Pathogenesis Of Non-small Cell Lung Cancer Research Articles

MiR-650 promotes non-small cell lung cancer cell proliferation and invasion by targeting ING4 through Wnt-1/β-catenin pathway.

Non-small cell lung cancer (NSCLC) is the most frequent cancer worldwide with a poor 5-year survival. miR-650 acts as an oncogene and regulates tumor progress in various cancers. Molecular mechanisms of miR-650 in NSCLC cell proliferation and invasion was studied. The mRNA levels of miR-650 and special genes were calculated using RT-qPCR. MTT and transwell assays were applied to measure the proliferative and invasive ability. Kaplan-Meier method was used to assess the survival of NSCLC patients. miR-650 was upregulated in NSCLC and upregulation of miR-650 was associated with a poor overall survival of NSCLC, while the results of ING4 demonstrated the opposite results. miR-650 promoted proliferation and invasion through Wnt-1/β-catenin pathway by targeting inhibitor of growth 4 (ING4) in A549 cells. ING4 was a direct target gene of miR-650 and the expression of ING4 was mediated by exogenous altering the expression of miR-650. Remarkably, alterations of ING4 expression eliminated the functions of miR-650 on the proliferation and metastasis of NSCLC. miR-650 enhanced A549 cell proliferation and invasion through Wnt-1/β-catenin pathway by directly targeting the 3′-UTR of ING4 mRNA. The newly identified miR-650/ING4 axis provides a novel insight into the pathogenesis of NSCLC.

Chinese perspectives on clinical efficacy and safety of alectinib in patients with ALK-positive advanced non-small cell lung cancer.

The incidence of lung cancer is increasing in China, in contrast to trends in Western countries, due to the increasing numbers of smokers and high levels of air pollution. Non-small-cell lung cancer (NSCLC) is the most common form of lung cancer, accounting for approximately 85% of lung cancers. Better understanding of the pathogenesis of NSCLC has led to the identification of multiple genetic mutations and chromosomal translocations such as those in the anaplastic lymphoma kinase (ALK) gene. To facilitate the identification of treatment targets, multiple guidelines (European Society for Medical Oncology, National Comprehensive Cancer Network, and American Society of Clinical Oncology) now recommend screening for genetic factors to help guide treatment decisions. In recent years, multiple ALK inhibitors have been developed to treat NSCLC, including the first-generation tyrosine kinase inhibitor (TKI) crizotinib; second-generation TKIs such as ceritinib, ensartinib, brigatinib, and alectinib; the third-generation TKI lorlatinib; and the fourth-generation TKI repotrectinib. These agents differ in structure, potency, and activity, both systemically and their effects on central nervous system (CNS) metastases. Recently, alectinib was approved in China to treat patients with locally advanced or metastatic NSCLC that were ALK+. Alectinib has demonstrated activity against NSCLC, including metastases within the CNS, with better tolerability than crizotinib. These ALK inhibitors represent significant advances in the treatment of NSCLC and yet patients will likely still exhibit disease progression. Alectinib offers greater potency with greater specificity as well as a better toxicity profile than many other TKIs that are currently available. Here, we review the role of ALK as a therapeutic target in NSCLC, the testing methods for identifying ALK-rearranged NSCLC, and the various TKIs currently being used or explored for treatment in this setting, with a focus on alectinib from a Chinese perspective.

Regulatory Network Analysis to Reveal Important miRNAs and Genes in Non-Small Cell Lung Cancer.

Objective Lung cancer has high incidence and mortality rate, and non-small cell lung cancer (NSCLC) takes up approximately 85% of lung cancer cases. This study is aimed to reveal miRNAs and genes involved in the mechanisms of NSCLC. Materials and Methods In this retrospective study, GSE21933 (21 NSCLC samples and 21 normal samples), GSE27262 (25 NSCLC samples and 25 normal samples), GSE43458 (40 NSCLC samples and 30 normal samples) and GSE74706 (18 NSCLC samples and 18 normal samples) were searched from gene expression omnibus (GEO) database. The differentially expressed genes (DEGs) were screened from the four microarray datasets using MetaDE package, and then conducted with functional annotation using DAVID tool. Afterwards, protein-protein interaction (PPI) network and module analyses were carried out using Cytoscape software. Based on miR2Disease and Mirwalk2 databases, microRNAs (miRNAs)-DEG pairs were selected. Finally, Cytoscape software was applied to construct miRNA-DEG regulatory network. Results Totally, 727 DEGs (382 up-regulated and 345 down-regulated) had the same expression trends in all of the four microarray datasets. In the PPI network, TP53 and FOS could interact with each other and they were among the top 10 nodes. Besides, five network modules were found. After construction of the miRNA-gene network, top 10 miRNAs (such as hsa-miR-16-5p, hsa-let-7b-5p, hsa-miR-15a-5p, hsa-miR-15b-5p, hsa-let-7a-5p and hsa-miR-34a- 5p) and genes (such as HMGA1, BTG2, SOD2 and TP53) were selected. ConclusionThese miRNAs and genes might contribute to the pathogenesis of NSCLC.

