ABSTRACT
Diabetic nephropathy (DN) is one of the leading causes of mortality in diabetic patients. Long non-coding RNA zinc finger E-box binding homeobox 1 antisense 1 (ZEB1-AS1) plays a crucial role in the development of various diseases, including DN. However, the molecular mechanism of ZEB1-AS1 in DN pathogenesis remains elusive. An in vitro DN model was established by treating HK-2 cells with high glucose (HG). Quantitative polymerase chain reaction (qRT-PCR) was utilized to detect the expression levels of ZEB1-AS1, microRNA-216a-5p (miR-216a-5p), and bone morphogenetic protein 7 (BMP7). Western blot assay was used to evaluate the protein levels of BMP7, epithelial-to-mesenchymal transition (EMT)-related proteins, and fibrosis markers. Additionally, the interaction among ZEB1-AS1, miR-216a-5p, and BMP7 was predicted by MiRcode (http://www.mircode.org) and starBase 2.0 (omics_06102, omicX), and confirmed by luciferase reporter assay. ZEB1-AS1 and BMP7 were down-regulated, while miR-216a-5p was highly expressed in kidney tissues of DN patients. Consistently, HG treatment decreased the levels of ZEB1-AS1 and BMP7, whereas HG increased miR-216a-5p expression in HK-2 cells in a time-dependent manner. ZEB1-AS1 upregulation inhibited HG-induced EMT and fibrogenesis. Furthermore, ZEB1-AS1 directly targeted miR-216a-5p, and overexpression of miR-216a-5p restored the inhibitory effects of ZEB1-AS1 overexpression on EMT and fibrogenesis. BMP7 was negatively targeted by miR-216a-5p. In addition, ZEB1-AS1 suppressed HG-induced EMT and fibrogenesis by regulating miR-216a-5p and BMP-7. lncRNA ZEB1-AS1 inhibited high glucose-induced EMT and fibrogenesis via regulating miR-216a-5p/BMP7 axis in diabetic nephropathy, providing a potential target for DN therapy.
Subject(s)
Humans , Diabetic Nephropathies/metabolism , Bone Morphogenetic Protein 7/metabolism , Epithelial-Mesenchymal Transition/physiology , RNA, Long Noncoding/physiology , Zinc Finger E-box-Binding Homeobox 1/metabolism , Down-Regulation , Up-Regulation , Cells, Cultured , MicroRNAs/metabolism , Diabetic Nephropathies/genetics , Real-Time Polymerase Chain ReactionABSTRACT
Diabetic nephropathy (DN) is one of the leading causes of mortality in diabetic patients. Long non-coding RNA zinc finger E-box binding homeobox 1 antisense 1 (ZEB1-AS1) plays a crucial role in the development of various diseases, including DN. However, the molecular mechanism of ZEB1-AS1 in DN pathogenesis remains elusive. An in vitro DN model was established by treating HK-2 cells with high glucose (HG). Quantitative polymerase chain reaction (qRT-PCR) was utilized to detect the expression levels of ZEB1-AS1, microRNA-216a-5p (miR-216a-5p), and bone morphogenetic protein 7 (BMP7). Western blot assay was used to evaluate the protein levels of BMP7, epithelial-to-mesenchymal transition (EMT)-related proteins, and fibrosis markers. Additionally, the interaction among ZEB1-AS1, miR-216a-5p, and BMP7 was predicted by MiRcode (http://www.mircode.org) and starBase 2.0 (omics_06102, omicX), and confirmed by luciferase reporter assay. ZEB1-AS1 and BMP7 were down-regulated, while miR-216a-5p was highly expressed in kidney tissues of DN patients. Consistently, HG treatment decreased the levels of ZEB1-AS1 and BMP7, whereas HG increased miR-216a-5p expression in HK-2 cells in a time-dependent manner. ZEB1-AS1 upregulation inhibited HG-induced EMT and fibrogenesis. Furthermore, ZEB1-AS1 directly targeted miR-216a-5p, and overexpression of miR-216a-5p restored the inhibitory effects of ZEB1-AS1 overexpression on EMT and fibrogenesis. BMP7 was negatively targeted by miR-216a-5p. In addition, ZEB1-AS1 suppressed HG-induced EMT and fibrogenesis by regulating miR-216a-5p and BMP-7. lncRNA ZEB1-AS1 inhibited high glucose-induced EMT and fibrogenesis via regulating miR-216a-5p/BMP7 axis in diabetic nephropathy, providing a potential target for DN therapy.
ABSTRACT
Background and purpose:MicroRNA (miRNA) belongs to a class of 19 to 30 nucleotide-long, endogenous noncoding RNA expressed in eukaryotes and predominantly inhibits gene expression at the post-transcriptional level. The miRNAs play critical roles in cell proliferation and differentiation, apoptosis, metabolism, and immune regulation. This study aimed to detect the expression of miR-216a-5p in lung cancer tissues and lung cancer cell lines, and to discuss the effects of miR-216a-5p on the invasion ability of lung cancer cells and the mechanism.Methods:Quantitative real-time PCR (qRT-PCR) was used to detect the expression of miR-216a-5p in lung cancer tissues of 55 cases and 7 lung cancer cell lines. Three lung cancer cell lines of A549, 95D and H460 were transiently transfected by miR-216a-5p, and Transwell was used to detect the effects of miR-216a-5p on the invasion of lung cancer cell lines. The dual luciferase reporter plasmids containing the miR-216a-5p candidate target gene and the gene of matrix metalloproteinase 16 (MMP16) were predicted and constructed. qRT-PCR and Western blot were used to detect the changes in mRNA and protein levels of target geneMMP16 by miR-216a-5p. The interference of MMP16 by siRNA and up-regulation miR-216a-5p by transfection were compared on the invasion of lung cancer cells.Results:The miR-216a-5p expression levels were all signiifcantly reduced in 90.91% (50 of 55 patients) tumor tissues compared with corresponding adjacent normal lung tissues (P<0.05). The miR-216a-5p expression levels were only 7.00%-32.00%in 7 lung cancer cells compared with the control group (P<0.05). Up-regulation of the expression of miR-216a-5p inhibited the invasion of lung cancer cells; interference of MMP16 by siRNA, as well as up-regulating miR-216a-5p by transfection, inhibited the expression of MMP16 in lung cancer leading to inhibition of the invasion of lung cancer cells. Conclusion:miR-216a-5p can be a candidate marker in clinical diagnosis and it can inhibit the invasion of lung cancer cells by down-regulating the expression of MMP16.