Expression Silence of DNA Repair Gene hMGMT Induced by RNA Interference

Objective： MGMT protein expression has been associated with tumor resistance to alkylating agents. The objective of this paper is to construct the RNA interference vector which can specifically induce the expression silence of human DNA repair gene hMGMT. Methods： The hMGMT specific siRNA expression cassette was made by two steps PCR, linked with pUCI 9 to get pU6-MGMTi, co-transfected with pEGFP-CI into 16HBE and screened by G418. The MGMT mRNA and protein levels were detected by RT-PCR and Western Blot respectively. Results： hMGMT specific RNA interfere vector pU6-MGMTi was constructed successfully. In transfected 16HBE cells MGMT mRNA level could hardly be detected and the protein level was only 10% of control. Conclusion： MGMT specific RNAi expression cassette can effectively inhibit MGMT expression. MGMT silence cell line was built by co-transfection technology, which offered condition for studying the gene function of MGMT.

MiR-34a Inhibits Cell Proliferation and Migration in Osteosarcoma by Downregulating DNMT1

Objective To investigate the effects of DNA methyl transferase 1(DNMT1)gene regulation by miR 34a in osteosarcoma(OS)MG63 cells and to determine the possible mechanisms.Methods A miR-34a mimic,mimic negative control(NC),miR-34a inhibitor,or inhibitor-NC were transfected into MG63 cells using Lipofectamine 3000.Cell viability was measured using the MTT assay.The mRNA levels of DNMT1 in MG63 cells were detected by qPCR.The proliferation of MG63 cells was detected by the CCK8 method.The protein expression of DNMT1 was measured by Western blotting.The effects of DNMT1 on the migration of MG63 cells were examined by a cell migration assay.The apoptosis of MG63 cells was detected by flow cytometry.Results The mRNA levels of DNMT1 were up-regulated in MG63 cells treated with the miR-34a inhibitor compared to other groups.The growth of MG63 cells was significantly increased in these cells.The protein expression of DNMT1 and the cell proliferation and migration decreased in the miR-34a mimic-treated group compared to the cells treated with the miR-34a inhibitor or the negative control(both P<0.05).Conclusion By targeting the DNMT1 gene,miR-34a reduces the expression level of DNMT1 in MG63 cells,thereby affecting the viability,migration,proliferation,and apoptosis of MG63 cells.Interventions targeting the miR-34a/DNMT1 axis may represent a novel targeted therapy for OS.

Histone deacetylase inhibitors inducing human cervical cancer cell apoptosis by decreasin~ DNA-methyltransferase 3B

Background Histone deacetylase （HDAC） inhibitors are a group of small chemical molecules that inhibit histone deacetylase. At cell level, HDAC inhibitors have multiple biological effects such as cell cycle arrest, apoptosis, cell differentiation and auotophagy. At molecular level, HDAC inhibitors cause histone and nonhistone acetylation and induce gene expression. HDAC inhibitors are widely used in cancer therapy because of its function of inducing apoptosis. However, the mechanisms of apoptosis effect are not fully understood. TSA is a classical HDAC inhibitor and widely used in epigenetic and anti-cancer research. In this study, we selected Trichostatin A （TSA） to investigate the mechanisms of HDAC inhibitors apoptotic effect on cancer cells. Methods Cervical cancer cell lines such as Hela, Caski and normal human keratinocyte line HaCaT were treated with various concentrations of TSA. Crystal violent assay and 3-（4,5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay were performed to determine cell number. PARP cleavage and FITC-AnexinV were performed to determine apoptosis. DNA-methyltransferase （DNMT）I, DNMT3A and DNMT3B were determined by regular PCR, qPCR and Western Blotting. Small interfering RNA （SiRNAi） was used to knock down DNMT3B. Results HDAC inhibitors only induce cervical cancer cell apoptosis. At 1 Iumol/L of TSA, 86% of Hela cell and 76% of Caski went apoptosis. For normal cells, HDAC inhibitors have no cytotoxic effect at therapeutic dosage, （90.0＋8.4）% of normal cell survive after treated with 1 IJmol/L of TSA. We compared 1 pmol/L group with untreated control with t-test. There was no significance between 1 pmol/L group and untreated control for normal cell （P 〉0.05）. HDAC inhibitors decreased DNMT3B in cancer cell but not in normal cell. Manually knock-down of DNMT3B induced Hela and Caski cell apoptosis. More than 99% of Hela and Caski cell went apoptosis after deprived of DNMT3B. Conclusions DNMT3B was essential to cervical cancer cell survival. Down-regulated DNMT3B by HDAC inhibitors may play an important role in the toxicity of HDAC inhibitors on cervical cancer cells.

Insights into the Interactions Between Corrinoid Iron-sulfur Protein and Methyl Transferase from Human Pathogen Clostridium difficile

The human pathogen Clostridium difficile infection（CDI） is one of the most important healthcare- associated infections. Methyltransferase（MeTrca） and corrinoid iron-sulfur protein（CoFeSPca） are two key proteins in the acetyl-coenzyme A synthesis pathway of Clostridium difficile, which is essential for the survival of the pathogen and is absent in humans. Hence, the interaction between MeTrca and CoFeSPcd can become innovative targets for the treatment of human CDI. In this study, the interaction between MeTrca and CoFeSPca was verified by fluorescence resonance energy transfer measurements. The influence of the interaction on the tertiary structure of MeTrcd and CoFeSPcd was studied by ANS-labeled fluorescence measurements. Molecular docking was also performed to understand the mechanism of the protein interactions. These results provide a molecular basis for innovative drug design and development to treat human CDI.