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Shaheen Khan, Fei Wu, Shengxi Liu, Qian Wu, and Stephen Safe

Abstract

Deletion analysis of several 17β-estradiol (E2)-responsive genes have identified GC-rich sites that are associated with hormone-induced transactivation in MCF-7 breast cancer cells. However, the role of individual specificity proteins (Sps) in mediating hormone-induced gene expression has not been unequivocally determined. In transient transfection studies using E2-responsive GC-rich promoters from the E2F1, carbamoylphosphate synthetase/aspartate transcarbamylase/dihydroorotase (CAD), and retinoic acid receptor α (RARα) genes, RNA interference using small inhibitory RNAs for Sp1 (iSp1), Sp3 (iSp3), and Sp4 (iSp4) decreased both basal and E2-induced transactivation. The contributions of individual Sp proteins to basal and E2-induced activity were promoter dependent. iSp1, iSp3, and iSp4 also significantly inhibited hormonal induction of E2F1, CAD, and RARα mRNA levels; however, the enhanced inhibitory effects of the latter two small inhibitory RNAs suggest that Sp3 and Sp4 play a major role in estrogen receptor α/Sp-mediated gene expression in MCF-7 cells.

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Qun Cheng, Weipin Dong, Lei Qian, Jingcheng Wu, and Yongde Peng

Visfatin is an adipocytokine that plays an important role in attenuating insulin resistance by binding to insulin receptor. It has been suggested that visfatin plays a role in the regulation of cell apoptosis and inflammation by an as yet unidentified mechanism. This study investigated the protective effects of visfatin on palmitate-induced islet β-cell apoptosis in the clonal mouse pancreatic β-cell line MIN6. The cells were treated with palmitate and/or recombinant visfatin. An 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan assay was used to detect cell proliferation, V-FITC/propidium iodide staining was used to measure cell apoptosis and necrosis, and western blot analysis was used to detect the expression of proapoptotic proteins. The incubation of the cells with visfatin led to a concentration-dependent increase of cell proliferation (1.55-fold at 10−7 M and 24 h compared with control, P<0.05). Visfatin significantly reduced the cell apoptosis induced by palmitate and caused a significant change in the expression of several proapoptotic proteins, including upregulation of Bcl-2 and a marked downregulation of cytochrome c and caspase 3. Visfatin also activated the ERK1/2 and the phosphoinositide 3-kinase (PI3K)/AKT signaling pathways in a time- and concentration-dependent manner, and the effect of visfatin on apoptosis was blocked by the specific ERK1/2 and PI3K/AKT inhibitors, PD098059 and LY294002. We conclude that visfatin can increase β-cell proliferation and prevent apoptosis, activate intracellular signaling, and regulate the expression of proapoptotic proteins. The antiapoptotic action of visfatin is mediated by activation of mitogen-activated protein kinase-dependent and PI3K-dependent signaling pathways.

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Jing Lu, Cheng Cheng, Zhen-chao Cheng, Qian Wu, Han Shen, Ming-xia Yuan, Bo Zhang, and Jin-Kui Yang

RFX6 transcription factor is believed to play a central role in directing cell development of insulin-producing pancreatic islet. RFX6 homozygous mutations cause syndromic neonatal diabetes with hypoplastic pancreas. However, RFX6 heterozygous mutations cause maturity-onset diabetes of the young (MODY) with normal pancreas development. Here, we show that RFX6 may control islet cell development and insulin production in different manners. The rfx6 knockout zebrafish generated by CRISPR/Cas9 exhibited an overt diabetes phenotype. Pancreatic islet failed to form compact structures in the knockout fish. While endocrine pancreatic islet non-β-cells were absent, insulin-producing β-cells were present in the knockout fish. Although insulin mRNA level was normal in the β-cells of the knockout fish, insulin protein level was decreased. High-throughput RNA sequencing (RNAseq) showed that differentially expressed genes were enriched in the translation term in islet β-cells from the knockout fish. Chromatin immunoprecipitation sequencing (ChIPseq) of normally developed islet β-cells from mice demonstrated that rfx6 interacted with translation initiation factors and controlled insulin translation. Our data indicate that Rfx6 may act as a transcription factor regulating the transcription of genes involved in mRNA translation, which may represent a new mechanism and treatment strategy for diseases.

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Ting Qi, Yanming Chen, Honggui Li, Ya Pei, Shih-Lung Woo, Xin Guo, Jiajia Zhao, Xiaoxian Qian, Joseph Awika, Yuqing Huo, and Chaodong Wu

Metformin improves obesity-associated metabolic dysregulation, but has controversial effects on adipose tissue inflammation. The objective of the study is to examine the direct effect of metformin on adipocyte inflammatory responses and elucidate the underlying mechanisms. Adipocytes were differentiated from 3T3-L1 cells and treated with metformin at various doses and for different time periods. The treated cells were examined for the proinflammatory responses, as well as the phosphorylation states of AMPK and the expression of PFKFB3/iPFK2. In addition, PFKFB3/iPFK2-knockdown adipocytes were treated with metformin and examined for changes in the proinflammatory responses. The following results were obtained from the study. Treatment of adipocytes with metformin decreased the effects of lipopolysaccharide on inducing the phosphorylation states of JNK p46 and on increasing the mRNA levels of IL-1β and TNFα. In addition, treatment with metformin increased the expression of PFKFB3/iPFK2, but failed to significantly alter the phosphorylation states of AMPK. In PFKFB3/iPFK2-knockdown adipocytes, treatment with metformin did not suppress the proinflammatory responses as did it in control adipocytes. In conclusion, metformin has a direct effect on suppressing adipocyte proinflammatory responses in an AMPK-independent manner. Also, metformin increases adipocyte expression of PFKFB3/iPFK2, which is involved in the anti-inflammatory effect of metformin.