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
Objective To detect altered levels and clinical significance of serum miR-216a and prognosis monitoring in acute pancreatitis (AP) patients.Methods Serum miR-216a levels were determined by quantitative reverse-transcription PCR (qRT-PCR) assay among 80 mild acute pancreatitis (MAP) patients,80 severe acute pancreatitis (SAP) patients and 74 healthy controls.And amylase (AMY),lipase (LPS),Ca2+,glucose (Glu),hematocrit (HCT),triglyceride (TG),total cholesterol (TC) and C reactive protein (CRP) were measured by biochemical analyzer.The clinical usefulness of miR-216a for AP patients was assessed by ROC curve analysis and correlation analysis.Results Compared with healthy controls (11.12 × 10-5 [5.83 × 10-5,19.12 × 10-5],the serum miR216a levels were significantly increased in AP patients(38.49 × 10-5 [24.05 × 10-5,62.02 × 10-5],(U =-9.10,P ＜ 0.01) . The serum miR-216a levels in MAP and SAP patients were (36.46 × 10-5 [22.29 × 10-5,55.80 × 10-5] vs 40.44 × 10-5 [25.84 × 10-5,65.48 × 10-5]),there was no significant difference between MAP and SAP patients (U =-0.96,P ＞ 0.05).The areas under ROC curve (AUCROC) of miR-216a for differential healthy controls and AP patients was 0.870 (95％ CI:0.825-0.915),cut-off value is 0.61.AUCROC of miR-216a for differential healthy controls and MAP patients was 0.865 (95％ CI:0.808-0.921),cut-off value is 0.59.And AUCROC of miR-216a for differential healthy controls and SAP patients was 0.876 (95％ CI:0.822-0.930),cut-off value is 0.66.Moreover,after the clinical improvement of the patients,the levels of serum miR-216a were significantly lowered from (41.88 × 10-5 [24.24 × 10-s,64.44 × 10-5]) to (20.58 × 10-5 [11.01 × 10-5,41.91 × 10-5]),the differences was significant(U =5.24,P ＜ 0.0l).Correlation analysis showed that miR-216a was positively correlated with CRP (r =0.215,P =0.006) in AP patients.Conclusion The levels of miR-216a in serum of AP patients were increased,which is a potential biomarker for the diagnosis and prognosis monitoring of AP.
ABSTRACT
Objective To explore the correlation between serum miRNA-216a level and severity of acute pancretitis (AP) .Meth-ods 17 cases of mild acute pancreatitis (MAP group) ,23 cases of severe acute pancreatitis (SAP group) and 30 cases of healthy subjective (control group) were selected in our hospital from June 2015 to June 2016 .Blood amylase activity and serum miRNA-216a were detected ,Ranson value ,APACHEⅡvalue and modified CT severity index (MCTSI) were used to evaluate the severity of AP ,analyze the correlation between miR-216a level and other indexes .Results The serum amylase activity of MAP group and SAP group in acute stage were higher than those in convalescence in these groups and the control group (P<0 .05) ,and the serum amyl-ase activity in the acute phase SAP group was higher than that in the MAP group ,and the difference was statistically significant (P<0 .05) .The relative expressions of serum miR-216a in MAP group and SAP group in acute stage were significantly higher than those in convalescence in these groups and the control group (P<0 .05) ,and the relative expressions of serum miR-216a in SAP group was higher than that in the MAP group ,and the difference was statistically significant (P<0 .05) .The miR-216a expression was positively correlated with Ranson score ,APACHE score and MCTSI score (r=0 .667 ,P<0 .05 ;r=0 .396 ,P<0 .05 ,and r=0 .648 ,P<0 .05) .Conclusion The expression level of serum miR-216a of patients with AP was significantly higher than that of healthy people ,and the expression level of serum miR-216a was positively correlated with the severity of AP ,which was useful for the diagnosis and prognosis of SAP .
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.
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Background and purpose: MicroRNAs are 19–25-nucleotide noncoding RNA molecules that regulate gene expression at the level of transcription and translation. The study aimed to confirm whether miR-216a suppresses cell proliferation and invasion by targeting PRKCA, thus to reveal molecular mechanism that miR-216a functions as a tumor suppressor in glioma. Methods: PRKCA 3’ untranslated region (UTR)-luciferase vector was constructed and dual-luciferase reporter gene assay was employed to examine the effect of miR-216a on luciferase activity. U251 cells were transfected with miR-216a mimics, and next Western blotting was performed to detect the expression of PRKCA protein. The effects of PRKCA downregulation on cell proliferation and invasion were observed after PRKCA siRNA was transfected into U251 cells. U251 cell proliferation assays were performed when cotransfected with miR-216a mimics. Results:The result demonstrated miR-216a could bind to the 3’UTR of PRKCA and inhibited the luciferase activity by 41%. PRKCA protein expressions were significantly down-regulated when miR-216a was overexpressed in U251. siRNA-mediated downregulation of PRKCA could suppress the potentials of cell proliferation and invasion. Conclusion:miR-216a suppresses cell proliferation and invasion by targeting PRKCA in glioma.
ABSTRACT
Objective To confirm whether miR-216a suppresses cell proliferation and induces cell apoptosis by targeting PKCα, thus to reveal molecular mechanism that miR-216a functions as a tumor suppressor in gastric cancer .Methods PKCα3′untranslat-ed region(UTR)-luciferase vector was constructed and dual-luciferase reporter gene assay was employed to examine the effect of miR-216a on luciferase activity .MGC-803 cells were transfected with miR-216a mimics ,and next Western blotting was performed to detect the expression of PKCαprotein .The effects of PKCαdownregulation on cell proliferation and apoptosis were observed after PKCαsiRNA were transfected into MGC-803 cells .MGC-803 cell proliferation assays were performed when cotransfected with miR-216a mimics .Results The result demonstrated miR-216a could bind to the 3′UTR of PKCαand inhibited the luciferase activi-ty ,cut the 41% .PKCαprotein expressions were significantly down-regulated when miR-216a was overexpressed in MGC-803 .siR-NA-mediated downregulation of PKCα could suppress the potentials of cell proliferation and induce apoptosis .Conclusion miR-216a suppresses cell proliferation and induces apoptosis by targeting PKCαmRNA 3′UTR in gastric cancer .