Systems biology has become an effective approach for understanding the molecular mechanisms underlying the development of lung cancer. In this study, sequences of 100 non-small cell lung cancer (NSCLC)-related proteins were downloaded from the National Center for Biotechnology Information (NCBI) databases. The Theory of Coevolution was then used to build a protein-protein interaction (PPI) network of NSCLC. Adopting the reverse thinking approach, we analyzed the NSCLC proteins one at a time. Fifteen key proteins were identified and categorized into a special protein family F(K), which included Cyclin D1 (CCND1), E-cadherin (CDH1), Cyclin-dependent kinase inhibitor 2A (CDKN2A), chemokine (C-X-C motif) ligand 12 (CXCL12), epidermal growth factor (EGF), epidermal growth factor receptor (EGFR), TNF receptor superfamily, member 6(FAS), FK506 binding protein 12-rapamycin associated protein 1 (FRAP1), O-6-methylguanine-DNA methyltransferase (MGMT), parkinson protein 2, E3 ubiquitin protein ligase (PARK2), phosphatase and tensin homolog (PTEN), calcium channel voltage-dependent alpha 2/delta subunit 2 (CACNA2D2), tubulin beta class I (TUBB), SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 2 (SMARCA2), and wingless-type MMTV integration site family, member 7A (WNT7A). Seven key nodes of the sub-network were identified, which included PARK2, WNT7A, SMARCA2, FRAP1, CDKN2A, CCND1, and EGFR. The PPI predictions of EGFR-EGF, PARK2-FAS, PTEN-FAS, and CACNA2D2-CDH1 were confirmed experimentally by retrieving the Biological General Repository for Interaction Datasets (BioGRID) and PubMed databases. We proposed that the 7 proteins could serve as potential diagnostic molecular markers for NSCLC. In accordance with the developmental mode of lung cancer established by Sekine et al., we assumed that the occurrence and development of lung cancer were linked not only to gene loss in the 3p region (WNT7A, 3p25) and genetic mutations in the 9p region but also to similar events in the regions of 1p36.2 (FRAP1), 6q25.2-q27 (PARK2), and 11q13 (CCND1). Lastly, the invasion or metastasis of lung cancer happened.
Biomarkers, Tumor ; metabolism ; Carcinoma, Non-Small-Cell Lung ; genetics ; metabolism ; pathology ; Databases, Factual ; Humans ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Protein Interaction Maps

MicroRNAs (miRNAs) regulate the proliferation and metastasis of cancer cells. Here, we showed that miR-152 was downregulated in non-small-cell lung cancer (NSCLC) tissues and cell lines. Overexpression of miR-152 suppressed cell proliferation and colony formation and also limited migration and invasion. Fibroblast growth factor 2 (FGF2) was confirmed as a direct target of miR-152. FGF2 knockdown suppressed cell proliferation, colony formation, migration and invasion, whereas FGF2 overexpression partially reversed the suppressive effect of miR-152. Furthermore, the presence of miR-152 was inversely correlated with FGF2 in NSCLC tissues. Overall, this study demonstrated that miR-152 suppressed the proliferation and invasion of NSCLC cells by downregulating FGF2. These findings provide novel insights with potential therapeutic applications for the treatment of NSCLC.
Carcinoma, Non-Small-Cell Lung/*genetics/metabolism/pathology ; Cell Movement ; Cell Proliferation ; Female ; Fibroblast Growth Factor 2/*genetics ; *Gene Expression Regulation, Neoplastic ; Humans ; Lung/metabolism/*pathology ; Lung Neoplasms/*genetics/metabolism/pathology ; Male ; MicroRNAs/*genetics ; Middle Aged ; Neoplasm Invasiveness/genetics/pathology

OBJECTIVETo investigate the biological behaviors and chemosensitivity of non-small cell lung cancer (NSCLC) cell line A549 after IGF-IR gene silencing by RNA interference (RNAi) in vitro.

METHODSTwo plasmids siRNA 1 and 2 expressing IGF-IR siRNA with human U6 promoter were constructed,and an unrelated siRNA was used as negative control. NSCLC A549 cells were transfected with sequence-specific siRNA or unrelated siRNA as control. Quantitative RT-PCR and Western blot were used to detect the expression of IGF-IR. NSCLC A549 cells were transfected with siRNA and treated with DDP. MTT assay and flow cytometry were used to assess the effects of IGF-IR silencing on tumor cell proliferation and chemosensitivity.

RESULTTransfection of NSCLC cells with siRNA resulted in reduction of IGF-IR mRNA expression by 78.9 % and protein production by 89.8%. The decrease in IGF-IR levels caused significant growth inhibition of A549 cells both at 48 h and at 72 h, and decrease of the IC50 of DDP at 24 h, 48 h and at 72 h. Flow cytometry showed that 77.5% of A549 cells retained in G0/G1 phase.

