors. CircCCDC134 knockdown also inhibited NSCLC tumor growth. Our study revealed that circCCDC134 was involved in regulating NSCLC progression through the miR-625-5p/NFAT5 pathway, confirming that circCCDC134 might function as the diagnostic and therapeutic target for NSCLC.

Serum Level of CEACAM1 in Patients with Nonsmall Cell Lung Cancer and Its Clinical Significance in Cancer Tissue.

Objective To analyze the expression of CEACAM1 in serum of patients with nonsmall cell lung cancer (NSCLC) and to explore the correlation and clinical significance between the expression of CEACAM1 and pathological parameters of NSCLC tissue. Methods A total of 100 patients with NSCLC who underwent tumor resection were screened. Another 100 healthy patients in physical examination department were selected as control group. Venous blood and cancer tissue samples were collected. The expression of CEACAM1, TGF-β, VEGF-A, and IL-8 was detected. Results The results of various indicators in the lung cancer group were much higher than those in the healthy group; CEACAM1 was significantly positively correlated with TGF-β expression. The later the clinical stage and the higher the degree of differentiation of cancer tissue specimens, the more the expression of CEACAM1 in serum samples. Conclusions The expression level of CEACAM1 in the serum of NSCLC patients is strongly correlated with TGF-β, VEGF-A, and IL-8, indicating that serum CEACAM1 and TGF-β levels can predict the occurrence, progression, and prognosis of lung cancer and provide a target for future targeted therapy of lung cancer.

A comprehensive profiling of soluble immune checkpoints from the sera of patients with non-small cell lung cancer.

BackgroundImmunotherapy was widely used for the treatment of non‐small cell lung cancer (NSCLC). However, whether inhibition of immune checkpoints individually or simultaneously could improve the therapeutic efficacy of NSCLC remains to be investigated. Here, we explored the aberrant levels of several checkpoints and evaluated their potential diagnostic values for NSCLC.MethodsSerum samples of 89 NSCLC patients and 57 healthy donors were collected from Nanjing Drum Tower Hospital between November 2019 and July 2020. Fourteen human immune checkpoints were quantified by Procarta‐Plex Human Immuno‐Oncology Checkpoint Panel.ResultsThe expression levels of sTIM‐3, sCD137, sCD27, sLAG‐3, sIDO, sPD‐L2, sCD152, sCD80, and sPD‐1 were all significantly increased in serum of NSCLC patients. Especially, sLAG‐3 was significantly elevated in serum of NSCLC patients at early‐stage (stages I and II), TIM‐3, CD137, and CD27 were significantly higher in the advanced NSCLC patients (stages III and IV) than in the early‐stage groups. Receiver operating characteristics (ROC) results showed that except for PD‐1, all the other immune checkpoint proteins had potential diagnostic values for NSCLC. sTIM‐3 had the highest diagnostic accuracy, followed by sLAG‐3. Combining sTIM‐3, sLAG‐3, and sCD137 could increase the accuracy to a higher level. Moreover, sCD27 was correlated with NSCLC cancer type, age, sex, and disease stage, while sCD137 was correlated with age and disease stage. sTIM‐3 and sIDO were correlated with stage and age, respectively.ConclusionsTIM‐3 and LAG‐3 were independent biomarkers for the early diagnosis of NSCLC. The combination of TIM‐3, LAG‐3, and CD137 could increase the diagnostic accuracy.

Role and Mechanism of Exosome-Derived Long Noncoding RNA HOTAIR in Lung Cancer.

