Non-small Cell Lung Cancer Tissue Specimens Research Articles

The Advantage of Targeted Next-Generation Sequencing over qPCR in Testing for Druggable EGFR Variants in Non-Small-Cell Lung Cancer.

The emergence of targeted therapies in non-small-cell lung cancer (NSCLC), including inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase, has increased the need for robust companion diagnostic tests. Nowadays, detection of actionable variants in exons 18-21 of the EGFR gene by qPCR and direct DNA sequencing is often replaced by next-generation sequencing (NGS). In this study, we evaluated the diagnostic usefulness of targeted NGS for druggable EGFR variants testing in clinical NSCLC material previously analyzed by the IVD-certified qPCR test with respect to DNA reference material. We tested 59 NSCLC tissue and cytology specimens for EGFR variants using the NGS 'TruSight Tumor 15' assay (Illumina) and the qPCR 'cobas EGFR mutation test v2' (Roche Diagnostics). The sensitivity and specificity of targeted NGS assay were evaluated using the biosynthetic and biological DNA reference material with known allelic frequencies (VAF) of EGFR variants. NGS demonstrated a sufficient lower detection limit for diagnostic applications (VAF < 5%) in DNA reference material; all EGFR variants were correctly identified. NGS showed high repeatability of VAF assessment between runs (CV% from 0.02 to 3.98). In clinical material, the overall concordance between NGS and qPCR was 76.14% (Cohen's Kappa = 0.5933). The majority of discordant results concerned false-positive detection of EGFR exon 20 insertions by qPCR. A total of 9 out of 59 (15%) clinical samples showed discordant results for one or more EGFR variants in both assays. Additionally, we observed TP53 to be a frequently co-mutated gene in EGFR-positive NSCLC patients. In conclusion, targeted NGS showed a number of superior features over qPCR in EGFR variant detection (exact identification of variants, calculation of allelic frequency, high analytical sensitivity), which might enhance the basic diagnostic report.

EIF4A3-Induced CircDHTKD1 regulates glycolysis in non-small cell lung cancer via stabilizing PFKL.

Lung cancer (LC) is one of the malignancies with the highest incidence and mortality in the world, approximately 85% of which is non-small cell lung cancer (NSCLC). Circular RNAs (circRNAs) exert multiple roles in NSCLC occurrence and development. The sequencing results in previous literature have illustrated that multiple circRNAs exhibit upregulation in NSCLC. We attempted to figure out which circRNA exerts an oncogenic role in NSLCL progression. RT-qPCR evaluated circDHTKD1 level in NSCLC tissue specimens and cells. Reverse transcription as well as RNase R digestion assay evaluated circDHTKD1 circular characterization in NSCLC cells. FISH determined circDHTKD1 subcellular distribution in NSCLC cells. Loss- and gain-of-function assays clarified circDHTKD1 role in NSCLC cell growth, tumour growth and glycolysis. Bioinformatics and RIP and RNA pull-down assessed association of circDHTKD1 with upstream molecule Eukaryotic initiation factor 4A-III (EIF4A3) or downstream molecule phosphofructokinase-1 liver type (PFKL) and insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) in NSCLC cells. Rescue assays assessed regulatory function of PFKL in circDHTKD1-meidated NSCLC cellular phenotypes. CircDHTKD1 exhibited upregulation and stable circular nature in NSCLC cells. EIF4A3 upregulated circDHTKD1 in NSCLC cells. CircDHTKD1 exerted a promoting influence on NSCLC cell malignant phenotypes and tumour growth. CircDHTKD1 exerted a promoting influence on NSCLC glucose metabolism. CircDHTKD1 exerts a promoting influence on NSCLC glucose metabolism through PFKL upregulation. RIP and RNA pull-down showed that circDHTKD1 could bind to IGF2BP, PFKL could bind to IGF2BP2, and circDHTKD1 promoted the binding of PFKL to IGF2BP2. In addition, RT-qPCR showed that IGF2BP2 knockdown promoted PFKL mRNA degradation, suggesting that IGF2BP2 stabilized PFKL in NSCLC cells. CircDHTKD1 exhibits upregulation in NSCLC. We innovatively validate that EIF4A3-triggered circDHTKD1 upregulation facilitates NSCLC glycolysis through recruiting m6A reader IGF2BP2 to stabilize PFKL, which may provide a new direction for seeking targeted therapy plans of NSCLC.