Long non‑coding RNA SLNCR1 regulates non‑small cell lung cancer migration, invasion and stemness through interactions with secretory phospholipase A2.

Long non-coding RNA (lncRNA) SRA-like non-coding RNA (SLNCR1; also known as linc00673) is a recently identified oncogenic lncRNA. The role of SLNCR1 in non-small cell lung cancer (NSCLC), a common malignancy, remains poorly understood. The present study aimed to investigate the involvement of long non-coding RNA SLNCR1 in the pathogenesis of NSCLC. Reverse transcription-quantitative PCR (RT-qPCR) and ELISA were performed to measure the levels of lncRNA SLNCR1 and secretory phospholipase A2 (sPLA2) in lung biopsies, respectively. Correlations between expression levels of lncRNA SLNCR1 and sPLA2 were analyzed by Pearson's correlation analysis. Diagnostic value of lncRNA SLNCR1 for NSCLC was evaluated by receiver operating characteristic curve analysis. lncRNA SLNCR1 small interfering (si)RNA and sPLA2 overexpression vector were transfected into NSCLC cells. Cell migration, invasion and stemness were examined by Transwell migration assay, Matrigel invasion assay and flow cytometric assay, respectively. Following transfection, expression of lncRNA SLNCR1 and sPLA2 was detected by RT-qPCR and western blotting, respectively. The results demonstrated that lncRNA SLNCR1 expression levels were upregulated in tumor tissues compared with adjacent healthy tissues of patients with NSCLC. Plasma lncRNA SLNCR1 and sPLA2 expression levels were upregulated in patients with NSCLC compared with healthy controls. Plasma lncRNA SLNCR1 and sPLA2 were positively correlated in patients with NSCLC, but not in healthy controls. lncRNA SLNCR1 siRNA silencing inhibited, whereas sPLA2 overexpression promoted cell migratory and invasive abilities and stemness. lncRNA SLNCR1 siRNA silencing led to downregulated sPLA2 expression, whereas sPLA2 overexpression did not significantly affect lncRNA SLNCR1 expression. sPLA2 overexpression attenuated the inhibitory effects of lncRNA SLNCR1 siRNA silencing on cell migration, invasion and stemness. In conclusion, lncRNA SLNCR1 may regulate cancer cell migration, invasion and stemness in NSCLC through interactions with sPLA2.

MiR-296-3p may affect the proliferation and migration of non-small cell lung cancer cells via regulating RABL3.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. The pathogenesis of NSCLC has not yet been fully understood, and the therapeutic efficacy of current anti-NSCLC medication remains unsatisfactory. Previous studies indicated that miR-296-3p was down-regulated in NSCLC, suggesting that miR-296-3p may participate in the pathogenesis of NSCLC; however, the specific mechanisms still need to be further explored. The aim of this work is to investigate the roles of miR-296-3p in NSCLC and the related mechanism. Thirty NSCLC tissue and paired adjacent tissue were collected, and Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) was performed to examine the expression of miR-296-3p in cancer tissue and the adjacent tissue. Next, A549 cells were cultured and transfected with miR-296-3p mimics, and cell migration and invasion were determined using scratch wound-healing and transwell assays. Moreover, Western blot assay was performed to determine the effect of miR-296-3p on the expression of Rab-like 3 (RABL3), Matrix metallopeptidase (MMP)-2, Janus kinase (JAK) and Signal transducer and activator of transcription 3 (STAT3); next, Dual-Luciferase reporter assay has been conducted to prove the direct targeting relationship between miR-296-3p and RABL3. Finally, the cells of different treatments were subcutaneously implanted into nude mice to investigate the effect of miR-296-3p mimics in the xenograft mice tumor models. Our data indicated that miR-296-3p was significantly down-regulated and RABL3 was markedly up-regulated in NSCLC tissue compared with the adjacent tissue. Moreover, transient over-expression of miR-296-3p in A549 cells induced a significant decrease in the proliferation and invasion ability of A549 cells, as well as decreased expression of RABL3, MMP-2, JAK and STAT3. Furthermore, the Dual-Luciferase reporter assay confirmed that RABL3 is a direct target of miR-296-3p. Finally, the results of animal studies indicated that miR-296-3p can regulate the tumorigenesis of A549 cells in vivo. Our findings proved that miR-296-3p may play a role as a tumor suppressor in NSCLC both in vitro and in vivo, and we first reported that miR-296-3p can regulate the migration and invasion of A549 cells via targeting RABL3. Our data suggested that miR-296-3p may serve as a potential therapeutic target for treating NSCLC.

