ABSTRACT
Retinoblastoma binding protein 2 (RBP2), a newly found histone demethylase, is overexpressed in gastric cancer. We examined the upstream regulatory mechanism of RBP2 at the microRNA (miRNA) level and the role in gastric carcinogenesis. We used bioinformatics to predict that microRNA-212 (miR-212) might be a direct upstream regulator of RBP2 and verified the regulation in gastric epithelial-derived cell lines. Overexpression of miR-212 significantly inhibited the expression levels of RBP2, whereas knockdown of miR-212 promoted RBP2 expression. Furthermore, we identified the putative miR-212 targeting sequence in the RBP2 3' UTR by luciferase assay. MiR-212 inhibited the colony formation ability of cells by repressing RBP2 expression and increasing that of P21(CIP1) and P27(kip1), both critical in cell cycle arrest. In addition, the expression of RBP2 and miR-212 in tumor tissue and matched normal tissue from 18 patients further supported the results in vivo. MiR-212 directly regulates the expression of RBP2 and inhibits cell growth in gastric cancer, which may provide new clues to treatment.
Subject(s)
Gene Expression Regulation, Neoplastic/genetics , MicroRNAs/genetics , Retinoblastoma-Binding Protein 2/metabolism , Stomach Neoplasms/genetics , Cell Cycle/genetics , Cell Line, Tumor , Cell Proliferation , Gene Knockdown Techniques , Humans , MicroRNAs/metabolism , Protein Binding , Retinoblastoma-Binding Protein 2/genetics , Stomach Neoplasms/pathologyABSTRACT
Objective To evaluate the expression of epidermal growth factor-like domain 7 (Egfl7) in atherosclerotic plaques and effects of its small interference RNA (siRNA) on angiogenesis gene expression in human endothelial cell line (HUVEC). MethodsEgfl7 expression in atheroscleroticplaquesweredetectedinhumaniliacarteryandmousearteriaeusing immunohistochemistry and immunofluorescence stainings.The siRNA targeting Egfl7 was transfected into HUVEC by lipofectamine with non-transfected cells and unconcerned siRNA as controls.At 0 h,12 h,24 h and 48 h after intervention,the levels of mRNA and protein of Egf17,vascular endothelial growth factor(VEGF),platelet derived growth factor-A (PDGF-A),platelet derived growth factor-B (PDGF-B),vascular cell adhesion molecule(VCAM) and intercellular adhesion molecule (ICAM)were measured by RT-PCR and Western blotting,respectively. ResultsThe expressions of Egfl7 in human iliac artery and mouse arteriae were increased.The expressions of Egfl7 in HUVEC at the levels of mRNA were[(0.14±0.02),(0.09±0.01),(0.02±0.01)]and the levels of protein[(0.71±0.11),(0.39±0.09),(0.07±0.01)]at 12 h,24 h and 48 h after siRNA intervention,respectively,which were decreased as compared with 0 h intervention [(0.31 ±0.05) and (0.93±0.08) ].Other genes such as VEGF,PDGF-A and PDGF-B were reduced or silenced at the levels of protein and mRNA in HUVEC with siRNA longer interventions(all P＜0.05).ConclusionsThe expression of Egfl7 in atherosclerotic plaques is increased.The siRNA inhibiting Egfl7 gene expression results in silence of other factors involved in angiogenesis.
ABSTRACT
AIM:To investigate the effect of tumor-suppressor RUNX3 on the transcription of apoptosis-related genes bcl-2,bax,caspase-3,caspase-8,caspase-9 in human gastric carcinoma cells BGC823,and to reveal the apoptosis molecular mechanism promoted by RUNX3. METHODS:The eukaryotic expression vector of human Runx3 gene pcDNA3.1-Runx3 was constructed. pcDNA3.1-Runx3 and blank vector pcDNA3.1 were transfected into BGC823 cells,respectively. After 48 h,the total mRNA and protein were acquired and the expression level of Runx3 was determined by RT-PCR and Western blotting. Then,the mRNA and protein expression of bcl-2,bax,caspase-3,caspase-8 and caspase-9 was determined by RT-PCR and Western blotting. ?-actin was used as a control. RESULTS:The eukaryotic expression vector pcDNA3.1-Runx3 was constructed successfully and transfected into BGC823 cells. RT-PCR and Western blotting confirmed that RUNX3 level was higher in pcDNA3.1-Runx3 transfected BGC823 cells than that in blank vector-transfected cells (P
ABSTRACT
AIM:To establish the gastric cancerous multidrug resistance cell stain BGC823/5-FU and investigate the relationship between the resistance and the expression of apoptosis related protein Survivin, Bcl-2, Bax and caspase-3. METHODS: Human gastric cancer cell line BGC823 was induced into MDR cell line by intermittent administration of high dose of 5-FU. MTT assay was used to detect the sensitivity of these MDR cells to some chemotherapeutic agents. Flow cytometry was used to detect the expression of P-glucoprotein and the accumulative value of intracellular daunorubicin (DNR) in these MDR cells. Western blotting was used to detect the expression of Survivin, Bcl-2, Bax and caspase-3. RESULTS: The resistance cell stain BGC823/5-FU was established, which possessed the ability of 10.82 fold resistance to 5-FU and cross-resistance to adriamycin, mitomycin C and cisplatin. The expression of P-glucoprotein was higher in BGC823/5-FU cells than that in BGC823 cells, while the accumulative value of intracellular DNR was decreased in BGC823/5-FU cells. Compared with its parent cells, expressions of Bax and caspase-3 in BGC823/5-FU cells were significantly down-regulated, surviving and Bcl-2 were upregulated in BGC823/5-FU cells. CONCLUSION: Gastric cancer cell line BGC823 has been induced into MDR cell line BGC823/5-FU. P-glucoprotein, Survivin, Bcl-2/Bax ratio and caspase-3 may play an important role in MDR of BGC823/5-FU cells.