Glucose-Regulated Protein 94 Expression in Patients with Non- Small Cell Lung Cancer and Prognostic Significance

The researchers aimed to study the correlations of expression of glucose-regulated protein 94 (GRP94) with clinicopathological characteristics and prognosis of patients with non-small cell lung cancer (NSCLC). NSCLC tissue specimens were collected from January 2016 to January 2018. The prognostic factors of NSCLC patients were analyzed. Survival curves were plotted. Logistic multivariate regression analysis was performed to explore the factors influencing prognosis. GRP94 protein was mainly expressed in the nucleus in NSCLC and para-carcinoma tissues. The high expression rates of GRP94 in NSCLC tissues were significantly different in patients with and without tumor metastasis after surgery (P&lt;0.05). GRP94-positive NSCLC patients had significantly longer mean survival time than GRP94-negative ones (P&lt;0.01). Positive GRP94 expression, maximum diameter of tumor, pathological type, lymph node metastasis, T stage and postoperative TNM stage were independent risk factors affecting prognosis. GRP94 is an independent factor influencing the prognosis of NSCLC, as a potential target for treatment.

Abstract 2526: Immune infiltrates in Kita-Kyushu lung cancer antigen 1 (KK-LC-1) expressing non-small cell lung cancer (NSCLC) patients

Abstract Introduction: KK-LC-1 is a cancer testis antigen expressed notably in lung, breast, and gastric cancer making it an attractive target for T-cell receptor therapy. Here, we evaluate cell densities of NSCLC tissue specimens with known KK-LC-1 gene expression to better characterize the tumor immune microenvironment in such patients. Methods: A seven-plex mIF panel (FoxP3, PDL1, PanCK, PD1, CD8, KK-LC-1, and CD68) was developed based on a previously established mIF Lung-cancer panel and addition of KK-LC-1 following rigorous optimization of KK-LC-1 antibody titer and panel order with qualitative assessment of monoplex immunofluorescent assays. Serial sections from 23 NSCLC cases (14 biopsy samples and 9 excision specimens) with known KK-LC-1 gene expression values were collected. Tissue sections were stained and imaged on an Akoya Biosciences Vectra Polaris multispectral imaging platform. The regions of interest were identified and stamped with the Phenochart mIF image management software. Spectral unmixing, tissue segmentation and cell phenotyping were performed with Akoya Biosciences InForm image analysis software using the multispectral fields workflow. The 4-tier positive staining thresholds for PD-L1 and KK-LC-1 were determined by a pathologist (WDW). Cell densities in the tumor and adjacent stroma as well as the KK-LC-1 H-score were assessed by linear regression analysis. Student’s t-test was used to compare between the top quartile of tumors based on KK-LC-1 gene expression (Q4) and the remaining 3 quartiles (Q1-Q3). Results: The study consisted of 17/23 adenocarcinoma (73.9%), 4/23 squamous cell carcinoma (SCC) (17.3%), and 2/23 (8.6%) unclear or mixed histology tumors. Adenocarcinoma trended to have higher KK-LC-1 gene expression compared to SCC tumors (mean 17.34 vs. 0.65, p-value = 0.204). 13/23 (56.5%) were pan-wild type, 6/23 (26.0%) were EGFR mutated, 2/23 (8.7%) were KRAS mutated, and 1/23 (4.4%) were MET Exon 14 skip and multiple alterations. We saw significantly higher cell density of FoxP3 in tumor cells in Q4 versus Q1-Q3 (mean 7.17 vs. 0.8 cells/mm2, p-value = 0.004), and there was a trend towards a higher FoxP3/CD8 cell density ratio (mean 3.28 vs. 0.05, p = .110) and higher PD-1 cell density (mean 186.7 vs. 32.8 cells/mm2, p=0.1548) in tumor cells in Q4 versus Q1-Q3. There was significant correlation towards increased CD8+ T cell density ratio between tumor and stroma cells in cases with higher KK-LC-1 gene expression (r2= 0.307, p = 0.011). Conclusion: KK-LC-1 expression in lung cancer is seen in multiple molecular subsets and is associated with increase in both regulatory T cells in tumor and in CD8+ T-cells in tumor relative to stroma. Strategies to deplete regulatory T cells and to further amplify CD8+ T cells should be pursued in the development of T-cell receptor therapy targeting KK-LC-1. Citation Format: Ainaz Dory Barkhordarzadeh, Robert C. Hsu, Rongfu Wang, Sue E. Martin, Jorge J. Nieva, William Dean Wallace. Immune infiltrates in Kita-Kyushu lung cancer antigen 1 (KK-LC-1) expressing non-small cell lung cancer (NSCLC) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2526.

Spatial metabolomics for evaluating response to neoadjuvant therapy in non-small cell lung cancer patients.

