Long noncoding RNA RP11-909N17.2 presages a poor prognosis of non-small cell lung cancer

BACKGROUND: Long non-coding RNAs (lncRNAs) were detected extraordinarily expressed in various tumors and could combine with microRNAs (miRNAs) to play important role in tumor cells. This study is to explore the role of lncRNA RP11-909N17.2 in NSCLC and discuss in what way it functions in NSCLC. METHODS: 120 NSCLC patients were enlisted in this study. Expression levels of lncRNA RP11-909N17.2 and miR-767-3p were detected and the correlation between lncRNA RP11-909N17.2 expression and the clinical data characteristics was analyzed. Prognosis potential of lncRNA RP11-909N17.2 was inferred with Kaplan-Meier and multivariate Cox regression assays. Biological functions of NSCLC cells were accessed by cell counting Kit-8, transwell migration and invasion assay. Mechanism of RP11-909N17.2 action on NSCLC cells was investigated by luciferase activity assay with wide-type or mutation. RESULTS: LncRNA RP11-909N17.2 has an ascendant expression while miR-767-3p has descended one in NSCLC tissue specimens and cells. Over-expression of lncRNA RP11-909N17.2 can shorten the overall survival period of NSCLC patients when compared with low expression. Knockdown of lncRNA RP11-909N17.2 suppressed biology function of NSCLC cell including proliferation, migration, and invasion. CONCLUSION: LncRNA RP11-909N17.2 can be developed into a prognostic index for NSCLC. LncRNA RP11-909N17.2 plays a promoting role in NSCLC cells possibly by binding miR-767-3p as a sponge.

Curcumin increases crizotinib sensitivity through the inactivation of autophagy via epigenetic modulation of the miR-142-5p/Ulk1 axis in non-small cell lung cancer

Drug resistance is a critical factor responsible for the recurrence of non-small cell lung cancer (NSCLC). Previous studies suggest that curcumin acts as a chemosensitizer and radiosensitizer in human malignancies, but the underlying mechanism remains elusive. In the present study, we explored how curcumin regulates the expression of miR-142-5p and sensitizes NSCLC cells to crizotinib. We found that miR-142-5p is significantly downregulated in NSCLC tissue samples and cell lines. Curcumin could increase crizotinib cytotoxicity by epigenetically restoring the expression of miR-142-5p. Furthermore, curcumin treatment suppressed the expression of DNA methylation-related enzymes, including DNMT1, DNMT3A, and DNMT3B, in NSCLC cells. In addition, the upregulation of miR-142-5p expression increased crizotinib cytotoxicity and induced apoptosis in tumor cells in a similar manner to that of curcumin. Strikingly, miR-142-5p overexpression suppressed crizotinib-induced autophagy in A549 and H460 cells. Mechanistically, miR-142-5p inhibited autophagy in lung cancer cells by targeting Ulk1. Overexpression of Ulk1 abrogated the miR-142-5p-induced elevation of crizotinib cytotoxicity in A549 and H460 cells. Collectively, our findings demonstrate that curcumin sensitizes NSCLC cells to crizotinib by inactivating autophagy through the regulation of miR-142-5p and its target Ulk1.

Construction of ceRNA network to identify the lncRNA and mRNA related to non-small cell lung cancer

Background Non-small cell lung cancer (NSCLC) harms human health, but its pathogenesis remains unclear. We wish to provide more molecular therapeutic targets for NSCLC. Methods The NSCLC tissue and normal tissue samples were screened for genetic comparison in the TCGA database. The predicted lncRNA and mRNA in BEAS2B and A549 cells were detected. Results Volcano plot displayed differentially expressed lncRNAs and mRNAs in adjacent tissues and NSCLC tissues. The survival curve showed that the lncRNA and mRNA had a significant impact on the patient’s survival. The results of GO term enrichment analysis indicated that mRNA functions were enriched in cell cycle-related pathways. In the ceRNA interaction network, 13 lncRNAs and 20 miRNAs were found to have an interactive relationship. Finally, 3 significantly different lncRNAs (LINC00968, lnc-FAM92A-9 and lnc-PTGFR-1) and 6 mRNAs (CTCFL, KRT5, LY6D, TMEM, GBP6, and TMEM179) with potential therapeutic significance were screened out. And the cell experiment verified our results. Conclusion We screened out clinically significant 3 lncRNAs and 6 mRNAs involved in the ceRNA network, which were the key to our future research on the treatment of NSCLC.

