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Objective To explore the effect of microRNA-22-3p (miR-22-3p) regulating the expression of Kruppel-like factor 6 (KLF6) on the cardiomyocyte-like differentiation of bone marrow mesenchymal stem cell (BMSC). Methods Rat BMSC was isolated and cultured,and the third-generation BMSC was divided into a control group,a 5-azacytidine(5-AZA)group,a mimics-NC group,a miR-22-3p mimics group,a miR-22-3p mimics+pcDNA group,and a miR-22-3p mimics+pcDNA-KLF6 group.Real-time fluorescent quantitative PCR (qRT-PCR) was carried out to determine the expression of miR-22-3p and KLF6 in cells.Immunofluorescence staining was employed to detect the expression of Desmin,cardiac troponin T (cTnT),and connexin 43 (Cx43).Western blotting was employed to determine the protein levels of cTnT,Cx43,Desmin,and KLF6,and flow cytometry to detect the apoptosis of BMSC.The targeting relationship between miR-22-3p and KLF6 was analyzed by dual luciferase reporter gene assay. Results Compared with the control group,5-AZA up-regulated the expression of miR-22-3p (q=7.971,P<0.001),Desmin (q=7.876,P<0.001),cTnT (q=10.272,P<0.001),and Cx43 (q=6.256,P<0.001),increased the apoptosis rate of BMSC (q=12.708,P<0.001),and down-regulated the mRNA (q=20.850,P<0.001) and protein (q=11.080,P<0.001) levels of KLF6.Compared with the 5-AZA group and the mimics-NC group,miR-22-3p mimics up-regulated the expression of miR-22-3p (q=3.591,P<0.001;q=11.650,P<0.001),Desmin (q=5.975,P<0.001;q=13.579,P<0.001),cTnT (q=7.133,P<0.001;q=17.548,P<0.001),and Cx43 (q=4.571,P=0.037;q=11.068,P<0.001),and down-regulated the mRNA (q=7.384,P<0.001;q=28.234,P<0.001) and protein (q=4.594,P=0.036;q=15.945,P<0.001) levels of KLF6.The apoptosis rate of miR-22-3p mimics group was lower than that of 5-AZA group (q=8.216,P<0.001).Compared with the miR-22-3p mimics+pcDNA group,miR-22-3p mimics+pcDNA-KLF6 up-regulated the mRNA(q=23.891,P<0.001) and protein(q=13.378,P<0.001)levels of KLF6,down-regulated the expression of Desmin (q=9.505,P<0.001),cTnT (q=10.985,P<0.001),and Cx43 (q=8.301,P<0.001),and increased the apoptosis rate (q=4.713,P=0.029).The dual luciferase reporter gene experiment demonstrated that KLF6 was a potential target gene of miR-22-3p. Conclusion MiR-22-3p promotes cardiomyocyte-like differentiation of BMSC by inhibiting the expression of KLF6.
Subject(s)
Animals , Rats , Myocytes, Cardiac , Kruppel-Like Factor 6 , Connexin 43 , Desmin , Cell Differentiation , Azacitidine/pharmacology , Mesenchymal Stem Cells , RNA, Messenger , MicroRNAsABSTRACT
Objective To investigate the expression and clinical value of miRNA -22 in esophageal and gastric cancer tissues and cell lines.Methods Realtime -PCR was performed to detect the expression of miRNA -22 in human esophageal cancer cell lines ECA109,TE -1,TE -8,TE -13 and normal esophageal epithelial cells HEEC,48 cases of esophageal cancer tissues and matched adjacent normal tissues,human gastric cancer cell lines SGC -7901,MKN -45,NCI -N87,AGS,NUGC -3,SUN -1,normal human gastric mucosa cell line GES -1 and normal stomach fibroblastic cell NSFC,88 cases of gastric cancer tissues and matched adjacent normal tissues.Results The expression of miRNA -22 was much less in four esophageal cancer cell lines than that of immortalized normal esophageal epithelial cells HEEC,and the expression of miRNA -22 was much less in six gastric cancer cell lines than that of gastric mucosal epithelial cell line GES -1 and normal stomach fibroblastic cell line NSFC.The esophageal cancer tissues showed aberrant down regulation of miRNA -22 compared with the adjacent non -tumor tissues [(3.51 ±1.05)vs.(11.23 ±2.95),t =18.13,P <0.05].The gastric cancer tissues showed aberrant down regula-tion of miRNA -22 compared with the adjacent non -tumor tissues [(2.15 ±1.23)vs.(9.14 ±2.86),t =22.93, P <0.05].Conclusion The expression of miRNA -22 was much lower in esophageal and gastric cancer tissues and cell lines,which provided basement to nosogenesis,early diagnosis and create drug treatment of the cancers.