CONCLUSIONThe sequence specific suppression of IGF-IR gene expression by RNAi enhances sensitivity to DDP in NSCLC cell.

Carcinoma, Non-Small-Cell Lung ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Gene Silencing ; Humans ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Plasmids ; genetics ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Receptor, IGF Type 1 ; metabolism ; Transfection

OBJECTIVETo explore the impact of Line-1 methylation on clinical features of non-small cell lung cancer and its connection with smoking and other living habits.

METHODSPyrosequencing was used to determine the extent of Line-1 methylation in cancer and adjacent tissues derived from 197 patients with primary non-small cell lung cancer. Non-conditional logistic regression analysis was performed to correlate the level of Line-1 methylation with clinical features and living habits of the patients.

RESULTSLine-1 methylation for cancer tissue and adjacent tissue has measured 68.20±11.63 and 78.90±2.09, respectively (P < 0.01), and has been associated with TNM staging, smoking history and histopathological types.

CONCLUSIONLung cancer tissue Line-1 methylation level is closely related with clinical features and smoking. There is also a correlation between histopathological types of lung cancer and relative hypomethylation of Line-1.

Aged ; Aged, 80 and over ; Carcinoma, Non-Small-Cell Lung ; genetics ; metabolism ; pathology ; DNA Methylation ; Female ; Humans ; Long Interspersed Nucleotide Elements ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Male ; Middle Aged

Carcinoma, Non-Small-Cell Lung ; diagnosis ; genetics ; pathology ; Humans ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Lung Neoplasms ; diagnosis ; genetics ; pathology ; Oncogene Proteins, Fusion ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

BACKGROUND: There is a high morbidity, mortality, and poor clinical prognosis of lung squamous cell carcinoma (LUSC). However, there is currently no effective targeted treatment plan for LUSC. As a long non-coding RNA (lncRNA), lncRNA miR143HG has been proven to play an important role in the occurrence and development of various tumors. However, the biological role played by lncRNA miR143HG in LUSC cells is still unclear. Therefore, this study aimed to investigate the mechanism of lncRNA miR143HG on regulating the biological behavior of LUSC H520 cells. METHODS: Pan-cancer analysis and differential expression analysis of lncRNA miR143HG were performed based on The Cancer Genome Atlas (TCGA) database. The predictive effect of lncRNA miR143HG on the diagnosis and prognosis of LUSC was evaluated by adopting the receiver operating characteristic (ROC) curve and timeROC curve. The enrichment degree of each pathway to lncRNA miR143HG was determined. The expression of lncRNA miR143HG and miR-155 in BEAS-2B cells and H520 cells was detected using quantitative real-time polymerase chain reaction (qRT-PCR). H520 cells were randomly divided into blank control group (without any treatment), negative control group (transfected with lncRNA-NC), lncRNA miR143HG group (transfected with lncRNA miR143HG), and lncRNA miR143HG+miR-155 group (co-transfected with lncRNA miR143HG and miR-155). The approaches of CCK-8, wound healing test, Transwell assay, flow cytometry, qRT-PCR, and Western blot were respectively employed to detect the cell proliferation ability, cell migration ability, cell invasion ability, cell apoptosis rate, and expression level of related genes and proteins of the Wnt/β-Catenin pathway. RESULTS: The results of pan-cancer analysis and differential analysis collectively showed that except for renal clear cell carcinoma, the expression of lncRNA miR143HG in other cancer tissues was higher than that in healthy tissues, and the differences were significant in LUSC. The evaluation results of the ROC curve and timeROC curve suggested that lncRNA miR143HG was of great significance in the prediction of diagnosis and prognosis of LUSC. The pathways enriched in high expression of lncRNA miR143HG mainly included focal adhesion, vascular smooth muscle contraction, calcium signaling pathways, and so on; the pathways enriched in the low expression of lncRNA miR143HG embraced oxidative phosphorylation, cell cycle, basic transcription factors, etc. The qRT-PCR results showed that lncRNA miR143HG was low expressed but miR-155 was highly expressed in H520 cells when compared to BEAS-2B cells (P<0.05). Compared with the negative control group, the expression levels of the gene of lncRNA miR143HG, the gene and protein of Wnt, as well as the gene and protein of β-Catenin were significantly increased, while the gene expression of miR-155, the ability of cell proliferation, cell migration, and cell invasion were significantly reduced, but the cell apoptosis rate was dominantly elevated in cells of lncRNA miR143HG group (P<0.05). In addition, compared with the lncRNA miR143HG group, overexpression of miR-155 could reverse the biological behavior mediated by lncRNA miR143HG, and the difference was statistically significant (P<0.05). CONCLUSIONS LncRNA miR143HG was of great significance for the biological behavior of H520 cells. LncRNA miR143HG inhibited the ability of proliferation, migration, and invasion, as well as enhanced the apoptosis of H520 cells by downregulating miR-155 expression, which may be related to the Wnt/β-Catenin pathway.
.
Humans ; RNA, Long Noncoding/genetics* ; beta Catenin/metabolism* ; Lung Neoplasms/genetics* ; Carcinoma, Squamous Cell/genetics* ; Carcinoma, Non-Small-Cell Lung/genetics* ; MicroRNAs/genetics* ; Lung/pathology* ; Cell Proliferation/genetics* ; Cell Movement/genetics* ; Gene Expression Regulation, Neoplastic