Abstract 4384: Role of EIF4G1 in non-small cell lung cancer pathogenesis and targeted therapy

Abstract Non-small cell lung cancer (NSCLC) accounts for about 85-90% of lung cancer cases, which represents the number one killer among cancers in US. The majority of lung cancer patients doesn't respond well to conventional chemo-/radio-therapeutic regimens and have a dismal 5-year survival rate of ~15%. The recent introduction of targeted therapy and immunotherapy gives new hopes to NSCLC patients, but their outcome/prognosis is far from satisfactory. The translation initiation EIF4F complex has been shown to play important roles in cancer progression. As an important component of the EIF4F complex, EIF4G1 serves as a scaffold and interacts with several other initiation factors such as EIF4E and EIF4A, and help to initiate cap-dependent translation in mammalian cells. EIF4G1 is overexpressed in a variety of cancers, but its functional role, clinical relevance and potential role as a therapeutic target in NSCLC remain largely unknown. Our recent data demonstrate that EIF4G1 expression level is much higher in NSCLC primary tumors than in paired adjacent normal tissues as well as in NSCLC cell lines than in the normal bronchial epithelial cells. Directly targeting EIF4G1 by RNAi or selective small-molecule inhibitors significantly reduce NSCLC cell proliferation, anchorage-independent growth in vitro as well as tumor progression in vivo. Our recent protein array and proteomics analyses indicate that EIF4G1: 1) has a complex interactome network in NSCLC cells; 2) appears to control the expression of a unique subset of the cellular proteome when compared to normal cells, which are closely related to NSCLC cells survival. Taken together, our data support that EIF4G1 plays a critical role in NSCLC survival, oncogenesis and tumor progression, and may serve as a novel and promising therapeutic target for improving NSCLC treatment. Citation Format: Zhiqiang Qin, Lu Dai, Luis Del Valle. Role of EIF4G1 in non-small cell lung cancer pathogenesis and targeted therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4384.

MicroRNA‑877 inhibits cell proliferation and invasion in non‑small cell lung cancer by directly targeting IGF‑1R

MicroRNAs (miRNAs/miRs) are frequently differentially expressed in non-small cell lung cancer (NSCLC), and differential miRNAs expression may be closely associated with NSCLC genesis and development. Therefore, an in-depth investigation of the cancer-associated miRNAs that are crucial for NSCLC pathogenesis may provide effective therapeutic targets for patients with this aggressive malignant tumor type. The expression levels and roles of miR-877 have been well studied in hepatocellular carcinoma and renal cell carcinoma. However, the expression pattern and functions of miR-877 in NSCLC as well as associated underlying mechanisms, to the best of our knowledge, have not yet been investigated. The present study revealed that miR-877 expression was downregulated in NSCLC tissues and cell lines. Low miR-877 expression was significantly associated with TNM stage and distant metastasis in patients with NSCLC. Functional experiments demonstrated that recovery of miR-877 expression restricted the proliferation and invasion of NSCLC cells. In addition, bioinformatics analysis predicted insulin-like growth factor 1 receptor (IGF-1R) as a potential target of miR-877. Luciferase reporter assays, reverse transcription-quantitative PCR and western blot analysis further validated that IGF-1R was a direct target of miR-877 in NSCLC. Furthermore, IGF-1R expression was markedly upregulated in NSCLC tissues, and exhibited an inverse correlation with miR-877 expression. Additionally, IGF-1R overexpression reversed the inhibitory effects in NSCLC cells caused by miR-877 upregulation. These findings demonstrated that miR-877 attenuated NSCLC cell proliferation and invasion, at least partly, by downregulating IGF-1R expression, thereby providing an new candidate biomarker for the diagnosis and therapy of patients with NSCLC.

Hsa_circ_0001946 Inhibits Lung Cancer Progression and Mediates Cisplatin Sensitivity in Non-small Cell Lung Cancer via the Nucleotide Excision Repair Signaling Pathway.

