ABSTRACT
Since the discovery of microRNAs (miRNAs) as a class of important regulatory molecules, miRNAs are involved in the occurrence and development of tumors. In this paper, we aimed to identify the role of miR-1274a in non-small cell lung cancer (NSCLC). The miR-1274a expression levels in four NSCLC cells and tissues from 125 patients were determined by qRT-PCR assays. Kaplan-Meier survival curves and Cox regression analysis were used to examine the prognostic significance of miR-1274a in NSCLC patients. The CCK-8 and Transwell assays were performed to evaluate the cell proliferation, invasion, and migration ability of NSCLC cells. The miR-1274a expression levels were significantly higher in NSCLC tissues than in adjacent normal tissues, and overexpression of miR-1274a had a poor prognosis in NSCLC patients. Functional studies in two NSCLC cell lines have shown that overexpression of miR-1274a could promote cell proliferation, migration, and invasion. miR-1274a expression levels are upregulated in NSCLC tissues, and a high expression is associated with a poor prognosis in patients with NSCLC. Moreover, miR-1274a promotes cell proliferation, migration, and invasion. Based on our findings, miR-1274a may act as a tumor miRNA in the occurrence and development of NSCLC.
Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , Lung Neoplasms/genetics , MicroRNAs/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Female , Humans , Lung Neoplasms/pathology , Male , Middle Aged , Prognosis , Up-Regulation/geneticsABSTRACT
Lung adenocarcinoma (LUAD) is the primary epithelial tumor of the lung. The lack of clinical symptoms and specific molecular diagnostic indicators during the early stages of LUAD mean that the disease may not be detected until late stages, and the 5-year survival rate is only approximately 15%. Long non-coding RNA ALMS1 intronic script 1 (ALMS1-IT1) was previously reported to be correlated with the poor prognosis of head and neck squamous cell carcinoma patients. Here, we investigated whether ALMS1-IT1 has prognostic potential for LUAD. Bioinformatics analyses were performed to examine the expression and prognostic value of ALMS1 and AVL9 (for which gene expression is positively correlated with ALMS1-IT1 expression in LUAD) in LUAD based on TCGA and Oncomine databases. We report that ALMS1-IT1 and AVL9 were both highly expressed in LUAD and correlated with poor outcomes in LUAD patients. Of note, the prognosis of LUAD patients with low expression of both ALMS1-IT1 and AVL9 was superior to that of other patients. Furthermore, the proliferation, migration and invasion of LUAD cells were decreased in cells lacking ALMS1-IT1, and this decrease could be almost completely reversed through overexpression of AVL9. Gene set enrichment analysis revealed that expression of genes related to the cell cycle pathway is closely related to both the high expression of ALMS1-IT1 and AVL9 in LUAD. Finally, up-regulation of ALMS1-IT1 can activate the cyclin-dependent kinase pathway, whereas absence of AVL9 can reverse this activation, as shown by western blotting. In summary, ALMS1-IT1/AVL9 may promote the malignant progression of LUAD, at least in part by regulating the cyclin-dependent kinase pathway.
Subject(s)
Adenocarcinoma of Lung/genetics , Cell Cycle Proteins/genetics , Adenocarcinoma/genetics , Adenocarcinoma of Lung/metabolism , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , China , Cyclin-Dependent Kinases/metabolism , Databases, Genetic , Humans , Introns/genetics , Lung/pathology , Lung Neoplasms/genetics , Prognosis , RNA Interference , RNA, Long Noncoding/genetics , Signal Transduction/genetics , Vesicular Transport Proteins/metabolismABSTRACT
This study investigated the expression of nucleolin in tissue samples in patients with non-small cell lung cancer (NSCLC). Nucleolin was studied to determine whether it has a prognostic value and if its levels correlate with various clinicopathologic parameters. The relationship between nucleolin and expression of DNA-PKcs was also evaluated. Immunohistochemistry was used for detecting the expression levels of nucleolin and DNA-PKcs in tissues from 225 stage IA to IIIB NSCLC patients who underwent lung surgery. Nucleolin was observed predominantly in the cytoplasm, and some levels were observed in the nucleus. Nucleolin expression was higher in NSCLC tissues than adjacent normal lung tissues. Among 225 NSCLC patients, 117 (52.0 %) had high expression of nucleolin. The expression of nucleolin was significantly associated with pathologic stage (P = 0.013) and T status (P = 0.043). Multivariate analysis revealed that nucleolin, cytoplasmic nucleolin, and nuclear nucleolin expression were independent prognostic factors for both overall survival (OS) (P < 0.001) and disease-free survival (DFS) (P < 0.001). A high level of nuclear nucleolin served as an independent prognostic factor for better survival, while a high level of cytoplasmic nucleolin was closely associated with worse prognosis in NSCLC patients. The expression of nucleolin and cytoplasmic nucleolin positively correlated with DNA-PKcs (P < 0.001). These data suggest that nucleolin could be an effective treatment target and prognostic factor for patients with NSCLC.