Human tissue cultures of lung cancer predict patient susceptibility to immune-checkpoint inhibition

Despite novel immunotherapies being approved and established for the treatment of non-small cell lung cancer (NSCLC), ex vivo models predicting individual patients’ responses to immunotherapies are missing. Especially immune modulating therapies with moderate response rates urge for biomarkers and/or assays to determine individual prediction of treatment response and investigate resistance mechanisms. Here, we describe a standardized ex vivo tissue culture model to investigate individual tumor responses. NSCLC tissue cultures preserve morphological characteristics of the baseline tumor specimen for up to 12 days ex vivo and also maintain T-cell function for up to 10 days ex vivo. A semi-automated analysis of proliferating and apoptotic tumor cells was used to evaluate tissue responses to the PD-1 inhibitor nivolumab (n = 12), from which two cases could be successfully correlated to the clinical outcome. T-cell responses upon nivolumab treatment were investigated by flow cytometry and multispectral imaging. Alterations in the frequency of the Treg population and reorganization of tumor tissues could be correlated to nivolumab responsiveness ex vivo. Thus, our findings not only demonstrate the functionality of T cells in NSCLC slice cultures up to 10 days ex vivo, but also suggests this model for stratifying patients for treatment selection and to investigate in depth the tumor-associated T-cell regulation.

Correlation of quantitative measures of estrogen receptor α and β expression in non-small cell lung cancer tissue with clinical characteristics of the disease

The expression level of estrogen receptors (ERα and ERβ) in surgical tumor biopsy specimens from 167 patients with non-small cell lung cancer (NSCLC) was quantified by flow cytometry-based immunofluorescence analysis. ERα and ERβ expression was revealed in all the tumor samples investigated. The level of ERα expression in the tumors varied from 10 to 58% and ERβ — 12% to 80%, indicating significant heterogeneity of estrogen receptor expression in tumors of different patients. The mean ERβ expression level was approximately 2-times higher compared to ERα (42.1±15.3% vs 21.4±11.3%; p<0.001). It allows to consider ER>β as a major estrogen target in NSCLC tissue. The level of ERα and ERβ expression in NSCLC tissue is independent of gender, smoking status, and the histological type of the tumor. Increased ERβ expression was detected only in male patients with lung adenocarcinoma in comparison with squamous cell carcinoma (p=0.02). The assessment of the correlation between ERα and ERβ in both the whole cohort of patients and subgroups with clinically relevant disease parameters revealed that the level of one marker does not predict the other one’s expression. The coefficient of determination, which characterizes how differences in one variable can be explained by the difference in a second variable, was less than 25% in any comparison group. The authors consider that the high level of ERβ expression and ERα coexpression in NSCLC tissue substantiates the clinical perceptiveness of a new treatment option for the disease, namely, adjuvant hormone (antiestrogen) therapy.

LINC01207 promotes the progression of non-small cell lung cancer via regulating ARHGAP11A by sponging miR-525-5p

BACKGROUND: The disorder of LINC01207 has a significant regulatory effect on cancers, nevertheless its role in non-small cell lung cancer (NSCLC) have not been illustrated. This study investigated the regulatory effect of LINC01207 on NSCLC and clarify its molecular mechanism. METHODS: Bioinformatics analysis was used to find the target lncRNA, miRNA and mRNA. LncBase and TargetScan databases predicted the relationship between LINC01207, miR-525-5p and ARHGAP11A. Dual-luciferase reporter gene assay and RNA binding protein immunoprecipitation assay were used to verify the binding relationship between genes. Fluorescence in situ hybridization assay was used to localize the expression of LINC01207 in NSCLC tissue. qRT-PCR and Western blot assays were used to measure the expression of LINC01207, miR-525-5p and ARHGAP11A. CCK-8 assay, Transwell assay and flow cytometry assay were used to detect NSCLC cell abilities. Mouse xenograft models further determined the effect of LINC01207 on the growth of NSCLC in vivo. RESULTS: LINC01207 was up-regulated in NSCLC tissue and cells, which was mainly localized in the cytoplasm. LINC01207 knockdown could inhibit the proliferation, migration and invasion of cancer cells and induce cell apoptosis. In addition, silencing LINC01207 could suppress tumor growth in vivo. LINC01207 could sponge and inhibit the expression of miR-525-5p in NSCLC cells, and inhibiting LINC01207 and miR-525-5p simultaneously could reverse the effect of miR-525-5p on the progression of NSCLC cells. Further study on downstream target genes showed that miR-525-5p could restrain the expression of ARHGAP11A, and then affect the progression of NSCLC. LINC01207 acting as a competing endogenous RNA (ceRNA) could regulate the expression of ARHGAP11A by competitively binding with miR-525-5p. CONCLUSION: LINC01207 regulates the progression of NSCLC by regulating the miR-525-5p/ARHGAP11A axis as a ceRNA and plays a carcinogenic role. In conclusion, our study elucidates the mechanism of LINC01207 regulating the progression of NSCLC, and provides a new idea for the diagnosis and treatment of NSCLC guided by lncRNA.