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Objective To explore the role of microRNA (miR)-22 and miR-1825 in the diagnosis and differential diagnosis of juvenile systemic lupus erythematous (JSLE).Methods The cases of JSLE hospitalized in Capital Institute of Pediatrics Teaching Hospital Affiliated to Peking University from June 2013 to May 2014 were selected as study group.The cases with systemic juvenile idiopathic arthritis (sJIA),nephrotic syndrome (NS),Kawasaki disease (KD),Henoch-Schonlein purpura(HSP) were selected as patients control group.The healthy children were selected as healthy control group.The expression levels of miR-22 and miR-1825 in the plasma of JSLE,sJIA,NS,KD,HSP and healthy children were detected by using real-time PCR respectively.Receiver operating characteristic curve (ROC) analysis was performed to evaluate the value of miR-22 and miR-1825 miRNA as a biomarker with the sensitivity and specificity.Three data bases,included Targetscan,PicTar and miRanda,were applied to predict the target gene.The target gene was analyzed by adopting Gene Ontology (GO) in terms of molecular function,biological process and cellular component,and by adopting Kyoto Encyclopedia of Genes and Genomes (KEGG) in terms of pathway.Results Compared with healthy children,the amount of miR-22 and miR-1825 in JSLE patients were lower,and there were significant differences(t =-3.076,-9.054,P ＜0.01,0.000 1).The levels of the miR-22 and miR-1825 miRNAs in controls of sJIA,NS,KD,HSP were significantly higher than those of JSLE (t =-4.410,-4.477,-4.494,-2.971,all P ＜ 0.000 1;t =-9.043,-6.045,-10.416,-8.712,all P ＜ 0.000 1),but there was no difference compared with healthy children(all P ＞ 0.05).The area under ROC curve(AUC) of miR-22 between JSLE and healthy children was 0.777.The AUC of miR-1825 between JSLE and healthy children was 1.000.The AUCs between JSLE and controls of sJIA,NS,KD,HSP of miR-22 were 0.731-1.000.The AUCs between JSLE and controls of sJIA,NS,KD,HSP of miR-1825 were 0.939-1.000.There was positive relation between the amount of miR-22 and complement C3 in plasma(r =0.493,P =0.027).Conclusions The amount of miR-22 and miR-1825 in the plasma of JSLE embrace the potential of distinguishing JSLE from healthy children,sJIA,NS,KD,HSP.MiR-22 has the ability to predict the activity of JSLE.
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Objective To investigate the value of plasma miR-22 in diagnosis of idiopathic pulmonary arterial hypertension ( IPAH ),and its role of regulation mechanisms in the pathogenesis of the disease.Methods Circulating miR-22 levels of IPAH patients and healthy controls were evaluated by RTPCR.The silico analysis of targets for miR-22 was taken, and followed by eGFP reporter assay for verification of predicted target gene Myc binding protein (MYCBP). Results Compared with healthy controls,the expression of plasma miR-22 in IPAH patients was significantly decreased (P ＜ 0.01 ).The area under curve (AUC) of ROC curve was 0.744.MYCBP was a real target of miR-22 confirmed by silico analysis and eGFP reporter assay. Conclusions The expression of plasma miR-22 was significantly decreased in IPAH patients,and it could serve as a potential biomarker for diagnosis.The miR-22 might be involved in the pathogenesis of the disease through promoting its target gene MYCBP to activate the c-Myc pathway.