ABSTRACT
OBJECTIVE@#To investigate the function and mechanism of transcription factor of MEIS1 and miR-425 to the proliferation of chronic myeloid leukemia cell K562.@*METHODS@#Bioinformatic prediction was used to analyze the binding of MEIS1 in miR-425 promoter region. ChIP-qPCR coupled with dual luciferase assay was used to detect the combination of MEIS1 and the transcription activity of miR-425, and its regulative role in the transcription activity miR-425. CCK-8 was used to detect the effect of MEIS1 and miR-425 on cell proliferation. Flow cytometry with PI staining was used to detected the effect of MEIS1 and miR-425 on K562 cell cycle progression. Western blot was used to examine the effect of miR-452 on the expression level of MEIS1.@*RESULTS@#MEIS1 could bind the promoter of miR-425 and repressed its transcription. After K562 was transfected by shRNA, the K562 cell proliferation and cell cycle progression was significantly inhibitied. Moreover, after K562 cells were transfected by miR-425 mimic, cell proliferation and cell cycle was inhibited. The expression level of MEIS1 could be inhibited by the combination of miR-425 and MEIS1 3'UTR.@*CONCLUSION@#MEIS1 can inhibit the activity of miR-425 in transcriptional level, while the miR-425 can suppress the expression of MEIS1 protein in post-transnational level. Therefore, a regulatory circuit comprising from MEIS1 and miR-425 regulates K562 cell proliferation.
Subject(s)
Humans , Apoptosis , Cell Proliferation , K562 Cells , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Genetics , MicroRNAs , Genetics , Myeloid Ecotropic Viral Integration Site 1 Protein , GeneticsABSTRACT
(1) BACKGROUND: 12-lipoxygenase (12-LO) is involved in the development of diabetic nephropathy (DN). In the present study, we investigated whether 12-LO inhibition may ameliorate type-2 DN (T2DN) by interfering with insulin resistance (IR); (2) METHODS: Rat glomerular mesangial cells, glomeruli and skeletal muscles were isolated and used in this study. Kidney histological changes were confirmed by periodic-acid Schiff staining; mRNA expression was detected by competitive reverse transcription polymerase chain reaction; and the protein level was determined by Western blot and the enzyme-linked immunosorbent assay, respectively; (3) RESULTS: The inhibition of 12-LO attenuated microalbuminuria (MAU) increases in type-2 diabetic rats, but not in type-1 diabetic rats. Infusion of 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) significantly increased the expression of angiotensin II (Ang II) and Ang II type 1 receptor (AT1R), but decreased the expression of AT1R-associated protein (ATRAP) in rat glomeruli, compared to the control. An in vitro study revealed that both 12(S)-HETE and insulin upregulated AT1R expression in rat mesangial cells. In the presence of p38 mitogen-activated protein kinase (MAPK) inhibitor, SB202190, the 12(S)-HETE-induced ATRAP reduction was significantly abolished. Interestingly, 12-LO inhibition did not influence AT1R expression in type-1 diabetic rats, but significantly abolished the increased AT1R and Ang II expression in glomeruli of type-2 diabetic rats. Furthermore, the inhibition of 12-LO significantly corrected impaired insulin sensitivity and fast serum insulin level, as well as the p-AMP-activated protein kinase (AMPK) reduction in skeletal muscle of type-2 diabetic rats; (4) CONCLUSION: The inhibition of 12-LO potentially ameliorated MAU by preventing IR through the downregulation of glomerular AT1R expression in T2DN.
Subject(s)
Albuminuria/metabolism , Arachidonate 12-Lipoxygenase/metabolism , Diabetic Nephropathies/metabolism , Insulin Resistance , Receptor, Angiotensin, Type 1/metabolism , 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid/pharmacology , Albuminuria/etiology , Animals , Cells, Cultured , Diabetes Mellitus, Type 1/complications , Diabetes Mellitus, Type 1/metabolism , Diabetes Mellitus, Type 2/complications , Diabetes Mellitus, Type 2/metabolism , Down-Regulation , Kidney Glomerulus/drug effects , Kidney Glomerulus/metabolism , Lipoxygenase Inhibitors/pharmacology , Male , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , Protein Kinase Inhibitors/pharmacology , Rats , Rats, Sprague-Dawley , Receptor, Angiotensin, Type 1/geneticsABSTRACT
BACKGROUND: The 12-lipoxygenase (12-LO) and angiotensin II (Ang II) interaction plays an important role in diabetic nephropathy (DN). Proteinuria in DN is associated with decreased slit diaphragm proteins including nephrin and P-cadherin. Therefore, we investigated whether Ang II type 1 receptor (AT1) blocker (ARB) regulates 12-LO activity and slit diaphragm protein expression in diabetic rat glomeruli. METHOD: Glomeruli were isolated with the sieving method, and classified into small glomeruli (SG; 75-µm sieve) and large glomeruli (LG; 125-µm sieve). RESULTS: 12(S)-HETE, a lipid product of 12-LO, was increased by Ang II in the glomeruli. Infusion of 12(S)-HETE and Ang II significantly decreased nephrin expression in LG, but increased it in SG compared to control. Glomerular P-cadherin expression was reduced after Ang II and 12(S)-HETE treatment without differences between LG and SG. ARB did not influence glycemic levels but completely abolished the increases in 12(S)-HETE, AT1 expression, and proteinuria in diabetic rats. Nephrin expression was significantly reduced in LG but increased in SG in diabetic rats compared to control. P-cadherin expression decreased in both diabetic LG and SG. The abnormalities of nephrin and P-cadherin were partially but significantly reversed by ARB. CONCLUSION: ARB potentially ameliorates DN via the up-regulation of glomerular nephrin and P-cadherin expression through the inhibition of 12-LO activation in the glomeruli of rats with DN.
