ABSTRACT
<p><b>OBJECTIVE</b>To construct a recombinant short hairpin RNA (shRNA) expression vector targeting EZH2 gene, and to determine its effect on the proliferation of colon adenocarcinoma SW480 cells.</p><p><b>METHODS</b>The DNA sequence with short hairpin structure was designed according to the EZH2 cDNA sequence and cloned into PGFP-V-RS vector to construct a recombinant expression vector silencing EZH2 gene. After identification, the shRNA-expressing vector was then transfected into SW480 cells. RT-PCR and Western blot were used to detect the inhibitory effect at both mRNA and protein levels. MTT was used to detect cell viability due to the alteration of EZH2 gene activity.</p><p><b>RESULTS</b>At 48 h after transfection, the expression of EZH2 mRNA in the gene silencing group and negative control group were 0.339 ± 0.013 and 1.968 ± 0.072, respectively. The expression of EZH2 protein in the gene silencing group and negative control group were 0.229 ± 0.008 and 1.168 ± 0.053, respectively. The expression of EZH2 in the gene silencing group was significantly lower than that in the negative control group (P < 0.01, P < 0.05). At 48 and 72 h after transfection, the inhibition rate of cell growth in the gene silencing group was 30.7% and 25.9%, respectively, indicating that the cell growth was significantly inhibited in comparison with that in the blank control group (P < 0.05).</p><p><b>CONCLUSIONS</b>A recombinant shRNA expression vector targeting EZH2 gene has been successfully constructed in this study, with a significant inhibitory effect on the proliferation of SW480 cells. This lays an experimental foundation for further exploring the mechanism underlying the action of EZH2 gene on tumor biology.</p>
Subject(s)
Humans , Adenocarcinoma , Metabolism , Pathology , Cell Line, Tumor , Cell Proliferation , Colonic Neoplasms , Metabolism , Pathology , Enhancer of Zeste Homolog 2 Protein , Gene Silencing , Gene Targeting , Genetic Vectors , Plasmids , Polycomb Repressive Complex 2 , Genetics , Metabolism , RNA, Messenger , Metabolism , RNA, Small Interfering , Genetics , Recombinant Fusion Proteins , Genetics , Metabolism , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To evaluate the efficacy of three-dimensional anal and endorectal ultrasound in identifying the internal opening and tracing the tract of the anorectal fistula.</p><p><b>METHODS</b>From November 2008 to January 2010, 127 patients suffering anorectal fistula were managed with three-dimensional endoanal and endorectal ultrasound. The internal opening, the tract of the fistula and fistula trace were identified by the ultrasonography with three-dimensional imaging. All results were confirmed and compared with findings from the operation.</p><p><b>RESULTS</b>The internal opening of the fistula was specified in 116 patients, the accuracy rate was 91.3% (116/127). The internal opening of the fistula was located above the dentate line in 112 patients, and located in rectal ampulla in 4 patients. The main fistula tract was identified in all the patients, the accuracy rate was 100%. In this group, the fistula tunneled as follows: trans-sphincteric in 47 patients, intersphincteric in 75 cases, supra sphincteric in 2 cases, extra sphincteric in 3 patients. Secondary extension was found in 37 patients, the accuracy rate was 100% (37/37).</p><p><b>CONCLUSIONS</b>Three-dimensional anal and endorectal ultrasound is an effective way for localizing the internal opening and the tract of anorectal fistula. It can provide valuable information for curative operation.</p>