BackgroundThe response to neoadjuvant chemotherapy (NAC) differs substantially among individual patients with non‐small cell lung cancer (NSCLC). Major pathological response (MPR) is a histomorphological read‐out used to assess treatment response and prognosis in patients NSCLC after NAC. Although spatial metabolomics is a promising tool for evaluating metabolic phenotypes, it has not yet been utilized to assess therapy responses in patients with NSCLC. We evaluated the potential application of spatial metabolomics in cancer tissues to assess the response to NAC, using a metabolic classifier that utilizes mass spectrometry imaging combined with machine learning.MethodsResected NSCLC tissue specimens obtained after NAC (n = 88) were subjected to high‐resolution mass spectrometry, and these data were used to develop an approach for assessing the response to NAC in patients with NSCLC. The specificities of the generated tumor cell and stroma classifiers were validated by applying this approach to a cohort of biologically matched chemotherapy‐naïve patients with NSCLC (n = 85).ResultsThe developed tumor cell metabolic classifier stratified patients into different prognostic groups with 81.6% accuracy, whereas the stroma metabolic classifier displayed 78.4% accuracy. By contrast, the accuracies of MPR and TNM staging for stratification were 62.5% and 54.1%, respectively. The combination of metabolic and MPR classifiers showed slightly lower accuracy than either individual metabolic classifier. In multivariate analysis, metabolic classifiers were the only independent prognostic factors identified (tumor: P = 0.001, hazards ratio [HR] = 3.823, 95% confidence interval [CI] = 1.716–8.514; stroma: P = 0.049, HR = 2.180, 95% CI = 1.004–4.737), whereas MPR (P = 0.804; HR = 0.913; 95% CI = 0.445–1.874) and TNM staging (P = 0.078; HR = 1.223; 95% CI = 0.977–1.550) were not independent prognostic factors. Using Kaplan‐Meier survival analyses, both tumor and stroma metabolic classifiers were able to further stratify patients as NAC responders (P < 0.001) and non‐responders (P < 0.001).ConclusionsOur findings indicate that the metabolic constitutions of both tumor cells and the stroma are valuable additions to the classical histomorphology‐based assessment of tumor response.

Correlation between metastasis-associated gene 1 expression and tumor-associated macrophages in non-small cell lung cancer.

The role of metastasis-associated gene 1 (MTA1) in the metastasis of non-small cell lung cancer (NSCLC) has been proved, but its role in the tumor microenvironment is still insufficient. The study was performed to explore the correlation between MTA1 and tumor-associated macrophages (TAMs) in NSCLC. The expression profile data of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) were downloaded from TCGA database. The tumor-infiltrating immune cells in each LUAD and LUSC patient were estimated using the CIBERSORT method. Then, the online TIMER database containing multiple algorithms was used to analyze the relationship between MTA1 and TAMs. Besides, correlations between MTA1 and TAMs markers were also explored. Additionally, the immunohistochemistry staining of MTA1 protein and CD206 was performed in 75 NSCLC tissue specimens. Associations of MTA1 and CD206 with the clinicopathological characteristics were analyzed, as well as the correlation between MTA1 and CD206. Based on different algorithms, MTA1 expression was correlated with the distribution of infiltrating immune cells in the tumor microenvironment and negatively correlated with tumor immune-stromal score. MTA1 was associated with TAMs markers according to TCGA database. In 75 NSCLC tissue specimens, the positive rate of MTA1 was 60.00% (45/75), which of CD206 was 42.67% (32/75). The MTA1 expression was significantly correlated with T stage, lymph node metastasis, and TNM stage. The CD206 expression was significantly correlated with T stage, lymph node metastasis, TNM stage, and tumor type. Additionally, we found that MTA1 was positively correlated with CD206 in NSCLC and LUSC. In NSCLC, MTA1 expression was correlated with the infiltrations of different types of macrophages and the expression of TAMs markers, as well as the M2-TAMs marker CD206, suggesting that MTA1-promoting tumor metastasis may mediate the infiltration of different types of macrophages in the tumor microenvironment.

Circ_0016760 accelerates non-small-cell lung cancer progression through miR-646/AKT3 signaling in vivo and in vitro.

