RESUMO
@# Objective： ：To study the effect of silencing DKK1 (Dickkopf1) gene on the proliferation, cell cycle and apoptosis of gastric cancer AGS cells and the action mechanism. Methods： ：The DKK1-shRNA vector was constructed and transfected into AGS cells. The stably transfected cell lines were screened. The total protein and RNAof the transfected cells were extracted and the mRNAand protein expressions of DKK1 were detected by qPCR and WB, respectively. The experiment was divided into blank control group (Control), negative control group (shNC) and DKK1 silence group (DKK1-shRNA). CCK8 assay was used to detect the proliferation ofAGS cells of each group cultured for 0, 24, 48, 72, 96, 120 and 144 h, and flow cytometry was used to analyze the cell cycle and apoptosis in each group. The relationship between DKK1 and clinicopathological features of gastric cancer was analyzed after searching HPA database. Results：The gastric cancer AGS cells with stable DKK1 gene knockdown was successfully established, and it was confirmed that the mRNA and proteinexpressions of DKK1 in DKK1-shRNA group decreased by 72% and 47%, respectively, compared to shNC group (all P<0.05). The cell proliferation curve showed that, the cell proliferation in DKKl-shRNAgroup significantly decreased after 72 hour of culture compared with that in control and shNC groups (P<0.05). The cell number of S phase decreased from 32.06% to 25.87%, while the number of G2/M phase increased from 8.49% to 21.26% compared with shNC group (all P<0.05). The number of apoptotic cells also statistically increased from 10.34% to 20.65% (all P<0.05). The data of HPAdatabase showed that DKK1 mRNAlevel in gastric cancer tissues was significantly higher than that in normal tissues, and the high expression of DKK1 mRNAwas negatively correlat ed with the survival rate of gastric cancer patients. Conclusion: : Silencing DKK1 gene can inhibit the proliferation of gastric cancer cells, arrest cells in G2/M phase and promote cell apoptosis. DKK1 plays a pro-carcinogenic effect in gastric cancer.
RESUMO
To provide a novel resource for analysis of the genome of Biomphalaria glabrata, members of the international Biomphalaria glabrata Genome Initiative (biology.unm.edu/biomphalaria-genome.html), working with the Arizona Genomics Institute (AGI) and supported by the National Human Genome Research Institute (NHGRI), produced a high quality bacterial artificial chromosome (BAC) library. The BB02 strain B. glabrata, a field isolate (Belo Horizonte, Minas Gerais, Brasil) that is susceptible to several strains of Schistosoma mansoni, was selfed for two generations to reduce haplotype diversity in the offspring. High molecular weight DNA was isolated from ovotestes of 40 snails, partially digested with HindIII, and ligated into pAGIBAC1 vector. The resulting B. glabrata BAC library (BG_BBa) consists of 61824 clones (136.3 kb average insert size) and provides 9.05 × coverage of the 931 Mb genome. Probing with single/low copy number genes from B. glabrata and fingerprinting of selected BAC clones indicated that the BAC library sufficiently represents the gene complement. BAC end sequence data (514 reads, 299860 nt) indicated that the genome of B. glabrata contains ~ 63 percent AT, and disclosed several novel genes, transposable elements, and groups of high frequency sequence elements. This BG_BBa BAC library, available from AGI at cost to the research community, gains in relevance because BB02 strain B. glabrata is targeted whole genome sequencing by NHGRI.