PURPOSE: The purpose of this study was to investigate the biological role and mechanism of miR-373 targeting of TFIIB-related factor 2 (BRF2) in the regulation of non-small cell lung cancer (NSCLC) cells. MATERIALS AND METHODS: miRNA microarray chip analysis of four paired NSCLC and adjacent non-tumor tissues was performed. Quantitative real-time polymerase chain reaction (qRT-PCR) andwestern blotting were used to detect the expression levels of miR-373 and BRF2 in NSCLC tissues and cell lines. The dual-luciferase reporter method was performed to determine if BRF2 is a target of miR-373. MTT, wound-healing, Transwell, and flow cytometric assays were conducted to examine the proliferation, migration, invasion, and cell cycle progression of NSCLC A549 cells, respectively; western blotting was used to detect the expression of epithelial-mesenchymal transition (EMT)–related proteins. RESULTS: The miRNA microarray chip analysis demonstrated that miR-373 was down-regulated in NSCLC tissues, and this result was confirmed by qRT-PCR. Additionally, miR-373 was confirmed to target BRF2. Moreover, miR-373 expression was inversely correlated with BRF2 expression in NSCLC tissues and cell lines; both miR-373 down-regulation and BRF2 up-regulation were strongly associated with the clinicopathological features and prognosis of NSCLC patients. In vitro, overexpression of miR-373 markedly inhibited cell proliferation, migration, and invasion; up-regulated the expression of E-cadherin; and down-regulated the expression of N-cadherin and Snail in A549 cell. Knockdown BRF2 by siRNA resulted in effects similar to those caused by overexpression of miR-373. CONCLUSION: MiR-373 is decreased in NSCLC, and overexpression of miR-373 can suppress cell EMT, and inhibit the proliferation, migration, and invasion of NSCLC A549 cells by targeting BRF2.
Blotting, Western ; Cadherins ; Carcinoma, Non-Small-Cell Lung* ; Cell Cycle ; Cell Line* ; Cell Migration Inhibition ; Cell Proliferation* ; Down-Regulation ; Epithelial-Mesenchymal Transition ; Humans* ; In Vitro Techniques ; Methods ; MicroRNAs ; Prognosis ; Real-Time Polymerase Chain Reaction ; RNA, Small Interfering ; Snails ; Up-Regulation

Gambogic acid (GA) is an anticancer agent in phase ‖b clinical trial in China but its mechanism of action has not been fully clarified. The present study was designed to search the possible target-related proteins of GA in cancer cells using proteomic method and establish possible network using bioinformatic analysis. Cytotoxicity and anti-migration effects of GA in MDA-MB-231 cells were checked using MTT assay, flow cytometry, wound migration assay, and chamber migration assay. Possible target-related proteins of GA at early (3 h) and late stage (24 h) of treatment were searched using a proteomic technology, two-dimensional electrophoresis (2-DE). The possible network of GA was established using bioinformatic analysis. The intracellular expression levels of vimentin, keratin 18, and calumenin were determined using Western blotting. GA inhibited cell proliferation and induced cell cycle arrest at G2/M phase and apoptosis in MDA-MB-231 cells. Additionally, GA exhibited anti-migration effects at non-toxic doses. In 2-DE analysis, totally 23 possible GA targeted proteins were found, including those with functions in cytoskeleton and transport, regulation of redox state, metabolism, ubiquitin-proteasome system, transcription and translation, protein transport and modification, and cytokine. Network analysis of these proteins suggested that cytoskeleton-related proteins might play important roles in the effects of GA. Results of Western blotting confirmed the cleavage of vimentin, increase in keratin 18, and decrease in calumenin levels in GA-treated cells. In summary, GA is a multi-target compound and its anti-cancer effects may be based on several target-related proteins such as cytoskeleton-related proteins.
Antineoplastic Agents ; pharmacokinetics ; Apoptosis ; drug effects ; Breast Neoplasms ; drug therapy ; metabolism ; Calcium-Binding Proteins ; genetics ; Cell Line, Tumor ; Cell Migration Assays ; Cell Migration Inhibition ; drug effects ; Cell Proliferation ; drug effects ; Computational Biology ; methods ; Cytoskeleton ; metabolism ; Electrophoresis, Gel, Two-Dimensional ; Flow Cytometry ; Gene Expression ; Humans ; Keratin-18 ; genetics ; Oxidation-Reduction ; Protein Biosynthesis ; drug effects ; Protein Transport ; Proteomics ; methods ; Transcription, Genetic ; drug effects ; Ubiquitin-Specific Proteases ; pharmacokinetics ; Vimentin ; genetics ; Xanthones ; pharmacokinetics

Understanding of the pathophysiology of inflammatory bowel disease (IBD) is constantly evolving and, recently, a number of biologic agents have been developed. They selectively target specific molecule or pathways and correct the imbalance of the gut immune system. Among them, antibody to tumor necrosis factor (anti-TNF-alpha) is the first developed drugs, and it dramatically improved the IBD management. However, more than one-third of the patients do not respond to the drugs due to antibody formation. To increase treatment efficacy, enormous effort to develop novel anti-cytokines which can be an alternative to anti-TNF-alpha has been made. They are anti CD4+ T cell cytokine including interleukin (IL)-12/23 and IL-17 blockers, selective anti-adhesion molecule known as natalizumab, vedolizumab and alicaforsen, T-cell proliferation inhibitor, anti-inflammatory cytokine, immune stimulator, growth factor, and mitogen-activated protein kinase inhibitor. The efficacy and safety of each drugs are under investigation. Some drugs reported very promising data, however, others showed disappointing and different results. In addition, most of the trials were done in a very small number of patients, and there is no trial comparing to anti-TNF-alpha. The present paper reviews the action mechanism, short or long term efficacy and safety of variable drugs other than anti-TNF-alpha in IBD.
Antibodies, Monoclonal/therapeutic use ; Cell Migration Inhibition ; Colony-Stimulating Factors/therapeutic use ; Cytokines/antagonists & inhibitors ; Gene Therapy ; Humans ; Immunosuppressive Agents/therapeutic use ; Inflammatory Bowel Diseases/drug therapy/*therapy ; Intercellular Signaling Peptides and Proteins/therapeutic use ; Mitogen-Activated Protein Kinases/antagonists & inhibitors ; Stem Cell Transplantation ; Tumor Necrosis Factor-alpha/*antagonists & inhibitors/immunology