Background and purpose: HOX transcript antisense RNA (HOTAIR) is a long noncoding RNA (lncRNA) that promotes tumor growth and metastasis. Exosomes can mediate intracellular communication in cancer by transferring active molecules. However, the role and mechanism of HOTAIR in nonsmall cell lung cancer (NSCLC) are still unclear. This study mainly explores the role and mechanism of exosome-derived HOTAIR in NSCLC. Methods: after the material characterization of the CD63 immune lipid magnetic bead (CD63-IMB), the exosomes in serum of NSCLC patients were captured through CD63-IMB for the corresponding biological characterization. Real-time quantitative reverse transcription PCR (qRT-PCR) was performed to detect the expression level of HOTAIR in tumor tissues, serum, and serum exosome from NSCLC patients. Subsequently, exosome secreted by NCI-H1975 cells with highly expressed HOTAIR was selected to treat low-expression A549 cells and HOTAIR knockdown on NCI-H1975 cells. In this way, action mechanisms of HOTAIR can be investigated by means of qRT-PCR, colony formation assays, and flow cytometry. Results: exosomes can be isolated by CD63-IMB, and taken up by cells effectively; the qRT-PCR results demonstrate that HOTAIR expressions are significantly upregulated in tumor tissues, serums, and exosomes isolated from serums of NSCLC patients. Clinicopathological correlation analysis shows that the upregulation of HOTAIR is closely associated with lymphatic metastasis and tumor node metastasis (TNM) staging (P < 0.05). HOTAIR expressions show a significant increase in A549 cells treated with exosomes derived from NCI-H1975 cells, signifying that both proliferation and migration of A549 cells are promoted, and HOTAIR depletion could inhibit the proliferation and migration of lung cancer cells. Conclusions: HOTAIR is highly expressed in tumor tissues, serums, and serum exosomes of NSCLC patients and its expression has a significant correlation with lymphatic metastasis and TNM staging. Moreover, the exosome may promote NSCLC proliferation and migration through HOTAIR transportation. Therefore, exosome-derived HOTAIR is expected to be a new molecular marker for NSCLC diagnosis, and exosomal transmission of HOTAIR may provide a new approach to NSCLC diagnosis.

Pentraxin 3 acts as a functional effector of Akt/NF-κB signaling to modulate the progression and cisplatin-resistance in non-small cell lung cancer

Pentraxin 3 (PTX3) has been documented to be involved in the development of chemoresistance, however, the mechanisms by which it regulates cisplatin (DDP) resistance in non-small cell lung cancer (NSCLC) have never been elucidated. Quantitative reverse transcriptase polymerase chain reaction and Western blot were carried to determine the expression of PTX3, ATP-binding cassette sub-family B member 1 (ABCB1)/P-glycoprotein 1 (p-gp), protein kinase B (Akt), phosphorylated Akt and nuclear factor-kappa B (NF-кB) p65. The biological roles of PTX3 in NSCLC progression and NSCLC cell resistance to DDP were evaluated using enzyme-linked immunosorbent assay, cell count kit-8, colony formation assay, flow cytometry, as well as xenograft tumor assay. The expression of PTX3 was increased in the serum of NSCLC patients as well as in NSCLC cell lines. Lower PTX3 level was associated with longer overall survival in lung adenocarcinoma and lung squamous cell carcinoma patients. Furthermore, PTX3 expression was greatly higher in DDP-resistant NSCLC cells than that in NSCLC cells. Silencing of PTX3 restrained the proliferation and promoted the apoptosis of NSCLC cells, as well as sensitized DDP-resistant NSCLC cells to DDP. Additionally, knockdown of PTX3 inhibited the growth of NSCLC tumors in vivo. Upregulation of PTX3 expression was dependent on the activation of Akt/NF-κB signaling. The induction of apoptosis by PTX3 knockdown was enhanced by MK-2206 or JSH-23. In conclusion, knockdown of PTX3 restrained the progression of NSCLC and sensitized NSCLC cells towards DDP, which provides a potential target to restore DDP chemoresponse.

MicroRNA-let-7e in serum-derived exosomes inhibits the metastasis of non-small-cell lung cancer in a SUV39H2/LSD1/CDH1-dependent manner.

