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Wu H.,Nanjing Medical University | Huang M.,Nanjing Medical University | Lu M.,Nanjing Medical University | Zhu W.,Nanjing Medical University | And 3 more authors.
Cancer Chemotherapy and Pharmacology | Year: 2013

Background: Human miR-34c has been reported to be associated with various human malignancies; however, it remains unknown whether miR-34c is involved in chemoresistance in gastric cancer. The aim of this study was to investigate the role of miR-34c in gastric cancer. Materials and methods: The adenosine triphosphate-based tumor chemosensitivity assay was used to measure drug sensitivity in gastric cancer samples. The expression levels of miRNA were determined by reverse transcriptase polymerase chain reaction (PCR) and those of protein were by Western blot analysis. Luciferase activity assay was used to verify the target genes of miRNAs. MTT assay was used to test the drug-resistant phenotype changes in cancer cells via overregulation of miRNAs. The methylation status of neighboring CpG islands of miR-34c-5p was analyzed by Bisulfite Sequencing PCR and methylation-specific PCR. Results: Quantitative real-time polymerase chain reaction demonstrated that expression of miR-34c-5p was downregulated in paclitaxel-resistant gastric cancer samples (p < 0.01). Cells derived from gastric cancer tissues with low miR-34c-5p expression and high microtubule-associated protein tau (MAPT) protein expression tended to have increased chemoresistance to paclitaxel in vitro. Luciferase activity assay confirmed that the 3′-UTR of MAPT mRNA contains a functional miR-34c-5p binding site. Overexpression of miR-34c-5p significantly downregulated MAPT protein expression and increased the chemosensitivity of paclitaxel-resistant gastric cancer cells. Further investigation demonstrated that differential methylation of CpG islands neighboring the miR-34c promoter regulated the expression of miR-34c-5p in gastric cancer cell lines. Conclusions: DNA methylation, dysregulation of miR-34c-5p, and MAPT expression are critical factors in the chemoresistance of gastric cancer to paclitaxel. © 2013 Springer-Verlag Berlin Heidelberg.

To prevent protein aggregation, some proteins are usually expressed as fusion proteins from which target proteins can be released by proteolytic or chemical reagents. In this report, small ubiquitin-related modifier (SUMO) linked with a hexa-histidine tag was used as a fusion partner for the antitumor-analgesic peptide from the venom of Buthus martensii (Karsch) scorpion (AGAP). The optimal expression level of the soluble fusion protein, SUMO-AGAP, was up to 40% of the total cellular protein. The fusion protein was purified by Ni-NTA affinity chromatography and cleaved by a SUMO-specific protease (Ulp1) to obtain the recombinant AGAP (rAGAP), which was further purified by Ni-NTA affinity chromatography. The purified final product was >95% pure by SDS-PAGE stained with Coomassie brilliant blue R-250. Mass spectroscopic analysis indicated the protein to be 7142.63 Dalton, which equaled the theoretically expected mass. N-terminal sequencing of rAGAP showed the sequence corresponded to the native protein. MTT assay indicated the rAGAP could significantly inhibit the proliferation of Jurkat and Hut 78 T lymphoma cell lines. The further writhing experiment showed that the rAGAP had an intensive analgesic effect. The expression strategy presented in this study allows convenient high yield and easy purification of the rAGAP with native sequences. © 2010 American Institute of Chemical Engineers

Wu H.,Nanjing Medical University | Huang M.,Nanjing Medical University | Cao P.,Jiangsu Province Institute of Traditional Chinese Medicine | Wang T.,Nanjing Medical University | And 2 more authors.
Cancer Biology and Therapy | Year: 2012

