ABSTRACT
<p><b>OBJECTIVE</b>To study the expression of enoyl CoA hydratase 1 (ECH1) and the effect when down-regulation of ECH1 gene expression in mouse hepatocarcinoma cell.</p><p><b>METHODS</b>Immunofluorescence was used for detecting the expression of ECH1, and stably transfected Hca-F cells with pGPU6/GFP/Neo-shRNA-ECH1 expression plasmids. Cell proliferation was assessed by Cell counting kit-8 (CCK8) assay. The Boyden-transwell assay (8 µm pore size) was performed to analyze the inhibitory effect of shRNA on Hca-F cell migration and invasion.</p><p><b>RESULTS</b>ECH1 expression was obtained in the cytoplasm and upregulated expression in Hca-F cells than that in Hca-P cells. The down-regulation of ECH1 could inhibit the cell proliferation of Hca-F cells, decrease the number of cell pass through Transwell (27.07 ± 17.49) compared with scramble-negative (72.38 ± 18.83) and Hca-F controls (59.06 ± 30.33), decrease the migration capacities of Hca-F cells, increase the ratio of Hca-F cells in S phase (86.1%) compared with scramble-negative (75.8%) and Hca-F controls (66.2%) and decrease the ratio of G(1) phase (9.4%) compared with scramble-negative (24.2%) and Hca-F controls (30.3%).</p><p><b>CONCLUSION</b>ECH1 serves as a potential critical factor attributes to tumor lymphatic metastasis.</p>
Subject(s)
Animals , Mice , Cell Cycle , Cell Line, Tumor , Cell Movement , Cell Proliferation , Cytoplasm , Down-Regulation , Enoyl-CoA Hydratase , Genetics , Metabolism , Gene Expression Regulation, Neoplastic , Liver Neoplasms, Experimental , Pathology , Lymphatic Metastasis , Plasmids , RNA, Small Interfering , Genetics , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To prepare monoclonal antibodies (mAbs) against enoyl-CoA hydratase 1 (ECH1).</p><p><b>METHODS</b>Normal human liver tissues were homogenized, and the mitochondria were isolated by differential centrifugation. The total mitochondrial proteins were used to immunize BALB/c mice to prepare mAbs by routine hybridoma technique. The mAbs were characterized by ELISA, Western blotting and immunohistochemistry. The specificity of the antibody was identified by mass spectrometry (MS) following immunoprecipitation (IP) and confirmed by Uni-ZAP expression library screening.</p><p><b>RESULTS</b>One clone of the hybridoma BGB095 secreting specific mAb against ECH1 was obtained. The mAb was identified to belong to Ig subclass IgG1 and could be used in ELISA, Western blotting, immunohistochemistry, and immunoprecipitation.</p><p><b>CONCLUSION</b>A hybridoma cell line stably secreting specific mAb against ECH1 has been established. The specific mAb against ECH1 can be of great value for functional and distribution studies of ECH1.</p>
Subject(s)
Animals , Humans , Mice , Antibodies, Monoclonal , Allergy and Immunology , Antibody Specificity , Blotting, Western , Cell Line , Enoyl-CoA Hydratase , Allergy and Immunology , Enzyme-Linked Immunosorbent Assay , Immunohistochemistry , Liver , Cell Biology , Metabolism , Mice, Inbred BALB C , Mitochondria , MetabolismABSTRACT
Renal cell carcinoma (RCC) is one of the most malignant tumors in urology, and due to its insidious onset patients frequently have advanced disease at the time of clinical presentation. Thus, early detection is crucial in management of RCC. To identify tumor specific proteins of RCC, we employed proteomic analysis. We prepared proteins from conventional RCC and the corresponding normal kidney tissues from seven patients with conventional RCC. The expression of proteins was determined by silver stain after two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The overall protein expression patterns in the RCC and the normal kidney tissues were quite similar except some areas. Of 66 differentially expressed protein spots (p<0.05 by Student t-test), 8 different proteins from 11 spots were identified by MALDI-TOF-MS. The expression of the following proteins was repressed (p<0.05); aminoacylase-1, enoyl-CoA hydratase, aldehyde reductase, tropomyosin alpha-4 chain, agmatinase and ketohexokinase. Two proteins, vimentin and alpha-1 antitrypsin precursor, were dominantly expressed in RCC (p<0.05).
Subject(s)
Aged , Female , Humans , Male , Middle Aged , Aldehyde Reductase/analysis , Amidohydrolases/analysis , Carcinoma, Renal Cell/metabolism , Comparative Study , Electrophoresis, Gel, Two-Dimensional , Enoyl-CoA Hydratase/analysis , Fructokinases/analysis , Kidney Neoplasms/metabolism , Proteome/analysis , Proteomics/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tropomyosin/analysis , Ureohydrolases/analysis , Vimentin/analysis , alpha 1-Antitrypsin/analysisABSTRACT
<p><b>OBJECTIVE</b>To investigate the alteration of the gene HSD17B4 in esophageal squamous cell carcinoma and its potential significance.</p><p><b>METHODS</b>The mRNA expression and loss of heterozygosity (LOH) of HSD17B4 in 40 primary esophageal tumors were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and microsatellite analysis with the intragenic marker D5S1384 of the gene.</p><p><b>RESULTS</b>The frequencies of allelic loss of D5S1384 and the rate of down-regulation of gene HSD17B4 were 46.2% and 62.5%, respectively.</p><p><b>CONCLUSION</b>HSD17B4 may be a candidate tumor suppressor gene associated with esophageal squamous cell carcinoma.</p>
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , 17-Hydroxysteroid Dehydrogenases , Genetics , Carcinoma, Squamous Cell , Genetics , Down-Regulation , Enoyl-CoA Hydratase , Genetics , Esophageal Neoplasms , Genetics , Gene Expression , Gene Expression Regulation, Neoplastic , Genetics , Genes, Tumor Suppressor , Hydro-Lyases , Loss of Heterozygosity , Microsatellite Repeats , Multienzyme Complexes , Genetics , Peroxisomal Multifunctional Protein-2 , RNA, Messenger , Genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
<p><b>OBJECTIVE</b>To determine the physiological role of D-bifunctional protein (DBP) in bile acid biosynthesis through investigating the effect of increasing activity of DBP on bile acid biosynthesis.</p><p><b>METHODS</b>Twenty male Wistar rats were divided into two groups: diethylhexyl phthalate (DEHP) group (n = 10) and control group (n = 10). Serum triglyceride, total cholesterol, hepatic DBP activity, and fecal bile acids were assayed. The mRNA levels of hepatic peroxisome proliferator-activated receptor alpha (PPARalpha), DBP, and cholesterol 7alpha-hydroxylase (CYP7A1) were detected by RT-PCR.</p><p><b>RESULTS</b>Compared with control group, serum triglyceride level was decreased significantly and PPARalphamRNA level was increased significantly in DEHP group (P < 0.01). Together with a sharp induction of DBP mRNA expression and DBP activity in DEHP group (P < 0.01), the levels of CYP7A1 mRNA and fecal bile acids were significantly increased by 1.9 times and 1.6 times respectively compared to control group (P < 0.01). There was a significantly positive correlation between DBP mRNA level or DBP activity and CYP7A1 mRNA level (r = 0.89, P < 0.01; r = 0.95, P < 0.01).</p><p><b>CONCLUSION</b>The up-regulation of DBP mRNA and activity in liver can result in the increase in CYP7A1 mRNA expression and bile acid biosynthesis, suggesting that DBP may be involved in bile acid biosynthesis together with CYP7A1.</p>