Non-small-cell lung cancer (NSCLC) remains the leading cause of cancer-related death worldwide. Accumulating research has highlighted the ability of exosome-encapsulated microRNAs (miRNAs or miRs) as potential circulating biomarkers for lung cancer. The current study aimed to evaluate the clinical significance of serum-derived exosomal miR-let-7e as a biomarker in the metastasis of NSCLC. Initially, the expression of miR-let-7e, SUV39H2, and CDH1 in human NSCLC tissues and exosomes isolated from the serum of NSCLC patients was determined by RT-qPCR, demonstrating that miR-let-7e was downregulated in NSCLC tissues and serum-derived exosomes, while SUV39H2 was upregulated in NSCLC tissues. Kaplan-Meier method revealed that both lower miR-let-7e expression and higher SUV39H2 expression were correlated with a lower survival rate of NSCLC patients. Next, SUV39H2 was predicted and validated to be a target of miR-let-7e using dual-luciferase reporter assay. NSCLC H1299 cells following ectopic expression and depletion experiments of miR-let-7e and SUV39H2 were treated with serum-derived exosomes, after which the viability, migration, and invasion of H1299 cells were detected using CCK-8 and Transwell assays. Further, in vivo experiments were conducted to elucidate the effect of exosomal miR-let-7e on tumorigenesis. Results revealed that miR-let-7e overexpression in serum-derived exosomes inhibited SUV39H2, resulting in impaired cell viability, migration, and invasion in vitro as well as delayed tumor growth in vivo. In conclusion, the key findings of the current study demonstrate that exosomal miR-let-7e from serum possesses anticarcinogenic properties against NSCLC via the SUV39H2/LSD1/CDH1 axis.

Tumor-Derived Exosomal miR-620 as a Diagnostic Biomarker in Non-Small-Cell Lung Cancer.

Background Evidence has suggested the functional role of exosomal miRNAs in cancer diagnosis. This study aimed to determine whether the serum exosomal biomarkers can improve the diagnosis of patients with non-small-cell lung cancer (NSCLC). Materials and Methods The exosomes were extracted from the serum of NSCLC patients (n = 235) and healthy donors (n = 231) using ultracentrifugation and then were evaluated by using transmission electron microscopy, qNano, and western blotting. The serum exosomal miRNA expression was validated using qPCR. Results Exosomal miR-620 was significantly reduced in NSCLC and early-stage NSCLC patients (P < 0.0001) when compared to that of healthy controls, with an area under the curve (AUC) of 0.728 and 0.707, respectively. Exosomal miR-620 expression showed an association with drinking (P=0.008) and distant metastasis (P=0.037). Additionally, the downregulated exosomal miR-620 showed association with chemotherapeutic effect (P=0.044). Conclusion These findings suggest the serum exosomal miR-620 as a promising diagnostic and prognostic noninvasive biomarker in NSCLC patients.

Highly sensitive detection of cytochrome c in the NSCLC serum using a hydrophobic paper based-gold nanourchin substrate.

Cytochrome c (Cyt c) is a biomarker of early apoptosis that plays a critical role in the diagnosis and therapy of non-small cell lung cancer (NSCLC). In this work, we proposed a novel surface-enhanced Raman scattering (SERS)-based biosensor to implement the ultrasensitive detection of Cyt c in the serum of NSCLC patients. The SERS-supporting substrates based on hydrophobic filter paper were composed of gold nanourchins (GNUs) surface-functionalized with the Cyt c aptamer and the cyanine 5-labeled complementary DNA. In the existence of Cyt c, it could specifically bind to its aptamer, which leads to the detachment of complementary strands modified with Cy5 and the great weakness of SERS signal. The finite-difference time domain (FDTD) simulation showed that the excellent SERS performance of GNUs aggregation was strongly dependent on a large number of "hot spots" at the tips and between the nanogaps of aggregated GNUs. Alkyl ketene dimer (AKD) was used to make the filter paper modify its property from hydrophilic to hydrophobic, which consequently increased the density of GNUs and extended the retention time of the analyte. SERS biosensors based on hydrophobic paper exhibited prominent reproducibility and selectivity. The detection limit of Cyt c in PBS was 1.148 pg/mL, while the detection limit in human serum was 1.79 pg/mL. Moreover, the analysis of the serum samples of healthy subjects and NSCLC patients confirmed the feasibility of its clinical application. The results were consistent with enzyme-linked immunosorbent assay results. This method can be a powerful strategy for quantitative detection of extracellular Cyt c, and it is expected that the SERS-based biosensors could be applied in the practical clinical diagnoses of NSCLC.