Background: Despite great advances in the diagnosis and treatment of non-small cell lung cancer (NSCLC), early diagnosis remains a challenge because patients usually have advanced lung cancer at the time they are diagnosed. The limited efficacy of conventional chemotherapy is another major problem in the treatment of NSCLC. Based on a published set of sequencing data, we find that hsa_circ_0001946 is a circRNA molecule with a significantly different expression level in three cell lines (human normal lung fibroblasts cell line MRC-5, human NSCLC cell line A549, cisplatin-resistant cell line A549/DDP), NSCLC tissues and paired adjacent normal tissues. We believe that hsa_circ_0001946 may have an effect on the progression of NSCLC and its sensitivity to cisplatin.Methods: We focused on investigating the circular RNA, hsa_circ_0001946. RNA interference of hsa_circ_0001946 was carried out in A549 cell lines to determine the effect of reduced hsa_circ_0001946 expression on lung cancer progression and was analyzed by Cell Counting Kit-8 (CCK-8), 5-ethynyl-20-deoxyuridine, clone formation, Hoechst, wound healing, and transwell assays. The nucleotide excision repair (NER) signaling pathway was identified by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Moreover, cellular responses to cisplatin were assessed through CCK-8 and flow cytometry assays. Western blot analysis and host-cell reactivation assay were used to determine the effect of hsa_circ_0001946 on NER signaling.Results: In this study, we found that the reduced expression of hsa_circ_0001946 promoted the viability, proliferation, migration, and invasion of NSCLC cells, as well as inhibition of cell apoptosis. Our findings suggest that hsa_circ_0001946 can affect the sensitivity of NSCLC cells to the chemotherapeutic drug cisplatin via modulation of the NER signaling pathway.Conclusions: Our study demonstrated the role of hsa_circ_0001946 in NSCLC pathogenesis, development, and chemosensitivity, and suggests that hsa_circ_0001946 may serve as a novel biomarker for the diagnosis and prediction of platinum-based chemosensitivity in patients with NSCLC.

YAP mediates the positive regulation of hnRNPK on the lung adenocarcinoma H1299 cell growth.

Lung cancer is the leading cause of cancer death worldwide, and non-small cell lung cancer (NSCLC) accounts for 80%-85% of diagnostic cases. The molecular mechanisms of NSCLC pathogenesis are not well understood. Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is a multifunctional protein that regulates gene expression and signal transduction and closely associated with tumorigenesis, but its mechanism of action in the pathogenesis of NSCLC is unclear. In this study, we observed that the expression pattern of hnRNPK in H1299 lung adenocarcinoma cells varied depending on the cell density in culture. Moreover, hnRNPK stimulated the ability of proliferation and colony formation of H1299 cells, which is important for the multilayered cell growth in culture. We further investigated whether there is an association between hnRNPK and the elements involved in the cell contact inhibition pathway. By using quantitative reverse transcriptase-polymerase chain reaction assay and a YAP activity reporter system, we found that hnRNPK upregulated the mRNA and protein levels and transcriptional activity of Yes-associated protein 1 (YAP), a master negative regulator of Hippo contact inhibition pathway. Furthermore, YAP knockdown with siRNA abolished the stimulatory effect of hnRNPK on H1299 cell proliferation. These results suggested that YAP could be one of the effectors of hnRNPK. Our data may provide new clues for further understanding the biological functions of hnRNPK, particularly in the context of lung adenocarcinoma oncogenesis.

Upregulated expression of ROCK1 promotes cell proliferation by functioning as a target of miR-335-5p in non-small cell lung cancer.

Lung cancer is regarded as one of the dominant causes in cancer patients among men and women all over the world. Rho-associated coiled-coil forming protein kinase l (ROCK1) is characterized as pivotal downstream effectors of the small GTPase RhoA and reported to participate in tumor metastasis. miR-335-5p acts as tumor suppressor microRNA and is identified to be downregulated in tumor tissues. miR-335-5p/ROCK1 axis has been demonstrated to promote cell proliferation and metastasis in gastric cancer, hepatocellular carcinoma and so on. However, the role it plays in promoting cell proliferation in non-small cell lung cancer (NSCLC) is poorly understood. Here, we demonstrated that the upregulated expression of ROCK1 was highly correlated with downregulated expression of miR-335-5p in NSCLC tissues and cell lines. Mechanistically, Knockdown of ROCK1 inhibited cell proliferation in vitro, accompanied by cell cycle change confirmed by flow analysis. Furthermore, miR-335-5p can downregulate the ROCK1 expression by directly binding to the 3'-untranslated region in posttranscriptional level. In vivo animal model showed similar results. Our findings highlighted the crucial role that miR-335-5p acted as a tumor suppressor to modulate cell proliferation and cell cycle progression via downregulating ROCK1 expression. And this miR-335-5p/ROCK1 axis contributed to NSCLC pathogenesis and might be promising targets for NSCLC therapy.

Host transcriptomic signatures associated with dysbiosis in a preclinical model of lung cancer.