Targeted gene therapy has become a promising approach for lung cancer treatment. In our previous work, we reported that the targeted expression of microRNA-7 (miR-7) operated by thyroid transcription factor-1 (TTF-1) promoter inhibited the growth of human lung cancer cells in vitro and in vivo; however, the intervention efficiency needed to be further improved. In this study, we identified the core promoter of TTF-1 (from -1299 bp to -871 bp) by 5' deletion assay and screened out the putative transcription factors nuclear factor-1 (NF-1) and activator protein-1 (AP-1). Further analysis revealed that the expression level of NF-1, but not AP-1, was positively connected with the activation of TTF-1 core promoter in human non-small-cell lung cancer (NSCLC) cells. Moreover, the silencing of NF-1 could reduce the expression level of miR-7 operated by TTF-1 core promoter. Of note, we optimized four distinct sequences to form additional NF-1-binding sites (TGGCA) in the sequence of TTF-1 core promoter (termed as ^optTTF-1 promoter), and verified the binding efficiency of NF-1 on the ^optTTF-1 promoter by electrophoretic mobility shift assay (EMSA). As expected, the ^optTTF-1 promoter could more effectively drive miR-7 expression and inhibit the growth of human NSCLC cells in vitro, accompanied by a reduced transduction of NADH dehydrogenase (ubiquinone) 1α subcomplex 4 (NDUFA4)/protein kinase B (Akt) pathway. Consistently, ^optTTF-1 promoter-driven miR-7 expression could also effectively abrogate the growth and metastasis of tumor cells in a murine xenograft model of human NSCLC. Finally, no significant changes were detected in the biological indicators or the histology of some important tissues and organs, including heart, liver, and spleen. On the whole, our study revealed that the optimized TTF-1 promoter could more effectively operate miR-7 to influence the growth of human NSCLC cells, providing a new basis for the development of microRNA-based targeting gene therapy against clinical lung cancer.
Animals ; Humans ; Mice ; Carcinoma, Non-Small-Cell Lung/therapy* ; Lung Neoplasms/metabolism* ; MicroRNAs/metabolism* ; Nuclear Proteins/metabolism* ; Thyroid Gland/pathology* ; Thyroid Nuclear Factor 1/genetics* ; Transcription Factors/metabolism*

OBJECTIVEData obtained from a differentially expressed cDNA library constructed previously in this laboratory demonstrated that the extracellular matrix molecule osteopontin (OPN) is one of most considerably over-expressed genes in non-small cell lung cancers (NSCLCs). The purpose of the present study was to explore the expression status of OPN in a large scale NSCLC tissue samples, and estimate its significance in progression of the malignant disease.

METHODSRT-PCR was performed with the tumor and adjacent normal tissues from 35 patients with NSCLC, at transcriptional levels of OPN. To determine the expression of OPN protein in the tumor tissues, immunohistochemical (IHC) staining was subsequently carried out on paraffin-embedded sections in tissue microarrays containing 662 samples derived from NSCLC cases. The correlation between the expression level of OPN and clinical characteristics was analyzed statistically.

RESULTSComparing with the paired normal lung tissue, high level RNA of OPN was detected in 80.0% (28/35) of the NSCLC tumor tissues by RT-PCR, which confirmed the information obtained previously by our differentially expressed cDNA library. The results of IHC analysis showed that positively stained OPN protein was observed in 59.6% (331/555) of the tumor tissues, which was remarkably higher than that (25.2%, 27/107) detected in the normal control tissues (P < 0.001). Among the NSCLCs investigated, over-expressed OPN was more frequently found in squamous cell carcinomas (SCCs) than in adenocarcinomas. A further analysis on SCCs demonstrated that the rate of over-expressed OPN was significantly different between the primary tumors with and without lymphatic metastases (68.6% vs. 49.7%, P = 0.001), but similar in the primary tumors and their corresponding metastases in lymph nodes (68.6% vs. 75.5%, P = 0.171).

CONCLUSIONExpression of OPN protein is distinctly increased in NSCLCs, particularly in SCCs. OPN over-expression is considerably correlated with lymph node metastasis, increasing the risk of tumor metastasis (OR = 2.212). The resulting data suggest that OPN facilitates the progression of NSCLCs.