Tumor-Derived Exosomal miRNAs as Diagnostic Biomarkers in Non-Small Cell Lung Cancer.

BackgroundDelayed diagnosis is the main obstacle to improve prognosis of non-small cell lung cancer (NSCLC). Novel biomarkers for the diagnosis of NSCLC are urgently needed. This study aimed to identify the specific exosomal miRNAs with diagnostic and prognostic potential in NSCLC patients.Materials and MethodsTransmission electron microscopy (TEM), qNano and western blots were used to characterize the exosomes isolated from the serum of NSCLC patients (n=330) and healthy donors (n=312) by ultracentrifugation. Exosomal miRNAs were profiled by miRNA microarrays and verified by quantitative PCR (qPCR). The diagnostic accuracy was determined by receiver operating characteristic (ROC) analysis.ResultsA total of differential 22 miRNAs were screened out based on P < 0.05 and fold difference>2.0 by miRNA microarrays, among which, exosomal miR-5684 and miR-125b-5p were significantly down-regulated in NSCLC patients compared to healthy donors, processing favorable diagnostic efficiency for (early) NSCLC. Importantly, the exosomal miR-125b-5p were associated with metastasis (P < 0.0001), chemotherapeutic effect (P=0.007) and survival (P=0.008).ConclusionExosomal miR-5684 and miR-125b-5p levels are significantly down-regulated in NSCLC patients, and serve as the promising diagnostic and prognostic biomarkers for NSCLC.

Analysis of the expression levels and clinical value of miR‑365 and miR‑25 in serum of patients with non‑small cell lung cancer

The present study aimed to investigate the expression levels and clinical value of miR-365 and miR-25 in serum of patients with non-small cell lung cancer (NSCLC). Patients (180) diagnosed with NSCLC at the Affiliated Hospital of Guangdong Medical University from July 2011 to December 2013 were used as the experimental group. Volunteers (90) undergoing health examinations were used as the control group. The serum of the patients was collected after fasting in the morning. The expression levels of miR-365 and miR-25 in the serum of patients was assessed by quantitative real-time PCR (qRT-PCR), and the relationship among miR-365, miR-25 and the postoperative survival rate of NSCLC patients was analyzed. The relative expression level of miR-25 of patients with peripheral infiltration was significantly higher than that of patients without peripheral infiltration (P<0.05). There were significant differences in the relative expression level of miR-25 in different pathological grades and TNM stages, as well as with lymph node metastasis (P<0.05). The survival rate of NSCLC patients with high expression of miR-25 was significantly lower than that of NSCLC patients with low expression of miR-25 (P<0.05). The relative expression level of miR-365 of patients with peripheral infiltration was significantly lower than that of patients without peripheral infiltration (P<0.05). There were significant differences in the relative expression level of miR-365 in different pathological grades and TNM stages, as well as with lymph node metastasis (P<0.05). The survival rate of NSCLC patients with high expression of miR-365 was significantly higher than that of NSCLC patients with low expression of miR-365 (P<0.05). In conclusion, the expression levels of miR-25 and miR-365 were different in the serum of NSCLC patients, and they were closely related to certain clinical characteristics such as peripheral infiltration, pathological grade, tumor diameter, TNM stage and lymph node metastasis. Moreover, it was revealed that miR-25 and miR-365 affected the 5-year survival rate of patients. miR-25 and miR-365 could be used as important tumor markers to evaluate the prognosis of NSCLC patients.