BackgroundCurrently, the prognosis of non‐small‐cell lung cancer (NSCLC) patients remains dismal due to recurrence and metastasis. The purpose of our study was to explore the role of circular RNA_0016760 (circ_0016760) in NSCLC progression and its associated mechanism.MethodsQuantitative real‐time polymerase chain reaction (qRT‐PCR) was implemented to measure the expression of circ_0016760, microRNA‐646 (miR‐646) and AK strain thymoma serine/threonine kinase 3 (AKT3). The protein level of AKT3 was examined by Western blot assay. Cell Counting Kit 8 assay, transwell assays, and flow cytometry were conducted to analyze cell proliferation, metastasis, and apoptosis. Dual‐luciferase reporter assay was used to confirm the interactions that were predicted by bioinformatics software (Circular RNA Interactome and TargetScan). A xenograft tumor model was built to investigate the role of circ_0016760 in vivo.ResultsCirc_0016760 and AKT3 were highly expressed in NSCLC tissue specimens and cell lines. Circ_0016760 interference suppressed cell proliferation, migration, and invasion and promoted the apoptosis of NSCLC cells. Circ_0016760 interacted with miR‐646 and negatively regulated its expression. MiR‐646 silencing partly counteracted circ_0016760 knockdown‐mediated influences in NSCLC cells. MiR‐646 bound to the AKT3 3′ untranslated region in NSCLC cells, and miR‐646 overexpression‐induced effects in NSCLC cells were partly overturned by the addition of AKT3 overexpression plasmid. Circ_0016760 silencing reduced the expression of AKT3 through enhancing miR‐646 expression. Circ_0016760 knockdown suppressed NSCLC tumor growth in vivo.ConclusionCirc_0016760 played an oncogenic role to promote the proliferation, migration, and invasion and restrained the apoptosis of NSCLC cells via miR‐646/AKT3 signaling.

Quantification of HER family dimers by proximity ligation assay and its clinical evaluation in non–small cell lung cancer patients treated with osimertinib

ObjectivesThe epidermal growth factor receptor (EGFR, also known as Her1) is a member of the human epidermal growth factor receptor (HER) family of proteins and a target of tyrosine kinase inhibitors (TKIs) in the treatment of non–small cell lung cancer (NSCLC) positive for activating mutations ofEGFR. Signal transduction by HER family proteins is dependent on their homo- or heterodimerization, but little is known of the relation between the relative proportions of such dimers of Her1 and sensitivity to EGFR-TKIs. We here investigated the feasibility of assessing this relation with the in situ proximity ligation assay (PLA) technique, which is able to detect the interaction of two proteins of interest when they are in close proximity. Materials and methodsIn situ PLA was applied to detect Her1 homodimers and Her1 heterodimers in NSCLC cell lines and tissue specimens positive for EGFR activating mutations. ResultsIn situ PLA allowed visualization and quantitative assessment of Her1 homodimers as well as of Her1 heterodimers with Her2, Her3, or Her4 not only in NSCLC cell lines but also in NSCLC tissue specimens obtained from various anatomic sites and by different collection methods. Treatment of NSCLC cell lines with EGFR-TKIs resulted in a decrease in the number of Her1 dimers, with the effect on homodimers being greater than that on heterodimers. A high ratio of Her1 heterodimers to homodimers was associated with poor progression-free survival in NSCLC patients treated with osimertinib. ConclusionIn situ PLA allows the detection of HER family dimers in NSCLC tissue, and quantitative assessment of Her1 homo- and heterodimers may prove informative for prediction of the response of NSCLC patients to EGFR-TKI treatment.

MZT2A promotes NSCLC viability and invasion by increasing Akt phosphorylation via the MOZART2 domain.

Mitotic spindle organizing protein 2A (MZT2A) is localized at the centrosome and regulates microtubule nucleation activity in cells. This study assessed the role of MZT2A in non–small‐cell lung cancer (NSCLC). Differential MZT2A expression was bioinformatically assessed using TCGA database, the GEPIA database, and Kaplan‐Meier survival data to determine the association between MZT2A expression and NSCLC prognosis. Furthermore, NSCLC tissue specimens were evaluated by immunohistochemistry. MZT2A was overexpressed or knocked down in NSCLC cells using cDNA and siRNA, respectively. The cells were subjected to various assays and treated with the selective Akt inhibitor LY294002 or co‐transfected with galectin‐3‐binding protein (LGALS3BP) siRNA. MZT2A mRNA and protein levels were upregulated in NSCLC lesions and MTZ2A expression was associated with poor NSCLC prognosis. MZT2A protein was also highly expressed in NSCLC cells compared with the expression in normal bronchial cells. MZT2A expression promoted NSCLC cell viability and invasion, whereas MTZ2A siRNA had the opposite effect on NSCLC cells in vitro. At the protein level, MZT2A induced Akt phosphorylation, promoting NSCLC proliferation and invasion (but the selective Akt inhibitor blocked these effects) through upregulation of LGALS3BP via the MTZ2A MOZART2 domain, whereas LGALS3BP siRNA suppressed MTZ2A activity in NSCLC cells. The limited in vivo experiments confirmed the in vitro data. In conclusion, MZT2A exhibits oncogenic activity by activating LGALS3BP and Akt in NSCLC. Future studies will assess MTZ2A as a biomarker to predict NSCLC prognosis or as a target in the control of NSCLC progression.

C-MYC-induced upregulation of LINC01503 promotes progression of non-small cell lung cancer.