The role of tumor suppressors and cell cycle factors in gastric carcinogenesis are well understood; however, the posttranscriptional changes that affect gene expression in gastric cancer are poorly characterized. MiR-135a has been shown to play a role in Hodgkin lymphoma. The aim of this study was to investigate the expression and role of miR-135a in gastric cancer. Quantitative real-time PCR demonstrated that miR-135a expression is downregulated in the majority of human primary gastric cancer tissues (8/11; 73%), compared with pair-matched adjacent non-tumor tissues. Furthermore, compared with the nonmalignant gastric cell line, GES -1, miR-135a expression was substantially downregulated in gastric cancer cell lines of various degrees of differentiation. Target analysis indicated miR-135a directly regulates Janus kinase 2 (JAK2), a cytoplasmic tyrosine kinase involved in cytokine receptor signaling pathways. Overexpression of miR-135a significantly downregulated the expression of JAK2 protein and also reduced gastric cancer cell proliferation and colony formation in vitro. MiR-135a-mediated JAK2 downregulation also reduced p-STAT3 activation and cyclin D1 and Bcl-x Lprotein expression. This study suggests that miR-135a may function as a tumor suppressor via targeting JAK to repress p-STAT3 activation, reduce cyclin D1 and Bcl-x L expression and inhibit gastric cancer cell proliferation. These results imply that novel treatment approaches targeting miR-135a may potentially benefit patients with gastric cancer. © 2012 Landes Bioscience.

Fan H.,Tongji University | Cao P.,Jiangsu Province Institute of Traditional Chinese Medicine | Game D.S.,Kings College London | Dazzi F.,Imperial College London | And 3 more authors.
Seminars in Immunology | Year: 2011

The pursuit of transplantation tolerance is the holygrail in clinical organ transplantation. It has been established that regulatory T cells (Tregs) can confer donor-specific tolerance in mouse models of transplantation. However, this is crucially dependent on the strain combination, the organ transplanted and most importantly, the ratio of Tregs to alloreactive effector T cells. The ex vivo expansion of Tregs is one solution to increase the number of alloantigen specific cells capable of suppressing the alloresponse. Indeed, ex vivo expanded, alloantigen specific murine Tregs are shown to preferentially migrate to, and proliferate in, the graft and draining lymph node. In human transplantation it has been proposed that depletion of the majority of direct pathway alloreactive T cells will be required to tip the balance in favour of regulation. Ex vivo expansion of alloantigen specific, indirect pathway human Tregs, which can cross regulate the residual direct pathway has been established. Rapid expansion of these cells is possible, whilst they retain antigen specificity, suppressive properties and favourable homing markers. Furthermore, considerable progress has been made to define which immunosuppressive drugs favour the expansion and function of Tregs. Currently a series of clinical trials of adoptive Treg therapy in combination with depletion of alloreactive T cells and short term immunosuppression are underway for human transplantation with the aim of minimizing immunosuppressive drugs and completely withdrawal. © 2011 Elsevier Ltd.

Zhou X.,State Key Laboratory of Coordination Chemistry | Cao P.,Jiangsu Province Institute of Traditional Chinese Medicine | Tian Y.,State Key Laboratory of Coordination Chemistry | Zhu J.,State Key Laboratory of Coordination Chemistry
Journal of the American Chemical Society | Year: 2010

Innovation in molecular diagnostics ultimately requires the conceptually distinct design of detection architectures. The diagnostic strategies reported thus far (planar/suspension arrays) suffer from either mass transport issues or limitations on the maximum number of targets that can be simultaneously detected. We report herein an expressed peptide assay scheme, by using nanoparticle probes, for detecting DNA hybridization events. The method exploits plasmid-encoded peptide tags as surrogate molecules for the matrix-assisted laser desorptlon/lonization time-of-flight mass spectrometry identification of target DNA. The binding of target DNA Is achieved through its recognition with a gold nanoparticle probe (functlonallzed with peptide-encoding plasmid and oligonucleotide complementary to part of the target sequence) and a microparticle probe (derivatized with oligonucieotide complementary to the rest of the target sequence). The magnetic separation of the three-component complex and expression of the peptide allows for the target identification by mass spectrometry. The detection of two DNA targets has been demonstrated through the selection of each individual tag for the respective target. Importantly, the modular nature of the probe design, by decoupling molecular binding events from peptide expression processes, should enable the ready extension of the methodology to the analysis of other species. An assay on a protein target has confirmed the efficacy of the conceptual framework proposed herein beyond the detection of DNA. The vast choice of mass tags offered by mass spectrometry provides significant advantages over previously documented assay systems. © 2010 American Chemical Society.

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