Abstract 3180: Development of a fully automated immunoassay system for detection of serum NY-ESO-1 and XAGE1 antibodies as biomarkers predicting the clinical efficacy of anti-PD-1 therapy

Abstract Background: Programmed cell death-1 (PD-1) and PD-ligand1 (PD-L1) inhibitors have become standard therapies for advanced non-small-cell lung cancer (NSCLC) due to prolonged patient survival. However, extended survival with anti-PD-1/PD-L1 therapy is limited to a small subset of NSCLC patients. Therefore, predictive biomarkers are needed to determine which patients will benefit. Recently, we reported that serum antibodies (Abs) against NY-ESO-1 and/or XAGE1 cancer-testis antigens are useful biomarkers that predict the clinical efficacy of anti-PD-1 monotherapy for NSCLC. The serum Abs were measured with enzyme-linked immunosorbent assay (ELISA); however, the ELISA method has several technical limitations for clinical applications. To overcome limitations in Ab measurement, we have developed a fully automated immunoassay system (HISCLTM) using chemiluminescent magnetic immunoassay technology. HISCLTM is widely used in many clinical fields because it is superior to ELISA due to its rapid reaction (17 minutes), wide dynamic ranges, and high reproducibility. Patients and Methods: Sera of NSCLC patients were obtained before anti-PD-1 monotherapy (nivolumab or pembrolizumab) as a part of standard care. NY-ESO-1/XAGE1 serum Abs were measured by ELISA and HISCLTM and their clinical performance was evaluated. In HISCLTM system, serum Abs were captured by magnetic beads coated with antigens, and the antigen-Ab complex was incubated with anti-human IgG conjugated with alkaline phosphatase. The chemiluminescent intensity acquired was then used for analyses. Results: Twenty-eight NSCLC patients were selected as the initial cohort for this study. Half of the patients responded to anti-PD-1 monotherapy and the remaining half did not. There were strong correlations between HISCLTM chemiluminescence signal intensity and titers of NY-ESO-1 and XAGE1 serum Abs in ELISA (r&amp;gt;0.93 for both). Twelve of thirteen Ab-positive patients in HISCLTM responded to anti-PD-1 therapy, while only two of fifteen Ab-negatives did (92% vs. 13%, p&amp;lt;0.001). Notably, the signal intensities in HISCLTM correlated well with tumor reduction rates after anti-PD-1 therapy. Serum NY-ESO-1/XAGE1 Ab-positivity in HISCLTM predicted efficacy with an AUC of 0.898, resulting in a sensitivity of 86% and specificity of 93%. Conclusions: We have developed a rapid and highly reproducible immunoassay system for detecting the serum NY-ESO-1 and XAGE1 Abs as predictive biomarkers of clinical benefits with anti-PD-1 monotherapy in NSCLC. In the future, our system would be examined in large-scale clinical trials for various other cancers. Citation Format: Yumiko Sakai, Hirotaka Abo, Kanako Sakaeda, Yusuke Atarashi, Nobuyuki Ide, Takaaki Yamaoka, Koji Kurose, Kenta Noda, Toshiyuki Sato, Toru Oga, Mikio Oka. Development of a fully automated immunoassay system for detection of serum NY-ESO-1 and XAGE1 antibodies as biomarkers predicting the clinical efficacy of anti-PD-1 therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3180.

MicroRNA genetic signature in non-small cell lung cancer (NSCLC) Egyptian patients

BackgroundCancer development is associated with deregulated microRNA (miRNA) in body fluids including serum, plasma, and bronchoalveolar lavage (BAL). Early diagnosis and early treatment of lung cancer improve survival and response to treatment. So, finding an easy detectable biomarker is crucially important to improve the disease outcome. So, we analyzed the differential expression of miRNA using microarray both in serum and BAL of 37 non-small cell lung cancer (NSCLC) patients and 30 healthy control subjects (15 non-smokers and 15 smokers).ResultsA total of 32 miRNAs were significantly differentially expressed in serum of NSCLC patients versus controls (13 up-regulated and 19 down-regulated), whereas 14 miRNAs were significantly differentially expressed in BAL of NSCLC patients relative to control (12 upregulated and 2 downregulated). The accuracy of MiRNAs to detect lung cancer patients versus control was 94.3% with a specificity of 97.8% and a sensitivity of 92.3%.ConclusionsExpression of miRNAs is specific in both serum and BAL of NSCLC patients, indicating that they might be considered easy diagnostic biomarkers for early lung cancer detection.

Comparison of the diagnostic value of CEA combined with OPN or DKK1 in non-small cell lung cancer.