Diagnostic Performance of Electromagnetic Navigation Bronchoscopy-Guided Biopsy for Lung Nodules in the Era of Molecular Testing

Electromagnetic navigation bronchoscopy (ENB) is an emerging technique used to evaluate peripheral lung lesions. The aim of this study was to determine the diagnostic yield, safety profile, and adequacy of specimens obtained using ENB for molecular testing. This single-center, prospective pilot study recruited patients with peripheral pulmonary nodules that were not suitable for biopsy via percutaneous transthoracic needle biopsy methods. The possibility of molecular testing, including epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), and programmed death ligand 1 (PD-L1), was identified with non-small cell lung cancer (NSCLC) tissue obtained using ENB. ENB-guided biopsy was performed on 30 pulmonary nodules in 30 patients. ENB-guided biopsy was successfully performed in 96.6% (29/30) of cases, but one case failed to approach the target lesion. The diagnostic accuracy of ENB-guided biopsy was 68.0% (17/25). Biopsy-related pneumothorax occurred in one patient and there was no major bleeding or deaths related to the procedure. Among 13 patients diagnosed with NSCLC, molecular testing was successfully performed in 92.3% (12/13). ENB-guided biopsy demonstrated acceptable accuracy and excellent sample adequacy, with a high possibility of achieving molecular testing and a good safety profile to evaluate peripheral pulmonary nodules, even when the percutaneous approach was difficult and/or dangerous.

Bioinformatics Analysis Predicts hsa_circ_0026337/miR-197-3p as a Potential Oncogenic ceRNA Network for Non-small Cell Lung Cancers

Background: Circular RNAs (circRNAs) play an essential role in developing tumors, but their role in non-small cell lung cancer (NSCLC) is unclear. Thus, the present study explored the possible molecular mechanism of circRNAs in NSCLC. Methods: Three circular RNA (circRNA) microarray datasets were downloaded from the Gene Expression Omnibus (GEO) database. Differential expressions of circRNAs (DECs) were identified in NSCLC tissue and compared to adjacent healthy tissue. The online cancer-specific circRNA database (CSCD) was used for the analysis of the DECs function. Protein-protein interaction (PPI) network, Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), Cytoscape, and UALCAN were used to predict the critical nodes and perform patient survival analysis, respectively. The interaction between the DECs, the predicted miRNAs, and hub genes was also determined. Finally, the circRNA-miRNA-mRNA network was established. Results: The expression of hsa_circ_0049271, hsa_circ_0026337, hsa_circ_0043256, and hsa_circ_0008234 was decreased in NSCLC tissues. The Encyclopedia of RNA Interactomes (ENCORI) and CSCD database results showed that hsa_circ_0026337 was found to sponge with miR-1193, miR-197-3p, miR-3605-5p, miR-433-3p, and miR-652-3p, and hsa_circ_0043256 to sponge with miR-1252-5p, miR-494-3p, and miR-558, respectively. Subsequently, 100 mRNAs were predicted to bind with these seven miRNA response elements (MREs). The GO analysis and KEGG pathway revealed that these 100 MREs might be involved in “histone deacetylase binding” and “cellular senescence”. PPI network and Cytoscape identified the top ten hub genes. Survival analysis data showed that the low expression of hsa_circ_0026337 was significantly associated with shortened survival time in NSCLC (P=0.037), which increased the expression level of hsa-miR-197-3p, thereby inhibiting the translation of specific proteins. Conclusion: This study examined the circRNA-miRNA-mRNA regulatory network associated with NSCLC and explored the potential functions of DECs in the network to elucidate the mechanisms underlying disease progression in NSCLC.

