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Yu X.-D.,Taizhou Municipal Hospital | Yang R.,Linzi District Huangcheng Town Health Center | Leng C.-J.,Linzi District Peoples Hospital
Computational Biology and Chemistry | Year: 2016

Human epidermal growth factor receptor (EGFR) plays a central role in the pathological progression and metastasis of lung cancer; the development and clinical application of therapeutic agents that target the receptor provide important insights for new lung cancer therapies. The tumor-suppressor protein MIG6 is a negative regulator of EGFR, which can bind at the activation interface of asymmetric dimer of EGFR kinase domains to disrupt dimerization and then inactivate the kinase (Zhang X. et al. Nature 2007, 450: 741-744). The protein adopts two separated segments, i.e. MIG6segment 1 and MIG6segment 2, to directly interact with EGFR. Here, computational modeling and analysis of the intermolecular interaction between EGFR kinase domain and MIG6segment 2 peptide revealed that the peptide is folded into a two-stranded β-sheet composed of β-strand 1 and β-strand 2; only the β-strand 2 can directly interact with EGFR activation loop, while leaving β-strand 1 apart from the kinase. A C-terminal island within the β-strand 2 is primarily responsible for peptide binding, which was truncated from the MIG6segment 2 and exhibited weak affinity to EGFR kinase domain. Structural and energetic analysis suggested that phosphorylation at residues Tyr394 and Tyr395 of truncated peptide can considerably improve EGFR affinity, and mutation of other residues can further optimize the peptide binding capability. Subsequently, three derivative versions of the truncated peptide, including phosphorylated and dephosphorylated peptides as well as a double-point mutant were synthesized and purified, and their affinities to the recombinant protein of human EGFR kinase domain were determined by fluorescence anisotropy titration. As expected theoretically, the dephosphorylated peptide has no observable binding to the kinase, and phosphorylation and mutation can confer low and moderate affinities to the peptide, respectively, suggesting a good consistence between the computational analysis and experimental assay. © 2016 Elsevier Ltd. All rights reserved.

Zhang X.-M.,Shandong University | Shan N.-N.,Shandong University | Sun M.,Linzi District Peoples Hospital | Wang X.,Shandong University | And 5 more authors.
International Immunopharmacology | Year: 2014

Background The T-cell immunoglobulin and mucin domain-(Tim)-1 molecule and Tim-3 are mainly expressed on activated T helper (Th) 2 and Th1 cells, respectively, and have been implicated in the pathogenesis of some autoimmune diseases. Immune thrombocytopenia (ITP) is a common autoimmune disorder, and the complex dysregulation of cellular immunity has been observed; however, the relationship between Tims and excessive immune responses in ITP remains unclear. Methods Using real-time quantitative polymerase chain reaction (RT-PCR), the mRNA expression levels of Tim-1, Tim-3, T-box transcription factor (T-bet) and GATA binding protein 3 (GATA-3) were measured in the peripheral blood mononuclear cells (PBMCs) of 45 newly diagnosed patients with active ITP, 34 ITP patients in remission and 31 healthy volunteers. Results Tim-3 mRNA expression in PBMCs in newly diagnosed patients was significantly decreased. At the same time, Tim-1 mRNA was not significantly declined, which resulted in a decreased ratio of Tim-3 to Tim-1 in ITP patients with active disease. During the remission stages, the levels of these transcription factors were comparable with those observed in healthy controls. Conclusions The reduced levels of Tim-3/Tim-1 in PBMCs during active stages of the disease suggest a possible role in the pathogenesis and course of ITP. Regulating the balance of Tim-1 and Tim-3 in ITP patients could also be a therapeutic approach against ITP. © 2013 Elsevier B.V.

Zhang S.-L.,Linzi District Peoples Hospital | Liu L.,Central South University
Experimental and Therapeutic Medicine | Year: 2015

microRNA (miR)-148a has been shown to act as an important suppressor in numerous human malignancies and is markedly downregulated in hepatocellular carcinoma; however, the role of miR-148a in the regulation of hepatocellular carcinoma cell invasion, as well as the underlying mechanism, has never been studied. In the present study, the expression level of miR-148a was found to be significantly decreased in hepatocellular carcinoma tissues and HepG2 cells when compared with that in the normal adjacent tissues. Furthermore, a novel target of miR-148a was found, sphingosine-1-phosphate receptor 1 (S1PR1), whose expression was negatively regulated by miR-148a at a post-transcriptional level in hepatocellular carcinoma HepG2 cells. Upregulation of miR-148a by transfection with miR-148a mimics notably suppressed HepG2 cell invasion, similar to the effect of the SIPR1 downregulation induced by SIPR1-specific small interfering RNA, while the restoration of S1PR1 expression reversed the inhibitory effect of miR-148a upregulation on HepG2 cell invasion. Accordingly, the current study suggests that miR-148a plays an inhibitory role in the regulation of hepatocellular carcinoma cell invasion by directly targeting S1PR1. © 2014, Spandidos Publications. All rights reserved.

Wang R.,Linzi District Peoples Hospital | Zhang P.,Qingdao Municipal Hospital | Li J.,Qingdao Municipal Hospital | Guan H.,Qingdao University | Shi G.,Qingdao Municipal Hospital
Biochemical and Biophysical Research Communications | Year: 2016

The hypoxia-inducible factor (HIF) is recognized as the master regulator of hypoxia response. HIF-α subunits expression are tightly regulated. In this study, our data show that ts20 cells still expressed detectable E1 protein even at 39.5°C for 12h, and complete depletion of E1 protein expression at 39.5°C by siRNA enhanced HIF-1α and P53 protein expression. Further inhibition of E1 at 39.5 °C by siRNA, or E1 inhibitor Ube1-41 completely blocked HIF-1α degradation. Moreover, immunoprecipitations of co-transfection of HA-ubiquitin and FLAG-HIF-1α plasmids directly confirmed the involvement of ubiquitin in the hypoxic degradation of HIF-1α. Additionally, hypoxic HIF-1 α degradation is independent of HAF, RACK1, sumoylation or nuclear/cytoplasmic localization. Taken together, our data suggest that constitutive HIF-1α protein degradation in hypoxia is absolutely ubiquitination-dependent, and unidentified E3 ligase may exist for this degradation pathway. © 2016 Elsevier Inc. All rights reserved.

Shang H.,Shandong University | Wang T.,Linzi District Peoples Hospital | Shang F.,Chinese Peoples Liberation Army | Huang K.-M.,General Hospital of Zibo | Li Y.-Q.,Shandong University
Molecular Medicine Reports | Year: 2014

MicroRNAs (miRNAs) are small non-coding RNAs that inhibit the expression of target protein-coding genes, most often at the post-transcriptional level. miRNAs are often found to be misregulated in human cancer and they can act as potent oncogenes or tumor suppressor genes. In this study, we found that a germline mutation in the miR-125a coding region is associated with human gastric cancer. This mutation reduced the expression of mature miR-125a and alleviated its inhibitory effect on erythroblastic leukemia viral oncogene homolog 2 (ERBB2) gene expression and on gastric tumor cell proliferation. Thus, the data of this study suggested that this germline mutation in pri-miR-125a likely contributes to the genetic predisposition to gastric cancer by reducing the production of miR-125a, thereby interfering with the expression of miR-125a target genes.

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