Ruyong Yao

MiR‐200b expression in breast cancer: a prognostic marker and act on cell proliferation and apoptosis by targeting Sp1

MicroRNAs (miRNAs) have been identified as important post‐transcriptional regulators involved in various biological and pathological processes of cells. In the present study, we investigated the roles and mechanisms of miR‐200b in human breast cancer (BC). MiR‐200b expression was carried out by qRT‐PCR in human BC cell lines and clinical samples and the prognostic potential of miR‐200b expression was further evaluated. In vitro, effects of miR‐200b on BC cell proliferation, apoptosis and cell cycle distribution were tested by CCK‐8 kit, flow cytometric analysis respectively. Luciferase assay and Western blot analysis were performed to validate the potential targets of miR‐200b after the preliminary screening by employing open access software. We found that miR‐200b was significantly down‐regulated in both BC tissues and cell lines. The low expression of miR‐200b was correlated with late TNM stage, negative oestrogen receptor and positive HER‐2 status. Multivariate analysis showed that miR‐200b expression was an independent prognostic predictor for BC patients. Integrated analysis identified Sp1 as a direct and functional target of miR‐200b. Knockdown of Sp1 inhibited cell proliferation, induce apoptosis and act on cell cycle resembling that of miR‐200b high expression. Our data demonstrates that miR‐200b has potential to serve as prognostic biomarker and tumour suppressor for BC patients. As a direct and functional target of miR‐200b, Sp1 and miR‐200b both could be an exciting target for BC treatment strategy.

FOXM1 mediates resistance to docetaxel in gastric cancer via up‐regulating Stathmin

Docetaxel is commonly used as an effective chemotherapeutic drug for gastric cancer patients recently. With the increasing emergence of docetaxel resistance nowadays, identification of suitable biomarkers for predicting chemosensitivity to docetaxel may be a key role for improving therapeutic effects for gastric cancer patients. In this study, we investigated the correlation between the expression of transcription factor forkhead box protein M1 (FOXM1) and chemotherapy response to docetaxel in gastric cancer, the possible mechanism for which was further explored. As a result, FOXM1 overexpression was shown to mediate resistance to docetaxel in gastric cancers. It altered microtubule dynamics to protect tumour cells from docetaxel‐induced apoptosis. Mechanistic investigations revealed that tubulin‐destabilizing protein Stathmin, which mediated docetaxel resistance in FOXM1‐silenced gastric cancer cells, is a direct down‐stream target of FOXM1, whereas another microtubule dynamics protein mitotic centromere–associated kinesin (MCAK), shown to be related to docetaxel resistance in gastric cancer cells, is not associated with FOXM1 expression significantly. These results were further provided by immunohistochemical analysis, indicating that FOXM1 and Stathmin expression levels were correlated in 103 post‐operational gastric cancer specimens. Moreover, when we attenuated FOXM1 expression with FOXM1 inhibitor thiostrepton, docetaxel resistance in gastric cancers was found to be reversed, simultaneously with the down‐regulation of FOXM1 and Stathmin. Therefore, FOXM1 can be a useful marker for predicting and monitoring docetaxel response. Through the inhibition of FOXM1, docetaxel resistance can be reversed, and thus FOXM1 could be a new therapeutic target in docetaxel‐resistant gastric cancer.

miR-141 confers docetaxel chemoresistance of breast cancer cells via regulation of EIF4E expression

Resistance to docetaxel, a chemotherapy drug for breast cancer (BC) treatment, occurs in ~50% of patients, and the underlying molecular mechanisms of drug resistance are not fully understood. Gene regulation through miR-141 has been proven to play an important role in cancer drug resistance. The present study investigated the role of miR-141 expression in BC cells of acquired docetaxel resistance. Inhibition of miR-141 enhanced the response to docetaxel in docetaxel-resistant cells (MCF-7/DTX and MDA-MB-231/DTX, respectively), whereas overexpression of miR-141 confered resistance in docetaxel-sensitive cells (MCF-7 and MDA-MB-231, respectively). By directly targeting the eukaryotic translation initiation factor 4E (EIF4E) mRNA, miR-141 acts on genes that are necessary for drug induced apoptosis rendering the cells drug resistant. Modulation of miR-141 expression was correlated with EIF4E expression changes and a direct interaction of miR-141 with EIF4E was shown by a luciferase assay. Thus, the present study is the first to show an increased expression of miR-141 in an acquired model of docetaxel resistance in BC. This serves as a mechanism of acquired docetaxel resistance in BC cells, possibly through direct interactions with EIF4E, therefore presenting a potential therapeutic target for the treatment of docetaxel resistant BC.

