ABSTRACT
OBJECTIVE To explore downstream regulatory pathway of microRNA-21 (miR-21) in colon cancer cells (RKO) through detecting miR-21 and its target PDCD4, and the influence of miR-21 regulation on the sensitivity of RKO cells to 5-fluorouracil (5-FU). METHODS 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the effect of 5-FU on the viability of RKO cells with knockout of miR-21 or high expression of PDCD4. Real-time was used to determine the expression of PDCD4, ABCC5 and CD44 in RKO cell after knockout of miR-21. RESULTS MTT assay reveals that the IC50 of 5-FU in RKO-WT cells (52.82 ± 0.06 umol/L) was about 67% higher than in miR-21 knockout cells (32.23 ± 0.05 umol/L) (P < 0.05), and the apoptosis ratio elevated after knockout of miR-21. High expression of PDCD4, a target gene of miR-21, can negatively regulate the expression of ABC transporter ABCC5 and the stem cell marker CD44. CONCLUSION MiR-21 can mediate the drug resistance to 5-FU by inhibiting its target PDCD4, which can regulate the expression of ABCC5 and CD44 genes.
Subject(s)
Humans , ATP Binding Cassette Transporter, Subfamily G, Member 5 , ATP-Binding Cassette Transporters , Genetics , Antimetabolites, Antineoplastic , Pharmacology , Apoptosis Regulatory Proteins , Physiology , Cell Line, Tumor , Colonic Neoplasms , Drug Therapy , Pathology , Drug Resistance, Neoplasm , Fluorouracil , Pharmacology , Hyaluronan Receptors , Genetics , Lipoproteins , Genetics , MicroRNAs , Physiology , RNA-Binding Proteins , PhysiologyABSTRACT
<p><b>OBJECTIVE</b>To study the clinical features and ABCG5/ABCG8 gene mutations of three pedigrees of phytosterolemia presented with macrothrombocytopenia and hemolysis.</p><p><b>METHODS</b>Erythrocyte and platelet morphology were examined under light microscope. Plasma sterol levels were measured by high pressure/performance liquid chromatography method. All of ABCG5 and ABCG8 exons and intron-exon boundaries were directly sequenced to identify mutations, the corresponding gene mutation sites of three families members and healthy individuals were detected.</p><p><b>RESULTS</b>All the patients presented macrothrombocytopenia, hemolysis, splenomegaly and xanthomas. The blood smears showed large platelets, some as large as erythrocytes, and abnormal erythrocyte shapes, such as stomatocytes. Plasma concentrations of phytosterols, especially sitosterol were markedly elevated (30 fold) in the affected patients. Four mutations were identified in these three pedigrees, ABCG5 C20896T (R446X) and A20883G, ABCG8 del43683-43724 and del1938C-1939G/ins1938T. The latter three were novel mutations reported for the first time.</p><p><b>CONCLUSIONS</b>Phytosterolemia associated with macrothrombocytopenia and hemolysis is a new subtype of this disease. Plasma phytosterols and related gene analysis should be performed when ever an unexplained macrothrombocytopenia, especially combined with haemolysis or/and stomatocytosis.</p>