Carcinoma embryonic antigen (CEA), osteopontin (OPN), and Dickkopf-1 (DKK1) expressed in serum are associated with hypoxia in tumor progression. However, the role of these proteins in the plasma of patients with non-small cell lung cancer (NSCLC) is poorly understood. The diagnostic values of CEA combined with OPN or DKK1 were compared in non-small cell lung cancer. This study investigated the diagnostic value of CEA combined with OPN and DKK1, respectively, in NSCLC. Eighty patients with NSCLC (NSCLC group) and 60 patients with benign lung diseases (benign lung disease group) admitted to Shandong Provincial Third Hospital from May 2014 to January 2015 were selected as the study subjects. In addition, 60 healthy subjects undergoing normal physical examination were selected as healthy control group. The OPN and DKK1 in serum of the two groups were detected by enzyme linked immunosorbent assay (ELISA), and the CEA expression was measured by Electrochemical Photometric method. The diagnostic value of CEA combined with OPN and DKK1, respectively, in NSCLC was analyzed. The expression of CEA, OPN, and DKK1 in serum of NSCLC group was significantly higher than that of healthy control group and benign lung disease group (P<0.05). The expression of CEA, OPN and DKK1 in serum of NSCLC patients was correlated with tumor diameter, lymph node metastasis, degree of pathological differentiation and clinical stage (P<0.05). ROC curve for diagnosis of NSCLC was drawn by further combination of serum CEA and OPN. The AUC of combined diagnosis of CEA and OPN for NSCLC was 0.920 (95% CI, 0.875-0.964), and the diagnostic sensitivity and specificity were 87.50 and 86.67%, respectively; the AUC of combined diagnosis of CEA and DKK1 for NSCLC was 0.912 (95% CI, 0.866-0.958), and the diagnostic sensitivity and specificity were 92.50 and 76.67%, respectively. CEA, OPN and DKK1 may be involved in the occurrence and progression of NSCLC and have good sensitivity and specificity in the diagnosis of NSCLC and may be new biomarkers for the diagnosis of NSCLC.

Serum miR-1228-3p and miR-181a-5p as Noninvasive Biomarkers for Non-Small Cell Lung Cancer Diagnosis and Prognosis.

Background Lung cancer is the leading cause of cancer-related mortality worldwide, and non-small cell lung cancer (NSCLC) accounts for over 80% of all lung cancers. Serum microRNAs (miRNAs), due to their high stability, have the potential to become valuable noninvasive biomarkers. This present study was aimed to identify the serum miRNAs expression signatures for the diagnosis and prognosis of NSCLC using bioinformatics analysis. Methods A total of 12 miRNAs profiling studies have been identified in Pubmed, Gene Expression Omnibus (GEO), and ArreyExpress databases. Differentially expressed miRNAs (DEmiRNAs) were analyzed according to GEO2R online tool and RRA method from R. Then, prediction of DEmiRNAs' target genes from TargetScan, PicTar, miRDB, Tarbase, and miRanda database. Furthermore, we using reverse transcription– quantitative polymerase chain reaction (RT-qPCR) to evaluate the expression levels of DEmiRNAs in serum samples obtained from NSCLC patients and healthy controls. Subsequently, the clinical significance of the tested miRNAs was determined using receiver operating characteristic (ROC) analysis and Cox regression analysis. Results A total of 27 DEmiRNAs were identified and 5 of them (miR-1228-3p, miR-1228-5p, miR-133a-3p, miR-1273f, miR-545-3p) were significantly up-regulated and 4 of them (miR-181a-5p, miR-266-5p, miR-361-5p, miR-130a-3p) were significantly down-regulated in NSCLC patients compared with healthy controls. RT-qPCR validated that miR-1228-3p (P =0.006) and miR-181a-5p (P =0.030) were significantly differentially expressed in the serum of NSCLC patients and healthy controls. ROC analysis on miR-1228-3p and miR-181a-5p revealed the area under the curve (AUC) of 0.685 (95% confidence interval [CI], 0.563–0.806; P =0.006) and 0.647 (95% CI, 0.506–0.758; P =0.049). ROC analysis on miR-1228-3p combined miR-181a-5p revealed the AUC of 0.711 (95% CI, 0.593–0.828; P =0.002). Multivariate Cox regression analysis demonstrated that the high serum miR-1228-3p level was an independent factor for the poor prognosis of NSCLC patients. Conclusions Serum miR-1228-3p and miR-181a-5p are potential noninvasive biomarkers for the diagnosis and prognosis of NSCLC patients.