Multi-institutional TSA-amplified Multiplexed Immunofluorescence Reproducibility Evaluation (MITRE) Study

BackgroundEmerging data suggest predictive biomarkers based on the spatial arrangement of cells or coexpression patterns in tissue sections will play an important role in precision immuno-oncology. Multiplexed immunofluorescence (mIF) is ideally suited to such assessments. Standardization and validation of an end-to-end workflow that supports multisite trials and clinical laboratory processes are vital. Six institutions collaborated to: (1) optimize an automated six-plex assay focused on the PD-1/PD-L1 axis, (2) assess intersite and intrasite reproducibility of staining using a locked down image analysis algorithm to measure tumor cell and immune cell (IC) subset densities, %PD-L1 expression on tumor cells (TCs) and ICs, and PD-1/PD-L1 proximity assessments.MethodsA six-plex mIF panel (PD-L1, PD-1, CD8, CD68, FOXP3, and CK) was rigorously optimized as determined by quantitative equivalence to immunohistochemistry (IHC) chromogenic assays. Serial sections from tonsil and breast carcinoma and non-small cell lung cancer (NSCLC) tissue microarrays (TMAs), TSA-Opal fluorescent detection reagents, and antibodies were distributed to the six sites equipped with a Leica Bond Rx autostainer and a Vectra Polaris multispectral imaging platform. Tissue sections were stained and imaged at each site and delivered to a single site for analysis. Intersite and intrasite reproducibility were assessed by linear fits to plots of cell densities, including %PDL1 expression by TCs and ICs in the breast and NSCLC TMAs.ResultsComparison of the percent positive cells for each marker between mIF and IHC revealed that enhanced amplification in the mIF assay was required to detect low-level expression of PD-1, PD-L1, FoxP3 and CD68. Following optimization, an average equivalence of 90% was achieved between mIF and IHC across all six assay markers. Intersite and intrasite cell density assessments showed an average concordance of R2=0.75 (slope=0.92) and R2=0.88 (slope=0.93) for breast carcinoma, respectively, and an average concordance of R2=0.72 (slope=0.86) and R2=0.81 (slope=0.68) for NSCLC. Intersite concordance for %PD-L1+ICs had an average R2 value of 0.88 and slope of 0.92. Assessments of PD-1/PD-L1 proximity also showed strong concordance (R2=0.82; slope=0.75).ConclusionsAssay optimization yielded highly sensitive, reproducible mIF characterization of the PD-1/PD-L1 axis across multiple sites. High concordance was observed across sites for measures of density of specific IC subsets, measures of coexpression and proximity with single-cell resolution.

KRT6A Promotes Lung Cancer Cell Growth and Invasion Through MYC-Regulated Pentose Phosphate Pathway

Keratin 6A (KRT6A) belongs to the keratin protein family which is a critical component of cytoskeleton in mammalian cells. Although KRT6A upregulation in non-small cell lung cancer (NSCLC) has been reported, the regulatory mechanism and functional role of KRT6A in NSCLC development have been less well investigated. In this study, KRT6A was confirmed to be highly expressed in NSCLC tissue samples, and its high expression correlated with poor patient prognosis. Furthermore, overexpression of KRT6A promotes NSCLC cell proliferation and invasion. Mechanistically, KRT6A overexpression is sufficient to upregulate glucose-6-phosphate dehydrogenase (G6PD) levels and increase the pentose phosphate pathway flux, an essential metabolic pathway to support cancer cell growth and invasion. In addition, we discovered that lysine-specific demethylase 1A (LSD1) functions upstream to promote KRT6A gene expression. We also found that the MYC family members c-MYC/MYCN are involved in KRT6A-induced G6PD upregulation. Therefore, this study reveals an underappreciated mechanism that KRT6A acts downstream of LSD1 and functions as a pivotal driver for NSCLC progression by upregulating G6PD through the MYC signaling pathway. Together, KRT6A and LSD1 may serve as potential prognostic indictors and therapeutic targets for NSCLC.