The purpose of this study was to detect the expression of long intergenic non-protein-coding RNA 1503 (LINC01503) in non-small cell lung cancer (NSCLC), and to further study its biological function, as well as the regulatory relationships of c-MYC with LINC01503 and the extracellular signal regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) signaling pathway in NSCLC. Tissue specimens were collected from 36 NSCLC patients, and the relative expression level of LINC01503 in the 36 cases of NSCLC tissue specimens and NSCLC cells was then determined using quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Then, the effects of LINC01503 on the proliferation and apoptosis of NSCLC cells were detected in vitro via Cell-Counting Kit (CCK)-8 assay, colony-forming assay and flow cytometry. Besides, the possible LINC01503 promoter-binding transcription factor was predicted using bioinformatics. After interference with c-MYC expression, the changes in the expression of LINC01503 were examined through qRT-PCR. Finally, the changes in the expressions of the molecular markers in the ERK/MAPK signaling pathway after interference with LINC01503 and c-MYC expressions were evaluated using Western blotting. According to qRT-PCR results, the expression of LINC01503 was upregulated in 30 out of 36 cases of NSCLC tissues. Compared with that in human normal bronchial epithelial cells, the expression of LINC01503 was elevated in NSCLC cells. As shown by the CCK-8 assay and colony-forming assay, the proliferation ability of NSCLC cells was weakened after interference with LINC01503 expression, and the flow cytometry results revealed the apoptosis rate of NSCLC cells was raised after interference with LINC01503 expression. Moreover, the bioinformatics prediction showed that c-MYC might be the LINC01503 promoter-binding transcription factor. Additionally, it was found through the qRT-PCR that the expression of LINC01503 declined after interference with c-MYC expression. Finally, based on Western blotting results, the expressions of phosphorylated ERK1/2 (p-ERK1/2) and p-MAPK/ERK kinase (MEK), the molecular markers in the ERK/MAPK signaling pathway, were inhibited after interference with c-MYC and LINC01503 expressions. The transcription factor c-MYC promotes the expression of LINC01503 in NSCLC and activates the ERK/MAPK signaling pathway to drive the development and progression of NSCLC.

M6 A transferase METTL3-induced lncRNA ABHD11-AS1 promotes the Warburg effect of non-small-cell lung cancer.

N6 -methyladenosine (m6 A) and long noncoding RNAs (lncRNAs) are both crucial regulators in non-small-cell lung cancer (NSCLC) tumorigenesis. However, the pathological roles of m6 A and lncRNAs in NSCLC progression are still limited and undefined. Here, lncRNA ABHD11-AS1 was upregulated in NSCLC tissue specimens and cells and the ectopic overexpression was closely correlated with unfavorable prognosis of NSCLC patients. Functionally, ABHD11-AS1 promoted the proliferation and Warburg effect of NSCLC. Mechanistically, m6 A profile was analyzed by methylated RNA immunoprecipitation sequencing (MeRIP-Seq). MeRIP-Seq presented that there was m6 A modification site in ABHD11-AS1. m6 A methyltransferase-like 3 (METTL3) installed the m6 A modification and enhanced ABHD11-AS1 transcript stability to increase its expression. In conclusion, our findings highlight the function and mechanism of METTL3-induced ABHD11-AS1 in NSCLC and inspire the understanding of m6 A and lncRNA in cancer biology.

MiR-495 and miR-5688 are down-regulated in non-small cell lung cancer under hypoxia to maintain interleukin-11 expression.

BackgroundHypoxia is a hallmark of cancer and is associated with poor prognosis. However, the molecular mechanism by which hypoxia promotes tumor progression remains unclear. MicroRNAs dysregulation has been shown to play a critical role in the tumor and tumor microenvironment. Here, we investigated the roles of miR‐495 and miR‐5688 in human non‐small cell lung cancer (NSCLC) and their underlying mechanism.MethodsThe expression levels of miR‐495 and miR‐5688 in human NSCLC tissue specimens were measured by quantitative real‐time polymerase chain reaction (qRT‐PCR). Deferoxamine (DFO) was used to determine whether the regulation of miR‐495 and miR‐5688 under hypoxia was dependent on hypoxia‐inducible factor 1‐alpha (HIF‐1α). Furthermore, the functions of miR‐495 and miR‐5688 in tumor progression were evaluated using colony formation, 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium (MTS), wound healing, transwell assays, and xenograft model. Two algorithms, PicTAR and Targetscan, were used to predict the target gene of these two miRNAs, and dual‐luciferase reporter assay was conducted to confirm the target. The unpaired two‐tailed t test, Pearson correlation analysis, and Fisher's exact probability test were performed for statistical analyses.ResultsTwo miRNAs, miR‐495 and miR‐5688, were found to participate in NSCLC progression under hypoxia. They were down‐regulated in NSCLC tissues compared with normal tissues. We determined that hypoxia led to the down‐regulation of miR‐495 and miR‐5688 in NSCLC cells, which was independent of HIF‐1α and cellular metabolic energy. In addition, miR‐495 and miR‐5688 suppressed cell proliferation, migration, and invasion in vitro. The NSCLC xenograft model showed that miR‐495 and miR‐5688 inhibited tumor formation in vivo. Interestingly, we found that miR‐495 and miR‐5688 had the same target, interleukin‐11 (IL‐11). Recombinant human IL‐11 counteracted the effects of miR‐495 and miR‐5688 on NSCLC cells, suggesting that miR‐495 and miR‐5688 executed their tumor suppressive role by repressing IL‐11 expression.ConclusionWe found that hypoxia down‐regulated the expression levels of miR‐495 and miR‐5688 in NSCLC to enhance IL‐11 expression and tumor progression, indicating that the miR‐495/miR‐5688/IL‐11 axis may serve as a therapeutic target and potential biomarker for NSCLC.