Differential expression of circulating serum miR-1249-3p, miR-3195, and miR-3692-3p in non-small cell lung cancer.

Global deregulation in miRNA expression is a hallmark of cancer cell. An estimated 2300 mature miRNAs are encoded by human genome; role of many of which in carcinogenesis and as cancer biomarkers remains unexplored. In this study, we investigated the utility of miR-3692-3p, miR-3195, and miR-1249-3p as biomarkers in non-small cell lung cancer (NSCLC). For this prospective study, 115 subjects, including 75 NSCLC patients and 40 controls, were recruited. The expression of miR-3692-3p, miR-3195, and miR-1249-3p was checked using qRT-PCR. The miRNA expression was correlated with survival outcome and therapeutic response. There were no significant differences in the mean age of NSCLC patients and controls (56.2 and 55.3years, respectively; p = 0.3242). Majority of NSCLC patients (67%) were smokers. We observed a significant upregulation of miR-3692-3p expression (p < 0.0001), while the expression of miR-3195 (p = 0.0017) and miR-1249-3p was significantly downregulated (p < 0.0001) in the serum of NSCLC patients as compared to controls. The expression of miR-1249-3p was significantly upregulated in lung adenocarcinoma versus lung squamous cell carcinoma (p = 0.0178). Interestingly, patients who responded to chemotherapy had higher expression of miR-1249-3p than non-responders (p = 0.0107). Moreover, patients with higher expression of miR-3195 had significantly longer overall survival (p = 0.0298). In multivariate analysis, miR-3195 emerged as independent prognostic factor for overall survival. We conclude that the miR-3195 may have prognostic significance, while miR-1249-3p may predict therapeutic response in NSCLC. Further studies are warranted to elucidate the role of these miRNAs in lung carcinogenesis and their utility as candidate cancer biomarkers.

Prognostic value of serum soluble interleukin-23 receptor and related T-helper 17 cell cytokines in non-small cell lung carcinoma.

The signaling of interleukin (IL)‐23 and its receptor (IL‐23R) play a crucial role in the development of cancers. However, the clinical significance of human serum soluble IL‐23R (sIL‐23R) and its relationship with IL‐23 are still not explored in non‐small cell lung cancer (NSCLC). In our study, sIL‐23R was first identified in the serum of NSCLC patients, but not in healthy controls, by proteomics. The IL‐23R mRNA and protein were upregulated in NSCLC cell lines and tissues tested by quantitative PCR, western blot analysis and immunohistochemistry. The levels of sIL‐23R, IL‐23, and IL‐17 in 195 NSCLC patients’ serum were determined by ELISA, and high levels of sIL‐23R were significantly associated with advanced N stage (P = .039), clinical stage (P = .007), and poor 5‐year survival rate. In vitro, sIL‐23R was shown binding to IL‐23 and the balance could affect patients’ N and T stage, overall survival, and downstream cytokine IL‐17 in a potential antagonistic relationship. Although sIL‐23R, IL‐23, and IL‐17 were all associated with poor prognosis, only the sIL‐23R/IL‐23 ratio (hazard ratio, 1.945; 95% confidence interval, 1.147‐3.299; P = .014) was found to be an independent factor for prognosis. Therefore, we identified fragments of soluble cytokine receptor of IL‐23R with affinity ability to its natural ligand IL‐23 in NSCLC patients’ serum. The balance between the 2 antagonists can work as a potential prognostic serum marker.