CUGBP1 promotes cell proliferation and suppresses apoptosis via down-regulating C/EBPα in human non-small cell lung cancers

CUGBP1, which is involved in posttranscriptional regulatory networks, may control cell growth, activation and differentiation. Meanwhile, CCAAT/enhancer-binding protein α (C/EBPα) acts as a basic leucine zipper transcription factor which controls differentiation-dependent gene expression and inhibits cell proliferation. To date, very little is known about the association between CUGBP 1 and C/EBPα in regulating cell proliferation and apoptosis in non-small cell lung cancer (NSCLC). CUGBP1 and C/EBPα mRNA expressions were analyzed in NSCLC tumor and adjacent normal tissues, and the relationship in clinicopathological parameters was evaluated. Knockdown of CUGBP1 and C/EBPα regulated by CUGBP1 in NSCLC cell line was identified by real-time PCR and Western blot. The effect of depletion of CUGBP1 was evaluated by MTT assay and Annexin/Propidium Iodide Apoptosis assay. CUGBP1 is highly expressed and expression of C/EBPα is low in NSCLC tissues. The correlation analysis revealed that there was negative correlation between the expression of CUGBP 1 and C/EBPα. Knockdown of CUGBP1 effectively silenced the expression of CUGBP1 and up-regulated C/EBPα. Also, suppression of CUGBP1 inhibits proliferation and induces apoptosis in A549 cells. These observations suggest that the first proof the overexpression of CUGBP1 in NSCLC contributes to tumorigenesis through down-regulation of C/EBPα. Knockdown of CUGBP1 or up-regulation C/EBPα might be a potential therapeutic approach for human non-small cell lung cancers.

MOR1 Expression in Gastric Cancer: A Biomarker Associated With Poor Outcome

At present, the expression of MOR1 and its function in gastric cancer remains unclear with evidence suggesting that it is to be involved in tumor progression and metastasis. The study was to assess the clinicopathologic relevance and prognostic value of MOR1 expression in gastric cancer.

Lentiviral vector-mediated doxycycline-inducible USP39 shRNA or cDNA expression in triple-negative breast cancer cells

Triple-negative breast cancer (TNBC), characterized by distinct biological and clinicopathological features, has a poor prognosis due to lack of effective therapeutic targets. Our previous data revealed that high levels of USP39 were selectively present in TNBC samples compared with their normal breast tissue samples and USP39 was also expressed at different levels in cultured TNBC cells and normal breast cells. Yet, the underlying cellular and molecular mechanisms of USP39 remain unclear. In the present study, we describe a doxycycline (DOX)-regulated lentiviral vector system expressing shRNA or cDNA of the USP39 gene in the TNBC cell line MDA-MB-231. USP39 expression was knocked down by the miR-30-based inducible lentiviral short hairpin RNA (shRNA) delivery system or overexpressed by the inducible cDNA system. The inducible shRNA-mediated downregulation of USP39 expression markedly reduced the proliferation and colony-forming ability of MDA-MB-231 cells, while overexpression of USP39 by the inducible system did not promote cancer cell proliferation. The lentiviral vector-mediated Tet-on system demonstrated efficient and inducible knockdown of USP39 or overexpression of USP39 in TNBC cells, facilitating a wide variety of applications for gene knockdown and overexpression experiments in gene functional studies in vitro and in vivo.

Overexpression of Her-2 upregulates FoxM1 in gastric cancer

The transcription factor, forkhead box protein M1 (FoxM1), and the tyrosine kinase receptor, human epidermal growth factor receptor-2 (Her-2), are aberrantly expressed in a number of human malignancies, and are closely associated with the development of cancer. However, their regulatory mechanisms and their involvement in tumor development have not been extensively investigated, particularly in gastric cancer. In the present study, we examined the expression levels of FoxM1 and Her-2 in samples from patients with gastric cancer, as well as gastric cancer cell lines. Their regulatory mechanisms and the connection between them were also explored. We found that FoxM1 and Her-2 expression was markedly higher in the gastric cancer samples, while a strong association was found between them at the mRNA and protein level. When we regulated the levels of Her-2 in the gastric cancer cell lines, the expression of FoxM1 changed accordingly. Following the transfection of Her-2 expression vectors into the gastric cell lines, the luciferase activity of the FoxM1 promoter positively increased along with the concentration of pcDNA3.1-Her-2. However, Her-2-siRNA inverted this variation, which was further confirmed by treatment with the Her-2 inhibitor, trastuzumab, revealing that Her-2 regulates FoxM1 expression at the promoter level in gastric cancer cells. Our results suggest that FoxM1 and Her-2 are important diagnostic markers for gastric cancer. FoxM1 may be a potential cellular target for therapeutic intervention, particularly in gastric cancers resistant to Her-2-targeted therapy.

Construction and Expression of an Eukaryotic Expression Vector Containing the IER3 Gene

BACKGROUND More and more research indicate that the immediately early response gene 3 (IER3) is involved in many biological processes, such as apoptosis and immunoreaction, as well as viral infection, tumorigenesis and tumour progression. METHODS Here we describe the construction of an eukaryotic expression vector containing IER3 gene and its expression in A549 cells as assessed through fluorescence microscopyand Western- blotting. RESULTS Fluorescence detection displayed that GFP in cytoplasm was high during 48 and 72 hours post-transfection. In addition, Western blotting showed significant increase in IER3 gene expression in the transfected cells compared with controls. CONCLUSION The recombinate plasmid expression vector was constructed successfully, which may provide a basis for further exploration of function of IER3 in lung cancer.