3107 Background: Enrichment of the lower airway microbiota with oral commensals has been associated with transcriptomic changes affecting several inflammatory pathways associated with non-small cell lung cancer (NSCLC) development and progression. Using a mouse model of NSCLC, we evaluated the effects of lower airway dysbiosis on tumor progression and host transcriptomics. Methods: Preclinical model of lung cancer was constructed by introducing luminescence-tagged Kras mutated cells into C57/B6 mice, causing lung cancer to develop. Lower airway dysbiosis was induced by weekly intratracheal challenge with either PBS or Veillonella parvula in wild type and lung cancer mice. Experiments were repeated twice to evaluate for survival as well as lower airway host response using flow cytometry and RNA sequencing (HiSeq). Sequence data was processed using a validated mouse gene expression signature matrix with cibersort from https://cibersort.stanford.edu and DESeq using FDR correction. Results: In wild type mice, lower airway dysbiosis with Veillonella did not affect the survival, weight gain or airway lumen diameter. Among lung cancer mice, dysbiosis led to increased mortality, weight loss, and tumor burden. Multiple transcriptomic signatures were identified among the dysbiosis groups (both in WT and lung cancer mice). Unsupervised hierarchical clustering of immune cell profiles using cibersort on whole transcriptome showed near perfect separation between the four experimental conditions. Amongst the most differentially enriched immune cell subsets, we identified that lung dysbiosis upregulates genes annotated to Th1 and Th2 cells (p < 0.01, q < 0.2). Using flow cytometry, we identified that PD-1, IL-17, and ROR-gamma are differentially expressed in CD4+ cells in dysbiosis conditions, and these patterns are consistent in whole RNA transcriptome. Conclusions: Transcriptomic signatures reveal immune profiles associated with dysbiosis, an experimental condition associated with worse outcomes in lung cancer. This investigation provides novel insights into how disruption of the lower airway microbiome may contribute to the pathogenesis of NSCLC.

MiR‑497 suppresses malignant phenotype in non‑small cell lung cancer via targeting KDR.

The aim of the present study was to evaluate the expression of microRNA‑497 (miR‑497) in non‑small cell lung cancer (NSCLC) tissues and cell lines, and to investigate possible mechanisms associated with its regulatory role on cell behaviors. The expression level of miR‑497 was evaluated in 15 cases of NSCLC tissues and 8 adjacent normal tissues, and in 8 NSCLC cell lines, including H1975, A549, H358, H1650, H460, Calu‑1, H1299 and H292, by reverse transcription‑quantitative polymerase chain reaction. Effects of miR‑497 overexpression on cell proliferation, invasion, apoptosis and radiosensitivity were examined with a Cell Counting Kit‑8 assay, Matrigel assay, flow cytometry and a clone formation assay in vitro, respectively, and in an in vivo ectopic tumor nude mice model. A dual luciferase reporter assay was employed for interaction between miR‑497 and its target gene kinase insert domain receptor (KDR). A significantly decreased level of miR‑497 was determined in NSCLC tissues, compared with adjacent normal tissues, and Calu‑1 and H1975 exhibited the lowest miR‑497 expression among the 8 NSCLC cell lines. miR‑497 overexpression could inhibit cell proliferation and invasion, promote cancer cell apoptosis and decrease cell clone formation following radiation treatment in vitro, and decrease tumor growth in vivo. Furthermore, a dual luciferase reporter assay revealed that KDR as the target gene for miR‑497. It was demonstrated that miR‑497 was downregulated in NSCLC specimens. Additionally, miR‑497 directly targeted and downregulated KDR expression, and inhibited malignant behaviors of NSCLC cells. These data indicated that miR‑497 could serve as a tumor suppressor gene involved in NSCLC pathogenesis.

Identification of novel biomarkers and candidate small molecule drugs in non-small-cell lung cancer by integrated microarray analysis.

Background: Non-small-cell lung cancer (NSCLC) remains the leading cause of cancer morbidity and mortality worldwide. In the present study, we identified novel biomarkers associated with the pathogenesis of NSCLC aiming to provide new diagnostic and therapeutic approaches for NSCLC.Methods: The microarray datasets of GSE18842, GSE30219, GSE31210, GSE32863 and GSE40791 from Gene Expression Omnibus database were downloaded. The differential expressed genes (DEGs) between NSCLC and normal samples were identified by limma package. The construction of protein–protein interaction (PPI) network, module analysis and enrichment analysis were performed using bioinformatics tools. The expression and prognostic values of hub genes were validated by GEPIA database and real-time quantitative PCR. Based on these DEGs, the candidate small molecules for NSCLC were identified by the CMap database.Results: A total of 408 overlapping DEGs including 109 up-regulated and 296 down-regulated genes were identified; 300 nodes and 1283 interactions were obtained from the PPI network. The most significant biological process and pathway enrichment of DEGs were response to wounding and cell adhesion molecules, respectively. Six DEGs (PTTG1, TYMS, ECT2, COL1A1, SPP1 and CDCA5) which significantly up-regulated in NSCLC tissues, were selected as hub genes according to the results of module analysis. The GEPIA database further confirmed that patients with higher expression levels of these hub genes experienced a shorter overall survival. Additionally, CMap predicted the 20 most significant small molecules as potential therapeutic drugs for NSCLC. DL-thiorphan was the most promising small molecule to reverse the NSCLC gene expression.Conclusions: Based on the gene expression profiles of 696 NSCLC samples and 237 normal samples, we first revealed that PTTG1, TYMS, ECT2, COL1A1, SPP1 and CDCA5 could act as the promising novel diagnostic and therapeutic targets for NSCLC. Our work will contribute to clarifying the molecular mechanisms of NSCLC initiation and progression.