Adenocarcinoma ; metabolism ; pathology ; Adult ; Aged ; Aged, 80 and over ; Carcinoma, Non-Small-Cell Lung ; metabolism ; pathology ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Lung Neoplasms ; metabolism ; pathology ; Lymphatic Metastasis ; Male ; Middle Aged ; Osteopontin ; genetics ; metabolism ; Up-Regulation

Lung squamous cell carcinoma (LSCC) accounts for approximately 30% of non-small cell lung cancer (NSCLC) cases and is the second most common histological type of lung cancer. Anaplastic lymphoma kinase (ALK)-positive NSCLC accounts for only 2%-5% of all NSCLC cases, and is almost exclusively detected in patients with lung adenocarcinoma. Thus, ALK testing is not routinely performed in the LSCC population, and the efficacy of such treatment for ALK-rearranged LSCC remains unknown. Echinoderm microtubule associated protein like 4 (EML4)-ALK (V1) and TP53 co-mutations were identified by next generation sequencing (NGS) in this patient with advanced LSCC. On December 3, 2020, Ensatinib was taken orally and the efficacy was evaluated as partial response (PR). The progression-free survival (PFS) was 19 months. When the disease progressed, the medication was changed to Loratinib. To our knowledge, Enshatinib created the longest PFS of ALK-mutant LSCC patients treated with targeted therapy since literature review. Herein, we described one case treated by Enshatinib involving a patient with both EML4-ALK and TP53 positive LSCC, and the relevant literatures were reviewed for discussing the treatment of this rare disease.
.
Humans ; Carcinoma, Non-Small-Cell Lung/drug therapy* ; Lung Neoplasms/pathology* ; Anaplastic Lymphoma Kinase/metabolism* ; Carcinoma, Squamous Cell/genetics* ; Mutation ; Cytoskeletal Proteins/genetics* ; Lung/pathology* ; Oncogene Proteins, Fusion/genetics* ; Protein Kinase Inhibitors/therapeutic use* ; Tumor Suppressor Protein p53/genetics*

BACKGROUND: Circular RNAs (circRNAs) are considered to be important regulators in cancer biology. In this study, we focused on the effect of circRNA baculoviral inhibitor of apoptosis protein (IAP) repeat containing 6 (circBIRC6) on non-small cell lung cancer (NSCLC) progression. METHODS: The NSCLC and adjacent non-tumor tissues were collected at Shanghai Ninth People's Hospital. Quantitative real-time polymerase chain reaction was conducted for assessing the levels of circBIRC6, amyloid beta precursor protein binding protein 2 (APPBP2) messenger RNA (mRNA), baculoviral IAP repeat containing 6 mRNA (BIRC6), and microRNA-217 (miR-217). Western blot assay was adopted for measuring the protein levels of APPBP2, E-cadherin, N-cadherin, and vimentin. Colony formation assay, transwell assay, and flow cytometry analysis were utilized for evaluating cell colony formation, metastasis, and apoptosis. Dualluciferase reporter assay and RNA immunoprecipitation assay were carried out to determine the interaction between miR-217 and circBIRC6 and APPBP2 in NSCLC tissues. The murine xenograft model assay was used to investigate the function of circBIRC6 in tumor formation in vivo. Differences were analyzed via Student's t test or one-way analysis of variance. Pearson's correlation coefficient analysis was used to analyze linear correlation. RESULTS: CircBIRC6 was overexpressed in NSCLC tissues and cells. Knockdown of circBIRC6 repressed the colony formation and metastasis and facilitated apoptosis of NSCLC cells in vitro and restrained tumorigenesis in vivo. Mechanically, circBIRC6 functioned as miR-217 sponge to promote APPBP2 expression in NSCLC cells. MiR-217 inhibition rescued circBIRC6 knockdown-mediated effects on NSCLC cell colony formation, metastasis, and apoptosis. Overexpression of miR-217 inhibited the malignant phenotypes of NSCLC cells, while the effects were abrogated by elevating APPBP2. CONCLUSIONS CircBIRC6 aggravated NSCLC cell progression by elevating APPBP2 via sponging miR-217, which might provide a fresh perspective on NSCLC therapy.
Amyloid beta-Peptides/metabolism* ; Amyloid beta-Protein Precursor/metabolism* ; Animals ; Carcinoma, Non-Small-Cell Lung/pathology* ; Cell Movement/genetics* ; Cell Proliferation/genetics* ; China ; Gene Expression Regulation, Neoplastic/genetics* ; Humans ; Lung Neoplasms/pathology* ; Mice ; MicroRNAs/metabolism* ; RNA, Circular/genetics* ; RNA, Messenger