| Gastric cancer(GC)is one of the most common malignant tumors,which can harms human health and life seriously. A steady decline in stomach cancer incidence and mortality rates has been observed in the majority of more developed countries in Northern America and Europe with the development of economy and improvement of the meterial standard of living, but similar decreasing trends have been noted in more recent years in areas with historically high rates, including several countries in Asia and Latin America. The prognosis of GC also remains poor because the molecular mechanisms of GC progression are incompletely understood.Diallyl disulfide(DADS) is one of the organosulfur compounds that derive from Allium vegetables, such as garlic. Laboratory evidence has revealed that DADS exerts multiple antitumor effects on a variety of tumors. We have previously demonstrated that DADS can suppress cell proliferation, angiogenesis or invasion in gastric cancer cell lines, and the mechanism may be related to induce G2/M phase cell cycle arrest,activate P38, downregulate ERK signaling pathway and abnormity of other signaling pathway.MicroRNAs(mi RNAs) are noncoding RNAs that are associated with gastric carcinogenesis closely. MiRNAs contribute to gastric carcinogenesis by altering the expression of oncogenes and tumor suppressors, affecting cell proliferation, apoptosis, motility, and invasion.microRNA-7 is a 23 nucleotide miRNA and the reduced levels of miR-7have beens linked to the development of many cancers and metastasis,which acts as a tumor suppressor.X-linked inhibitor of apoptosis(XIAP), is a member of the IAP family of endogenous caspase inhibitors that blocks the execution of apoptosis, and plays important roles in tumor development, which is closely related to tumor genesis, growth, apoptosis, migration and metastasis.We have previously demonstrated DADS treatment altered the expression profile of mi RNAs in human gastric cancer cell. Microarray analysis was performed on the expression of DADS-treated and DMSO treated MGC-803 cells(mock). Mi R-7 were significantly up-regulated with a 1.54 fold change. To further confirm microarray data, quantitative real-time PCR(qRT-PCR) was used to measure expression levels of miR-7 to determine whether there were changes in their expression. We found miR-7 was down-regulated in gastric cancer tissue and human gastric cancer cells and significantly up-regulated after treated with DADS, which was consistent with the microarray analysis results.Moreover, to further explore potential molecular targets affected by DADS in gastric cancer, we screened and obtained some proteins through proteomics research approaches, which showed significantly differential expression between DADS ? treated and untreated gastric cancer cells.Twenty-three differentially expressed proteins which included XIAP were identified based on MALDI-TOF-MS and database query with MS-Fit.To further confirm proteomics data, qRT-PCR was used to measure expression levels of XIAP in human gastric cancer cells. Our findings showed that XIAP was up-regulated human gastric cancer cells and significantly down-regulated after treated with DADS.The experimental study was carried out in three parts to investigate the effect of miR-7 and XIAP on proliferation and invasion of human gastric cancer cells and to elucidate the relationship between the two.Part I Up-regulation of miR-7 inhibit proliferation and invasion in human gastric cancer cells induced by diallyl disufidePurpose: To investigate the effect of miR-7 and DADS on proliferation and invasion in human gastric cancer cells.Methods: To obtain stable miR-7 overexpressing or knockdown cell lines, we performed experiments using GV358 lentiviral vector.SGC-7901 and HGC-27 cells infected with miR-7 overexpressing or knockdown lentiviral vector. After 48 h of infection, cells were selected with puromycin and miRNA overexpression or knockdown was quantified at 7 days post infection. At the same time, cells in each group were treated with 30mg/l DADS. The effect of mi R-7 on proliferation and invasion of human gastric cancer cells were investigated using qRT-PCR, CCK-8 assay, clone formation assay, scrath assay, transwell migration assay, transwell invasion assay, apoptosis evalution and cell cycle evalution by flow cytometry, and xenograft tumor assay in vivo.Results: QRT-PCR verified that the stable miR-7 overexpression and knockdown SGC-7901 and HGC-27 cell lines were constructed successfully. The expression of miR-7 up-regulated in these cells treated with 30mg/l DADS. DADS and stable overexpression of mi R-7 using lentiviral vectors reduced proliferation properties and colony formationactivity in SGC-7901 and HGC-27 cells, and can induce the SGC-7901 cells apoptosis. Cell cycle progression analysis also showed a significant increase of G2/M cell fractions indicating a cell cycle arrest in SGC-7901.Furhtermore, DADS and miR-7 overexpression resulted in a significantly decrease of migratory and invasive capacity of SGC-7901 and HGC-27 cell lines. At last, DADS and stable miR-7 overexpression using lentiviral vectors in SGC-7901 cells inhibited tumor xenograft growth in immunodeficient mice. On the contrary, after miR-7 knockdown, the experimental result showed oppositive effects compared with miR-7overexpression. In addition, MiR-7 overexpression can synergize with DADS to inhibit cell proliferation, colony formation, invasion, migration,xenograft tumor growth, and induce cell apoptosis