Elevated Circulating CD4+CD25-Foxp3+ Regulatory T Cells in Patients with Nonsmall Cell Lung Cancer.

Purpose: CD4+CD25+Foxp3+ regulatory T (Treg) cell-mediated immunosuppression has been implicated as a crucial mechanism of tumor immune cell escape in nonsmall cell lung cancer (NSCLC). However, little is known concerning the specific role of CD4+CD25-Foxp3+ Treg cells in NSCLC. The aim of this study was to investigate the frequency of circulating CD4+CD25-Foxp3+ Treg cells and their role in NSCLC. Methods: The frequencies of Treg, T helper (Th)1, Th2, and Th17 cells in peripheral blood were separately measured in 36 NSCLC patients and 20 healthy controls (HCs) using flow cytometry. Serum cytokine concentrations were determined using cytometric bead arrays. Results: The frequencies of circulating CD4+CD25+ T cells and CD4+CD25+Foxp3+ and CD4+CD25-Foxp3+ Treg cells were significantly higher in advanced-stage NSCLC patients compared with patients with limited-stage NSCLC. The frequencies of circulating CD4+CD25+Foxp3+ and CD4+CD25-Foxp3+ Treg cells were negatively correlated with interleukin (IL)-17, but positively correlated with serum IL-10 levels. In addition, the Th17/CD4+CD25-Foxp3+ Treg cell ratios were negatively correlated with serum cytokeratin 19 fragment (CYFRA 21-1) concentrations in patients with NSCLC. Moreover, coculturing CD4+CD25-Foxp3+ Treg cells and CD14+ monocytes in vitro resulted in a higher frequency of CD206+CD14+ M2-like monocytes compared with CD14+ monocytes. Conclusions: Elevated circulating CD4+CD25-Foxp3+ Treg cells may be involved in the pathogenesis of NSCLC.

Investigation into the underlying molecular mechanisms of non-small cell lung cancer using bioinformatics analysis

Development of new therapeutic strategies has proven to be of little impact on non-small cell lung cancer (NSCLC), and unfortunately, its molecular mechanisms still remain poorly understood. Therefore, the current study aimed to diagnose the genes associated in the pathogenesis of NSCLC. The differentially expressed genes (DEGs) were diagnosed using the limma software package. The WebGestalt was used to perform pathway and Gene Ontology (GO) enrichment analysis of the DEGs. Protein-protein interaction (PPI) networks, extracted modules, miRNA-target genes regulatory network and miRNA-target genes regulatory network were used to obtain insight into the actions of DEGs. Survival analysis for DEGs carried out. A total of 118 DEGs, including 74 up-regulated and 44 down-regulated genes were diagnosed between serum starved Calu-6 lung cancer cells transfected with control siRNA and serum starved Calu-6 lung cancer cells transfected with siRNA against the LAPTM4B gene. The up-regulated genes were enriched in PPAR signalling pathway, angiogenesis, positive regulation of neuron projection development and extracellular space. The down-regulated genes were enriched in FoxO signalling pathway, plasminogen activating cascade, cardiac chamber morphogenesis and cell surface. The current study screened the genes in PPI network, extracted modules, miRNA-target genes regulatory network and TF-target genes regulatory network with higher degrees as hub genes, which included FGFR1, HEY1, SPP1, FOS, NDRG1, TFF3, CDK15, PDGFRB, IL7R, LYPD6B, IQGAP2, CEACAM1, VEGFA, SGK1, GPRC5B and WNT5A. Survival and expression analysis suggested that low expressed CACNA1C and GATA2, and high expressed MANSC1 and MSX2 in patients with NCSLC was linked with a positive prognosis for overall survival and all individual stages of NSCLC. In conclusion, the current study diagnosed DEGs between serum starved Calu-6 lung cancer cells transfected with control siRNA and serum starved Calu-6 lung cancer cells transfected with siRNA against the LAPTM4B gene, which could improve our understanding of the molecular mechanisms of small interference RNA (siRNA) against LAPTM4B in NSCLC, and up- and down-regulated genes, over and under expressed genes in NSCLC patients indicated a positive prognosis for overall survival and may be used as a prognostic biomarker for patients with NSCLC. Thus, we developed a signature based on seven DEGs with prognostic value for NSCLC patients using multi-steps bioinformatics methods, which may provide new insights and potential biomarkers for prognosis, as well as possibly serving as new therapeutic targets in clinical applications.