MicroRNA-612 suppresses the malignant development of non-small-cell lung cancer by directly targeting bromodomain-containing protein 4.

Background: Aberrant expression of microRNAs (miRNAs) in non-small-cell lung cancer (NSCLC) has been reported. Dysregulation of miRNAs exerts tumor-suppressing or tumor-promoting actions on the pathology and biological behaviors of NSCLC. miR-612 is associated with many types of human cancer; however, the expression, potential roles, and regulatory mechanisms of miR-612 in NSCLC remain unclear.Material and methods: Here, the expression level of miR-612 in NSCLC tissue specimens and a panel of cell lines were evaluated by RT-qPCR. Cell-Counting Kit 8, flow cytometry, Transwell migration and invasion, and in vivo tumor growth assays were performed to determine the functional role of miR-612 in malignant phenotypes of NSCLC cells. The molecular mechanism underlying the tumor-suppressive roles of miR-612 in NSCLC was investigated.Results: miR-612 was expressed at low levels in NSCLC, and low miR-612 expression was significantly correlated with TNM stage and lymph node metastasis. NSCLC patients with low miR-612 expression had shorter overall survival rate than those with high levels. Exogenous miR-612 expression decreased proliferation, migration, and invasion, and promoted apoptosis of NSCLC cells in vitro. miR-612 upregulation hindered NSCLC tumor growth in vivo. Bromodomain-containing protein 4 (BRD4) was confirmed as a direct target gene of miR-612 in NSCLC cells. BRD4 was obviously overexpressed in human NSCLC tissues and inverse correlated with miR-612 expression. Inhibition of BRD4 expression simulated the tumor-suppressive functions of miR-612 overexpression in NSCLC cells. Reintroduction of miR-612 expression abrogated the miR-612-mediated suppressive effects on NSCLC cells. BRD4 upregulation inhibited activation of the PI3K/Akt pathway in NSCLC cells in vitro and in vivo.Conclusion: This study supports the first evidence that miR-612 exerts tumor-suppressive roles in the aggressive behaviors of NSCLC cells in vitro and in vivo through direct targeting BRD4 and deactivating the PI3K/Akt pathway. Thus, miR-612 might be a promising target for anticancer therapies in patients with NSCLC.

SUMO1P3 is associated clinical progression and facilitates cell migration and invasion through regulating miR-136 in non-small cell lung cancer

Long non-coding RNA small ubiquitin-like modifier 1 pseudogene 3 (SUMO1P3) is located on chromosome 1q23.2, and has been suggested to serve as oncogenic lncRNA in many kinds of human malignancy. The role of SUMO1P3 in non-small cell lung cancer (NSCLC) was still unknown. In our study, we analyzed The Cancer Genome Atlas (TCGA) database, and observed SUMO1P3 expression was increased in both lung squamous cell carcinoma and lung adenocarcinoma. Then, we confirmed that SUMO1P3 expression was significantly increased in NSCLC cancer tissues and cell lines. Meanwhile, the expression levels of SUMO1P3 expression in metastatic lymph node specimens were strikingly elevated in comparison to primary NSCLC tissue specimens. Then, we found high SUMO1P3 expression was correlated with late clinical stage, lymph node metastasis, distant metastasis and poor differentiated degree. In the survival analysis of TCGA, we observed that SUMO1P3 expression had no association with overall survival and disease free survival in NSCLC patients. There was a statistically negative correlation between SUMO1P3 expression and miR-136 expression in NSCLC tissues. Moreover, miR-136 directly bound to SUMO1P3, and SUMO1P3 negatively regulated miR-136 expression in NSCLC cells. Furthermore, SUMO1P3 promoted NSCLC cell migration and invasion via regulating miR-136. In conclusion, SUMO1P3 functions as metastasis-associated lncRNA in NSCLC.