Extensive serum biomarker analysis in patients with non-small-cell lung carcinoma

Lung cancer is a common malignant disease, nearly 2.09 million new patients occurred last year. Approximately 85% of the patients are classified as non-small-cell lung cancer (NSCLC). It is therefore important to identify new diagnostic and prognostic biomarkers for the early detection of this disease. The presented study identifies biomarkers in the serum of NSCLC patients. The expression of 274 cytokines was measured by a novel antibody array methodology and ELISA was applied to validate the array results. The levels of MIP-1 α, IL-8, MIP-1 β, Resistin, GDF-15, HGF, CA125, FLRG, VCAM-1, DKK-3, sTNF-R1, CTACK, Acrp30, CXCL-16 and LYVE-1 were significantly higher in serum from NSCLC patients, while the level of TIMP-2 and IGFBP-6 were lower. More importantly, the validation supported the result of the antibody array. The result of the antibody array indicates that these cytokines might be novel auxiliary biomarkers in the diagnosis and prognosis of NSCLC.

High Aldehyde Dehydrogenase Levels Are Detectable in the Serum of Patients with Lung Cancer and May Be Exploited as Screening Biomarkers.

Objectives Since early detection improves overall survival in lung cancer, identification of screening biomarkers for patients at risk represents an area of intense investigation. Tumor liberated protein (TLP) has been previously described as a tumor-associated antigen (complex) present in the sera from lung cancer patients. Here, we set out to identify the nature of TLP to develop this as a potential biomarker for lung cancer screening. Materials and Methods Beginning from the peptide epitope RTNKEASI previously identified from the TLP complex, we produced a rabbit anti-RTNKEASI serum and evaluated it in the lung cancer cell line A549 by means of immunoblot and peptide completion assay (PCA). The TLP sequence identification was conducted by mass spectrometry. The detected protein was, then, analyzed in patients with non-small cell lung cancer (NSCLC) and benign lung pathologies and healthy donors, by ELISA. Results The anti-RTNKEASI antiserum detected and immunoprecipitated a 55 kDa protein band in the lysate of A549 cells identified as aldehyde dehydrogenase isoform 1A1, revealing the molecular nature of at least one component of the previously described TLP complex. Next, we screened blood samples from a non-tumor cohort of 26 patients and 45 NSCLC patients with different disease stages for the presence of ALDH1A1 and global ALDH. This analysis indicated that serum positivity was highly restricted to patients with NSCLC (ALDH p < 0.001; ALDH1A1 p=0.028). Interestingly, the global ALDH test resulted positive in more NSCLC samples compared to the ALDH1A1 test, suggesting that other ALDH isoforms might add to the sensitivity of the assay. Conclusion Our data indicate that ALDH levels are elevated in the sera of NSCLC patients, even with early stage disease, and may thus be evaluated as part of a marker panel for non-invasive detection of NSCLC.

Serum miR-210-3p as a Potential Noninvasive Biomarker of Lung Adenocarcinoma: A Preliminary Study.

Background: Development of noninvasive biomarkers could potentially contribute to extending the 5-year overall survival rate of nonsmall cell lung cancer (NSCLC) patients. Circulating microRNAs (miRNAs), due to their high stability, have the potential to become valuable cancer biomarkers. Methods: Using reverse transcription-quantitative polymerase chain reaction and testing three methods for data normalization, the expression levels of six miRNAs were evaluated in serum samples obtained from 50 NSCLC patients. Subsequently, the clinical significance of the tested miRNAs was determined. Results: Significant downregulation of miR-21-5p, miR-30a-5p, and miR-126-3p and upregulation of miR-210-3p and miR-486-5p in serum samples of NSCLC patients were identified in comparison to healthy controls. miR-205-5p appeared to be undetectable in all tested samples. Furthermore, miR-210-3p was differentially expressed between two subtypes of NSCLC. Receiver operating characteristic analysis for miR-210-3p revealed the area under the curve of 0.842 (95% confidence interval, 0.72-0.96; p = 0.0003) and demonstrated that miR-210-3p displayed considerable accuracy in discriminating between lung adenocarcinoma (AC) patients and healthy controls. Conclusions: Findings from this preliminary study indicate that five of six tested miRNAs were deregulated in the serum of NSCLC patients. Moreover, miR-210-3p appears to be a promising biomarker for diagnosis of lung AC.