Epigenetic Suppression of the T-box Subfamily 2 (TBX2) in Human Non-Small Cell Lung Cancer.

(1) The TBX2 subfamily of transcription factors (TBXs 2, 3, 4 and 5) are markedly down-regulated in human non-small cell lung cancer (NSCLC) and exert tumor suppressor effects in lung malignancy. Yet, mechanisms underlying suppressed expression of the TBX2 subfamily in NSCLC are elusive. Here, we interrogated probable epigenetic mechanisms in suppressed expression of the TBX2 subfamily in human NSCLC. (2) TBX2 subfamily gene expression and methylation levels in NSCLC and normal lung tissues were surveyed using publicly available RNA-sequence and genome-wide methylation datasets. Methylation β-values of the four genes were statistically compared between NSCLCs and normal lung tissues, correlated with gene expression levels, and interrogated with clinicopathological variables. Expression and methylation levels of TBXs were quantified in NSCLC cells using real-time PCR and methylation-specific PCR assays, respectively. Effects of the DNA methyltransferase inhibitor 5-azacytidine (Aza) on TBX2 subfamily expression were assessed in NSCLC cells. Impact of TBX2 subfamily expression on Aza-treated cells was evaluated by RNA interference. (3) All four TBXs were significantly hypermethylated in NSCLCs relative to normal lung tissues (p < 0.05). Methylation β-values of the genes, with exception of TBX2, were significantly inversely correlated with corresponding mRNA expression levels (p < 0.05). We found no statistically significant differences in hypermethylation levels of the TBX2 subfamily by clinicopathological features including stage and tobacco history. Expression levels of the TBX genes were overall suppressed in NSCLC cells relative to normal alveolar cells. Members of the subfamily were significantly hypermethylated in all tested NSCLC cell lines relative to normal alveolar cells. Treatment with Aza induced the expression of the TBX2 subfamily concomitant with NSCLC cell growth inhibition. Further, simultaneous knockdown of the four TBX genes markedly reduced anti-growth effects of Aza in NSCLC cells. (4) Our study sheds light on new epigenetic profiles in the molecular pathogenesis of human NSCLC.

Down-regulation of microRNA-144-3p and its clinical value in non-small cell lung cancer: a comprehensive analysis based on microarray, miRNA-sequencing, and quantitative real-time PCR data

BackgroundPrevious studies have shown that miR-144-3p might be a potential biomarker in non-small cell lung cancer (NSCLC). Nevertheless, the comprehensive mechanism behind the effects of miR-144-3p on the origin, differentiation, and apoptosis of NSCLC, as well as the relationship between miR-144-3p and clinical parameters, has been rarely reported.MethodsWe investigated the correlations between miR-144-3p expression and clinical characteristics through data collected from Gene Expression Omnibus (GEO) microarrays, the relevant literature, The Cancer Genome Atlas (TCGA), and real-time quantitative real-time PCR (RT-qPCR) analyses to determine the clinical role of miR-144-3p in NSCLC. Furthermore, we investigated the biological function of miR-144-3p by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Protein-protein interaction (PPI) network was created to identify the hub genes.ResultsFrom the comprehensive meta-analysis, the combined SMD of miR-144-3p was − 0.95 with 95% CI of (− 1.37, − 0.52), indicating that less miR-144-3p was expressed in the NSCLC tissue than in the normal tissue. MiR-144-3p expression was significantly correlated with stage, lymph node metastasis and vascular invasion (all P < 0.05). As for the bioinformatics analyses, a total of 37 genes were chosen as the potential targets of miR-144-3p in NSCLC. These promising target genes were highly enriched in various key pathways such as the protein digestion and absorption and the thyroid hormone signaling pathways. Additionally, PPI revealed five genes—C12orf5, CEP55, E2F8, STIL, and TOP2A—as hub genes with the threshold value of 6.ConclusionsThe current study validated that miR-144-3p was lowly expressed in NSCLC. More importantly, miR-144-3p might function as a latent tumor biomarker in the prognosis prediction for NSCLC. The results of bioinformatics analyses may present a new method for investigating the pathogenesis of NSCLC.