Circular RNA circ_0026134 regulates non-small cell lung cancer cell proliferation and invasion via sponging miR-1256 and miR-1287

Non-small cell lung cancer (NSCLC) is the major type of lung malignancy with increasing incidence worldwide. Despite progression in diagnosis and management of NSCLC, the patients’ survival rate is still unfavorable. Accumulating data have shown that circular RNAs (circRNAs) act as key roles in oncogenesis. In this project, circ_0026134 expression in NSCLC tissue specimens and cells was measured by RT-qPCR. The functional roles (cell growth, apoptosis, migration, and invasion) of circ_0026134 were investigated by gain and loss-of-function assays. In addition, luciferase reporter assay was used to uncover the mechanisms of circ_0026134. Circ_0026134 was frequently upregulated in NSCLC samples and cell lines. Furthermore, we observed that downregulation of circ_0026134 attenuated NSCLC cell proliferation and metastatic properties and induced cell apoptosis. The overexpression of circ_0026134 in NSCLC cells caused the opposite effect. For the part of mechanism exploration, miR-1256 and miR-1287 were proved to be sponged by circ_0026134. Rescue experiments further indicated that the oncogenic role of circ_0026134 was dependent on its regulation of miR-1256 and miR-1287. Taken together, this study may provide a new insight and treatment target for NSCLC.

MicroRNA-345 suppresses cell invasion and migration in non-small cell lung cancer by directly targeting YAP1.

To investigate the roles and the underlying mechanism of miR-345 in regulating non-small cell lung cancer (NSCLC) cell migration and invasion. Fifty-two pairs of NSCLC tissues and matched normal lung tissue samples were collected from NSCLC patients who had undergone surgical resection between June 2015 and August 2017 in our hospital. Human NSCLC cell lines (NCI-H460, NCI-H1299, A549 and GLC-82) and normal human bronchial epithelium cell BEAS-2B were cultured. NSCLC cell migration and invasion capacities were determined by transwell assays. The relative protein and mRNA expression level of yes-associated protein 1 (YAP1) were detected by Western blot and Quantitative Real-time polymerase chain reaction (qRT-PCR) analysis, respectively. MiR-345 was prominently downregulated in NSCLC tissue specimens. Results of transwell assays showed that miR-345 overexpression could dramatically inhibit NSCLC cell migration and invasion. Additionally, data in current study also identified YAP1 as a direct functional target of miR-345 in NSCLC cells. YAP1 expressions were negatively correlated with the miR-345 expressions in NSCLC tissue samples. Moreover, YAP1 was found to be involved in the functions of miR-345 in inhibiting NSCLC cell invasion and migration. All the above results indicated that miR-345 inhibited NSCLC cell migration and invasion by targeting YAP1, suggesting that miR-345/YAP1 axis might be a promising biomarker for NSCLC treatments.

Down-regulation of miR-5702 is associated with clinical progression and poor prognosis in patients with non-small-cell lung cancer.

Significant down-regulation of miR-5702 and its tumor-suppressive roles in non-small-cell lung cancer (NSCLC) have been reported previously. However, its clinical significance in NSCLC has not yet been reported. In this study, we aimed to identify the prognostic value of miR-5702 in NSCLC patients. The expression levels of miR-5702 were detected by Real-time quantitative polymerase chain reaction (RT-qPCR). The chi-square test was used for the examination of relationship between miR-5702 expression and clinicopathologic factors. The association between miR-5702 expression and overall survival in patients with NSCLC was statistically analyzed by Kaplan-Meier. Finally, univariate and multivariate models were conducted to determine the prognostic value of miR-5702 in NSCLC patients. MiR-5702 expression was lowly expressed in NSCLC tissue specimens compared with adjacent nontumor tissue (p < 0.01). It was observed that low expression of miR-5702 was associated with clinical stages (p = 0.029), lymph node metastasis (p = 0.016) and distant metastasis (p = 0.004). Moreover, Kaplan-Meier assay showed that patients with lower miR-5702 expression had worse overall survival time than that with higher miR-5702 expression (p = 0.0097). Finally, multivariate analysis showed that low expression of miR-5702 was independently associated with overall survival of NSCLC patients (HR = 3.128; 95 % CI: 1.237-5.668, p = 0.005). Our data, for the first time, indicated that miR-5702 expression may be considered as a prognostic biomarker in NSCLC patients.

