title: Regulation of Insulin Resistance by Multiple MiRNAs via Targeting the GLUT4 Signalling Pathway.
abstract: OBJECTIVE
  Type 2 Diabetes Mellitus (T2DM) is characterized by insulin resistance (IR), but the underlying molecular mechanisms are incompletely understood. MicroRNAs (miRNAs) have been demonstrated to participate in the signalling pathways relevant to glucose metabolism in IR. The purpose of this study was to test whether the multiple-target anti-miRNA antisense oligonucleotides (MTg-AMO) technology, an innovative miRNA knockdown strategy, can be used to interfere with multiple miRNAs that play critical roles in regulating IR.
  
  
  METHODS
  An MTg-AMO carrying the antisense sequences targeting miR-106b, miR-27a and miR-30d was constructed (MTg-AMO106b/27a/30d). Protein levels were determined by Western blot analysis, and transcript levels were detected by real-time RT-PCR (qRT-PCR). Insulin resistance was analysed with glucose consumption and glucose uptake assays.
  
  
  RESULTS
  We found that the protein level of glucose transporter 4 (GLUT4), Mitogen-activated protein kinase 14 (MAPK 14), Phosphatidylinositol 3-kinase regulatory subunit beta (PI3K regulatory subunit beta) and mRNA level of Slc2a4 (encode GLUT4), Mapk14 (encode MAPK 14) and Pik3r2 (encode PI3K regulatory subunit beta) were all significantly down-regulated in the skeletal muscle of diabetic rats and in insulin-resistant L6 cells. Overexpression of miR-106b, miR-27a and miR-30d in L6 cells decreased glucose consumption and glucose uptake, and reduced the expression of GLUT4, MAPK 14 and PI3K regulatory subunit beta. Conversely, silencing of endogenous miR-106b, miR-27a and miR-30d in insulin-resistant L6 cells enhanced glucose consumption and glucose uptake, and increased the expression of GLUT4, MAPK 14 and PI3K regulatory subunit beta. MTg-AMO106b/27a/30d up-regulated the protein levels of GLUT4, MAPK 14 and PI3K regulatory subunit beta, enhanced glucose consumption and glucose uptake.
  
  
  CONCLUSIONS
  Our data suggested that miR-106b, miR-27a and miR-30d play crucial roles in the regulation of glucose metabolism by targeting the GLUT4 signalling pathway in L6 cells. Moreover, MTg-AMO106b/27a/30d offers more potent effects than regular singular AMOs.
authors.forename: Tong
authors.initials: T
authors.lastname: Zhou
authors.forename: Xianhong
authors.initials: X
authors.lastname: Meng
authors.forename: Hui
authors.initials: H
authors.lastname: Che
authors.forename: Nannan
authors.initials: N
authors.lastname: Shen
authors.forename: Dan
authors.initials: D
authors.lastname: Xiao
authors.forename: Xiaotong
authors.initials: X
authors.lastname: Song
authors.forename: Meihua
authors.initials: M
authors.lastname: Liang
authors.forename: Xuelian
authors.initials: X
authors.lastname: Fu
authors.forename: Jiaming
authors.initials: J
authors.lastname: Ju
authors.forename: Yang
authors.initials: Y
authors.lastname: Li
authors.forename: Chaoqian
authors.initials: C
authors.lastname: Xu
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhang
authors.forename: Lihong
authors.initials: L
authors.lastname: Wang
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D000070416:Antagomirs / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D003921:Diabetes Mellitus, Experimental / Q000139:chemically induced / Q000378:metabolism / Q000473:pathology; D015536:Down-Regulation; D005947:Glucose / Q000378:metabolism; D051275:Glucose Transporter Type 4 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D007333:Insulin Resistance; D008297:Male; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D048308:Mitogen-Activated Protein Kinase 14 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D016415:Sequence Alignment; D015398:Signal Transduction
chemical_list: D020413:3' Untranslated Regions; D000070416:Antagomirs; D005947:Glucose; D051275:Glucose Transporter Type 4; D035683:MicroRNAs; D048308:Mitogen-Activated Protein Kinase 14; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D012333:RNA, Messenger; C546641:Pik3cb protein, rat