Subject(s)
Carcinoma, Non-Small-Cell Lung/pathology , DNA-Activated Protein Kinase/metabolism , Lung Neoplasms/pathology , Nuclear Proteins/metabolism , Phosphoproteins/biosynthesis , RNA-Binding Proteins/biosynthesis , Adult , Aged , Aged, 80 and over , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/mortality , Cell Nucleus/metabolism , Cytoplasm/metabolism , Disease-Free Survival , Female , Humans , Immunohistochemistry , Kaplan-Meier Estimate , Lung Neoplasms/metabolism , Lung Neoplasms/mortality , Male , Middle Aged , Prognosis , Retrospective Studies , NucleolinABSTRACT
Nucleolin (C23) is an important anti-apoptotic protein that is ubiquitously expressed in exponentially growing eukaryotic cells. In order to understand the impact of C23 in radiation therapy, we attempted to investigate the relationship of C23 expression with the radiosensitivity of human non-small cell lung cancer (NSCLC) cells. We investigated the role of C23 in activating the catalytic subunit of DNA-dependent protein kinase (DNA- PKcs), which is a critical protein for DNA double-strand breaks (DSBs) repair. As a result, we found that the expression of C23 was negatively correlated with the radiosensitivity of NSCLC cell lines. In vitro clonogenic survival assays revealed that C23 knockdown increased the radiosensitivity of a human lung adenocarcinoma cell line, potentially through the promotion of radiation-induced apoptosis and adjusting the cell cycle to a more radiosensitive stage. Immunofluorescence data revealed an increasing quantity of γ-H2AX foci and decreasing radiation-induced DNA damage repair following knockdown of C23. To further clarify the mechanism of C23 in DNA DSBs repair, we detected the expression of DNA-PKcs and C23 proteins in NSCLC cell lines. C23 might participate in DNA DSBs repair for the reason that the expression of DNA-PKcs decreased at 30, 60, 120 and 360 minutes after irradiation in C23 knockdown cells. Especially, the activity of DNA-PKcs phosphorylation sites at the S2056 and T2609 was significantly suppressed. Therefore we concluded that C23 knockdown can inhibit DNA-PKcs phosphorylation activity at the S2056 and T2609 sites, thus reducing the radiation damage repair and increasing the radiosensitivity of NSCLC cells. Taken together, the inhibition of C23 expression was shown to increase the radiosensitivity of NSCLC cells, as implied by the relevance to the notably decreased DNA-PKcs phosphorylation activity at the S2056 and T2609 clusters. Further research on targeted C23 treatment may promote effectiveness of radiotherapy and provide new targets for NSCLC patients.
Subject(s)
Adenocarcinoma/genetics , Carcinoma, Large Cell/genetics , Lung Neoplasms/genetics , Phosphoproteins/genetics , RNA-Binding Proteins/genetics , Radiation Tolerance/genetics , Apoptosis/genetics , Apoptosis/radiation effects , Carcinoma, Non-Small-Cell Lung/genetics , Cell Line, Tumor , Cell Proliferation/genetics , Cell Proliferation/radiation effects , Cell Survival/genetics , Cell Survival/radiation effects , DNA Breaks, Double-Stranded/radiation effects , DNA Repair/genetics , DNA Repair/radiation effects , DNA-Activated Protein Kinase , Gene Knockdown Techniques , Humans , Nuclear Proteins , Phosphorylation , RNA Interference , NucleolinABSTRACT
BACKGROUND: Nimotuzumab is a humanized IgG1 monoclonal antibody specifically targeting EGFR. In this study, we aimed to investigate the molecular mechanisms of nimotuzumab in its effects of enhancing cancer cell radiosensitivity. PRINCIPAL FINDING: Lung cancer A549 cells and breast cancer MCF-7 cells were pretreated with or without nimotuzumab for 24 h before radiation to perform the clonogenic survival assay and to analyze the cell apoptosis by flow ctyometry. γ-H2AX foci were detected by confocal microscopy to assess the effect of nimotuzumab on radiation induced DNA repair. EGFR activation was examined and the levels of DNA damage repair related proteins in A549 cells at different time point and at varying doses exposure after nimotuzumab and radiation treatment were examined by Western blot. Pretreatment with nimotuzumab reduced clonogenic survival after radiation, inhibited radiation-induced EGFR activation and increased the radiation-induced apoptosis in both A549 cells and MCF-7 cells. The foci of γ-H2AX 24 h after radiation significantly increased in nimotuzumab pretreated cells with different doses. The phosphorylation of AKT and DNA-PKcs were remarkably inhibited in the combination group at each dose point as well as time point. CONCLUSIONS: Our results revealed that the possible mechanism of nimotuzumab enhancing the cancer radiosensitivity is that nimotuzumab inhibited the radiation-induced activation of DNA-PKcs through blocking the PI3K/AKT pathway, which ultimately affected the DNA DSBs repair.