and cell cycle arrest.On the contrary, miR-7 knockdown can antagonize the role of DADS in human gatric cancer cells.Part II Down-regulation of mi R-7 inhibit proliferation and invasion in human gastric cancer cells induced by diallyl disufidePurpose: To investigate the effect of XIAP and DADS on proliferation and invasion in human gastric cancer cells.Methods: At first, XIAP expression was examined by immunohistochemistry(IHC) using gastric cancer tissue microarrays.Then, GV358 lentiviral vector of XIAP overexpressing and pcDNA6.2plasmids of XIAP knockdown were constructed. Thirdly, SGC-7901 and HGC-27 cells were transfected with the XIAP overexpressing lentiviral vector and reconstructed XIAP knockdown plasmids. To obtain stable XIAP overexpressing or knockdown cell lines, cells were selected with puromycin or Blasticidin S HCl. At the same time, cells in each group were treated with 30mg/l DADS. The effect of XIAP on proliferation and invasion of human gastric cancer cells wereinvestigated using qRT-PCR, Western blot analysis, immunofluorescence assay, CCK-8 assay, clone formation assay, scrath assay, migration assay,invasion assay, apoptosis evalution and cell cycle evalution by flow cytometry, and xenograft tumor assay in vivo.Results: The positive cytoplasm XIAP expression could be found in human gastric cancer cells, by contrast, XIAP expression was negative or part positive in normal human gastric epithelial cells and atypical hyperplasia epithelium cells. Cancer cells showed a significantly higher expression compared to normal and atypical hyperplasia epithelium cells(P<0.05). QRT-PCR verified that the stable XIAP overexpression and knockdown SGC-7901 and HGC-27 cell lines were constructed successfully. The expression of XIAP down-regulated in these cells treated with 30mg/l DADS. Western blot data showed obviously decreased XIAP expression levels in XIAP knockdown group and increased XIAP expression levels in XIAP overexpression group in both SGC-7901 and HGC-27 cells comparing to the negative control, and obviously decreased XIAP expression levels in any group treated with DADS. Similar results were obtained using immunofluorescence assay.DADS and XIAP stable knockdown reduced proliferation properties and colony formation activity in SGC-7901 and HGC-27 cells, and can induce the SGC-7901 cells apoptosis. Cell cycle progression analysis also showed a significant increase of G2/M cell fractions indicating a cell cycle arrest in SGC-7901. Furhtermore, DADS and XIAP knockdown resulted in a significantly decrease of migratory and invasive capacity of SGC-7901 and HGC-27 cell lines. At last, DADS and XIAP stable knockdown of SGC-7901 cells inhibited tumor xenograft growth in immunodeficient mice. These results were in accord with the results of the overexpression of mi R-7. On the contrary, we also discovered that when the target gene XIAP was over expressed, the cell viability and colony formation activity of SGC-7901 and HGC-27 cancer cells were suppressed apparently, the cell apoptosis was increased. Those meaned that the experimental result showed oppositive effects compared with XIAP knockdown. In addition, XIAP knockdown can synergize DADS o inhibit cell proliferation, colony formation, invasion, migration,xenograft tumor growth, and can induce cell apoptosis and cell cycle arrest. On the contrary, XIAP overexpression can antagonize the role of DADS in human gatric cancer cells.Part III XIAP downregulated by microRNA-7 in human gastric cancer cellsPurpose: The validation of miR-7 down-regulate XIAP.Methods: Sequences of mature miR-7 and XIAP 3’UTR were obtained from online website miRBase and GeneBank respectively.Subsequently, potential molecular targets of mi R-7 were analysed by boinformatic algorithms in the threen commonly used databases including DIANA-micro T-CDS, Targetscan and mi RANDA. Based on these, wild type and mutant cDNA fragments of XIAP 3’UTR were then cloned into luciferase reportervector GV306 to construct the fusional expression plasmids including the GV306-XIAP- 3’UTR-WT and GV306-XIAP-3’UTR-MT. Relative luciferase activity was detected in SGC-7901 cells cotransfected with lentiviral vector of mi R-7 overexpression and the fusional plasmids. Furthermore, the mRNA levels and protein levels of target genes in mi R-7 overexpressed or knockdown human gastric cancer cells were detected with Real-time PCR and Western blot, in order to confirm the regulative role of miR-7 on XIAP expression.Results: Bioinformatic analysis revealed the XIAP mRNA 3’UTR contained the potential binding site of mi R-7. The fluorescent reporter assay also confirmed that miR-7 can directly bind to the specific site of XIAP mRNA 3’UTR and negatively regulate the gene expression. When miR-7 was over-expressed in SGC-7901 and HGC-27 cells, mRNA level and protein level of XIAP were both depressed. On the contrary, when miR-7 was knockdown, mRNA level and protein level of XIAP were both up-regulated.Conclusions:1. Up-regulation of miR-7 induced by diallyl disulfide can inhibit proliferation, migration, invasion, xenograft tumor growth, induce cell apoptosis and cycle arrest in human gastric cancer cells2. Down-regulation of XIAP induced by diallyl disulfide can inhibit proliferation, migration, invasion, xenograft tumor growth, induce cell apoptosis and cycle arrest in human gastric cancer cells3. XIAP was validated as a target for mi R-7 and can been down-regulated by it... |
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