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Background: Previous investigation has demonstrated that CD4+ T cells play a crucial role in effective immunity against Helicobacter pylori (H.pylori) infection. It has been well proved that Lpp20 is one of major protective antigens that induce immune responses after H.pylori invades host. Therefore it is valuable to identify CD4+ T cell epitopes on Lpp20, which is uncharacterized. Methods: Putative epitopes of H-2d restricted CD4+ T cell on Lpp20 of H.pylori were predicted by the SYFPEITHI algorithm and then eight hypothetical epitope peptides were synthesized. After BALB/c mice were primed with recombinant Lpp20, splenic CD4+ T cells were isolated and stimulated with synthesized peptides to measure T cell proliferation and MHC restriction. Cytokine profile was determined by ELISA and real-time PCR. Two identified epitopes were used to immunize mice to investigate CD4+ T cell response by flow cytometry. Results: Two of eight peptides were able to stimulate CD4+ T cell proliferation and were mapped to residues 83-97aa and 58-72aa on Lpp20 respectively. These two peptides additively stimulated Th1 cells to secrete IFN-γ. The percentage of CD4+ T cell from mice immunized with two identified epitopes respectively was higher than the control group. Conclusion: The identification and characterization of two CD4+ T cell epitopes of Lpp20 helps understand the protective immunity of Lpp20 in H.pylori infection and design effective epitope vaccines against H.pylori. [ABSTRACT FROM AUTHOR]

Background: The potential mechanisms regarding how methylation of microRNA(miRNA) CpG Island could regulate cancer cell chemo-resistance remains unclear. This study aims to explore the epigenetic dysregulation mechanism of miRNA-493 and the ability to modulate lung cancer cell chemotherapy resistance., Methods: Real-time quantitative PCR (qRT-PCR) and In situ hybridization (ISH) were used to analyze the expression of miR-493 in lung cancer cell lines and tumor tissue, respectively. Bisulfite sequencing PCR (BSP) was used to exam the promoter CpG Island of miR-493. The effect of miR-493 on chemosensitivity was evaluated by cell viability assays, apoptosis assays and in vivo experiment. The DNA damage was measured by γ-H2AX immunofluorescence. Luciferase reporter assay was used to assess the target genes of miR-493. Expression of target proteins and downstream molecules were analyzed by Western blot., Results: miR-493 is silenced in resistant lung cancer cell due to the aberrant DNA methylation. Enforced expression of miR-493 in lung cancer cells promotes chemotherapy sensitivity to cisplatin through impairing the DNA damage repair and increasing the cells apoptosis in vitro and in vivo. Furthermore, we identify that TCRP1 is a direct functional target of miR-493. Ectopic expression of TCRP1 attenuated increased apoptosis in miR-493-overexpressing lung cancer cells upon cisplatin treatment. Meanwhile, miR-493 level is negatively correlated with TCRP1 expression in lung cancer patients and TCRP1 expression were correlated with poor survival., Conclusions: Our results highlight that hyper-methylation of miR-493CpG island might play important roles in the development of lung cancer chemo-resistance by targeting TCRP1, which might be used as a potential therapeutic target in preventing the chemo-resistance of lung cancer.

Background: Chemoresistance is a major obstacle in successfully treating cancers, and the mechanisms responsible for drug resistance are still far from understood. Carbonic anhydrase 9 (CA9) has been shown to be upregulated in the drug-resistant tongue cancer cell line Tca8113/PYM and to be associated with drug resistance. However, the mechanisms regulating CA9 expression and its role in drug resistance remain unclear., Methods: Bioinformatic and experimental analysis involving ChIP and luciferase reporter assays were used to validate Zinc finger E-box-binding homeobox 1 (ZEB1) as a transcriptional regulator of CA9. Gene expression and protein levels were evaluated by quantitative RT-PCR and western blotting, respectively. Sensitivity to chemotherapy was examined using the MTS assay and Hoechst staining and analysis caspase-3 activity to evaluate changes in apoptosis. Intracellular pH (pHi) was measured using fluorescent pH-indicator BCECF-AM. Protein expression in patient tissue samples was examined by immunohistochemistry and survival of tongue cancer patients from which these samples were derived was also analyzed., Results: ZEB1 bound to the promoter of CA9 to positively regulate CA9 expression in tongue cancer cells. Knockdown of CA9 using short interfering RNA (siRNA) abolished the chemoresistance resulting from ZEB1 overexpression in Tca8113 and SCC-25 cells, and CA9 overexpression attenuated chemosensitivity induced by ZEB1 knockdown in Tca8113/PYM cells. CA9 knockdown also prevented maintenance of pHi mediated by overexpression of ZEB1 in Tca8113 and SCC-25 cells following chemotherapy, associated with increased apoptosis and caspase-3 activation. Conversely, ectopic expression of CA9 suppressed decrease in pHi mediated by ZEB1 knockdown in Tca8113/PYM cells following chemotherapy, accompanied by decreased apoptosis and caspase-3 activation. Importantly, a positive correlation was observed between ZEB1 and CA9 protein expression in tongue cancer tissues, and expression of these proteins associated with a poor prognosis for patients., Conclusion: Our finding that tumor cells regulate pHi in response to chemotherapy provides new insights into mechanisms of drug resistance during cancer treatment. Identification of the ZEB1-CA9 signaling axis as a biomarker of poor prognosis in tongue cancer will be valuable in future development of therapeutic strategies aimed at improving treatment efficacy, especially in terms of drug resistance associated with this disease.