MRTF-A Regulates the Proliferation and Migration of Non-small Cell Lung Cancer Cells of A549 through HOTAIR

背景与目的非小细胞肺癌（non-small cell lung cancer, NSCLC）作为肺癌的一种，由于其高发病率一直备受关注。准确揭示其发病机制对于NSCLC的诊断以及治疗具有重要的指导意义。MATF-A作为转录调控因子在多种肿瘤的发生发展过程中发挥着重要作用，可以调控多种肿瘤细胞的迁移过程。HOTAIR是近年研究发现的一个长链非编码RNA，在多种肿瘤中异常表达并参与多种肿瘤的增殖及迁移的进程。本研究旨在探究MRTF-A通过HOTAIR调控NSCLC的增殖及迁移进程。方法构建MRTF-A的过表达质粒及干扰质粒，检测对A549细胞增殖及迁移的影响。设计合成HOTAIR的siRNA检测对A549细胞增殖及迁移的作用。qRT-PCR检测MRTF-A对HOTAIR表达的调控作用。构建HOTAIR的启动子检测MRTF-A对HOTAIR启动子活性的影响。结果过表达MRTF-A促进A549细胞的增殖，沉默MRTF-A的表达抑制A549细胞的增殖及迁移。干扰HOTAIR的表达抑制A549细胞的增殖及迁移。MRTF-A能够影响HOTAIR的表达，同时能够调控HOTAIR启动子的活性。结论MRTF-A通过HOTAIR调控NSCLCA549细胞的增殖及迁移进程。

Correlations of the ZEB1 expression with the incidence and prognosis of non-small cell lung cancer.

In order to investigate the role of the zinc finger E-box-binding homeobox 1 (ZEB1) expression in the incidence of non-small cell lung cancer (NSCLC), the effects of the ZEB1 expression on the pathogenesis and prognosis of NSCLC were investigated. Correlations of the expression of ZEB1 in 88 clinical patients with NSCLC with clinicopathological features were determined. The patients were followed up for 60 months to study the correlation between the ZEB1 expression and the prognosis of NSCLC patients. To further explore the role of the ZEB1 expression in the incidence of NSCLC, the expressions of ZEB1 and E-cadherin in three NSCLC cell lines (A549, NCI-H1299 and NCI-H1975) and human normal lung epithelial BESA-2B cell line were measured, and the cell invasion ability was detected. The role of ZEB1 in NSCLC cell invasion was verified through the knockdown of ZEB1 by the short hairpin ribonucleic acids (shRNAs). In addition, its effects on the proliferation and apoptosis of NSCLC cells were confirmed by the overexpression of ZEB1. The expression of ZEB1 in NSCLC tissues was remarkably higher than that in normal tissues, and the expression level of ZEB1 was significantly related to the cancer stage and tumor size. The lower the expression of ZEB1 was, the higher the overall survival rate and the longer the survival time would be. The expression of ZEB1 was negatively correlated with that of E-cadherin in cell lines, and ZEB1-shRNAs markedly reduced the invasion ability of NSCLC cells. The overexpression of ZEB1 resulted in an increase in the proliferation activity and a significant decrease in the apoptosis of A549 cells. The expression of ZEB1 is closely related to the incidence and prognosis of NSCLC. Increased expression of ZEB1 is helpful for promoting the invasion of NSCLC and enhancing the proliferation activity of NSCLC.

Significance of miR-15a-5p and CNKSR3 as Novel Prognostic Biomarkers in Non-Small Cell Lung Cancer

In recent years, targeted cancer treatment methods at various molecular levels have been developed for Non-Small Cell Lung Cancer (NSCLC), one of two major subtypes of lung cancer. miRNAbased clinical trials are currently the preferred targeted therapeutic strategy. Also, ceRNAs (competing endogenous RNA) would be the newest and the most effective approach to uncover novel interactions between mRNAs and miRNAs in NSCLC carcinogenesis. There are many factors influencing the efficiency of a miRNA to suppress or silence translation of the target mRNA. The most effective event is the presence of other RNAs showing ceRNA activity. These RNAs contain binding sites for specific miRNAs and enable miRNAs to bind these pseudo targets, instead of the original binding sites on the target mRNA. Therefore, the mRNA of the target gene is less affected by this miRNA, while the amount of miRNA remains the same in the media. For this project, we determined that five clinically important different oncogenes (PDL1, FGFR1, DDX3X, SLC1A5, FXR1 ) are involved in the pathogenesis of NSCLC. For this purpose, we transfected model NSCLC cell line, A549, with miRNAs (miR-150-5p, miR-15a-5p, miR-503-5p) targeting these oncogenes to investigate whether these oncogenes will be suppressed at the mRNA level and also how the suppression efficiency of these miRNA on the oncogenes will be affected by possible ceRNA (CNKSR3, POU2F1, HIPK2) activities. miR-15a-5p was determined to have the most suppressive effect on the five genes and three potential ceRNAs (p<0.05). Furthermore, CNKSR3 was the ceRNA most affected by all three miRNAs (p<0.05). CNKSR3 was affected more than the oncogenes known to act on NSCLC and this might make it a stronger and novel marker for use in possible treatment regimens designed using miR-15a-5p silencing effect on oncogenes in NSCLC pathogenesis. According to the literature, this is the first study associating NSCLC with miR-15a-5p and CNKSR3.