Ferritin heavy subunit enhances apoptosis of non-small cell lung cancer cells through modulation of miR-125b/p53 axis

Ferritin is a nanocage protein composed by the variable assembly of 24 heavy and light subunits. As major intracellular iron storage protein, ferritin has been studied for many years in the context of iron metabolism. However, recent evidences have highlighted its role, in particular that of the heavy subunit (FHC), in pathways related to cancer development and progression, such as cell proliferation, growth suppressor evasion, cell death inhibition, and angiogenesis. At least partly, the involvement in these pathways is due to the ability of FHC to control the expression of a repertoire of oncogenes and oncomiRNAs. Moreover, the existence of a feedback loop between FHC and the tumor suppressor p53 has been demonstrated in different cell types. Here, we show that ectopic over-expression of FHC induces the promoter hypermethylation and the down-regulation of miR-125b that, in turn, enhances p53 protein expression in non-small cell lung cancer (NSCLC) cell lines. Notably, analysis by absolute quantitative RT-PCR of FHC, miR-125b, and p53 strongly suggests that this axis might be active in human NSCLC tissue specimens. In vitro, FHC over-expression attenuates survival of NSCLC cells by inducing p53-mediated intrinsic apoptosis that is partially abrogated upon miR-125b re-expression. Overall, our findings demonstrate that FHC acts as a tumor suppressor gene, thus providing a potential molecular strategy for induction of NSCLC apoptotic cell death.

Positive feedback loop of lncRNA LINC01296/miR-598/Twist1 promotes non-small cell lung cancer tumorigenesis.

Emerging evidence has illustrated the vital roles of long noncoding RNAs (lncRNAs) in human cancers. However, the role of lncRNAs in non-small cell lung cancer (NSCLC) is still elusive and poorly understood. In the current study, our team conducted extensive experiments to identify the role oflong intergenic nonprotein coding (LINC01296) on NSCLC tumorigenesis. The results illustrated that the elevated LINC01296 expression in NSCLC tissue specimens and cell lines were closely correlated with the poor prognosis of patients with NSCLC. Functional studies revealed that LINC01296 knockdown silenced by small interfering RNAs inhibited proliferation, accelerated apoptosis in vitro, and impaired tumor growth in vivo. Mechanical studies showed that INC01296 harbored miR-598, acting as a microRNA "sponge." Besides, miR-598 targeted the 3'-UTR of Twist1. Interestingly, transcription factor Twist1 could bind with the promoter of INC01296 and activate its transcriptional level. In summary, we conclude that INC01296/miR-598/Twist1 constitutes a positive feedback loop to promote the tumorigenesis of NSCLC, providing a novel insight and a valuable therapeutic strategy.

Study on molecular mechanism of MiRNA-29a in promoting proliferation and invasion of non-small-cell lung cancer by inhibiting MTSS1.

To investigate the biological role of micro-ribonucleic acid (miR)-29a in non-small-cell lung cancer (NSCLC). 55 cases of NSCLC tissue specimens and paired normal lung tissue specimens collected in the Department II of Oncology, Ruikang Hospital Affiliated to Guangxi University of Chinese Medicine from July 2012 to April 2015 were randomly included. The fluorescent quantitative polymerase chain reaction, Western blotting, and immunohistochemistry were performed to detect the expression levels of miR-29a and metastasis suppressor 1 (MTSS1). Pearson correlation analysis was utilized to investigate the relationship between miR-29a expression and MTSS1 expression in NSCLC tissues, and the Kaplan-Meier survival curves were constructed to analyze the association of miR-29a expression with the survival time of NSCLC patients. A54 proliferation and invasion abilities were measured by means of plate clone formation assay, and transwell assay after the miR-29a was suppressed by miRNA inhibitor. Luciferase assay was used to detect the target gene of miR-29a. In NSCLC tissues, the miR-29a expression level was higher than that in normal lung tissues (p<0.05), while the expression level of MTSS1 protein was remarkably lower than that in normal lung tissues (p<0.05). The median survival time of the patients was 15.1 months in high miR-29a expression group and 18.3 months in low miR-29a expression group (p<0.05). The miR-29a expression was negatively correlated with the expression level of MTSS1 protein in NSCLC tissues (r=-0.762, p<0.05). Luciferase results suggest that miR-29a binds to the promoter region of MTSS1 and inhibits its transcription level. The expression of MTSS1 protein was up-regulated notably after miR-29a knockdown by an inhibitor. It was revealed in the results of transwell assay and plate clone formation assay that the proliferative and invasive capacity of A549 cells was significantly decreased after knockdown of miR-29a. The transcribed miR-29a down-regulates the protein level of MTSS1, suppressor of tumor proliferation and invasion, thereby promoting the proliferative and invasive capacity of NSCLC cells. Both miR-29a and MTSS1 are expected to become potential therapeutic targets for NSCLC.