| Objective:The LncRNA TCONS＿00017953 was significantly up-regulated in patients with atrial fibrillation compared with sinus rhythm(SR),which was screened by gene chip technology.In vitro experiments were conducted to explore whether LncRNA TCONS＿00017953 could regulate the expression of HCN4 gene,thus to demonstrate that LncRNA TCONS＿00017953 could regulates HCN4 expression in the occurrence of atrial fibrillation,and to find new targets for the prevention and treatment of atrial fibrillation.Methods:(1)From December 2014 to October 2015,10 patients(5 patients with permanent AF,5 patients with SR)with rheumatic heart valvular disease aged from 45 to55 years old were selected at the Cardiovascular Department of the affiliated hospital of Southwest Medical University.All patients had no history of taking anti-arrhythmic and anti-heart failure medicines.The RNase-free reagent was used to preserve right atrial appendages removed through the valve replacement surgery.(2)According to our previous research,differentially expressed lncRNAs in AF and SR had been screened,and all the coding genes(m RNAs)in the range of its upstream and downstream 300k had been searched.Through Pearson correlation calculation,the gene with significant co-expression with the LncRNA was selected,and the LncRNA and LncRNA-m RNA pair associating with atrial fibrillation were selected.(3)The total RNA of the right atrial appendage specimens were extracted from the AF group and the SR group,and the expressions of LncRNA TCONS＿00017953and HCN4 m RNA were detected by q PCR.(4)In vitro experiment was desired to verify whether LncRNA TCONS 00017953 could regulate the expression of HCN4.Firstly,H1 line cells(human embryonic stem cells,h ESCs)were induced to differentiate into cardiomyocyte-like cells.Secondly,gene transfection technique was used to up-regulate and down-regulate LncRNA TCONS＿00017953 expression in cardiomyocyte-like cells.The differentiated cardiomyocyte-like cells were assigned into three groups:the LncRNA TCONS＿00017953 up-regulation group,the LncRNA TCONS＿00017953down-regulation group and the blank transfection group.Thirdly,the expression of HCN4 in each group was detected by western blot.Finally,the whole cell patch clamp technique was employed to record the HCN4 channel current changes of the cells in three groups.Results:(1)A total of 333 LncRNAs which may be associated with permanent AF were screen out from 111062LncRNAs(Fold change>2 and P<0.05)by Arraystar human LncRNA microarrays(v3.0).Among them,114 LncRNAs expressions were significantly up-regulated while 219 LncRNAs expressions significantly down-regulated.Three LncRNA-m RNA pairs associated with AF were selected,namely TCONS＿00017953 and HCN4,NONHSAG011610 and KCNN2 as well as TCONS＿l2＿00005793 and KCNJ2.(2)The total RNA extracted from the right atrial appendage tissues were verified by q PCR,and the results showed that the expression of LncRNA TCONS＿00017953 in the AF group significantly up-regulated compared with the SR group(4.542±0.218 vs 1.463±0.126,P=0.009).And the expression of HCN4 in the AF group also significantly increased compared with the SR group(3.285±0.226 vs 1.594±0.164,P=0.039).(3)Firstly,in in-vitro experiment,H1 line cells were induced and successfully differentiated into cardiomyocyte-like cells.Through the immunofluorescence staining of the cardiac specific marker c Tn T,the expression of cardiac specific troponin was observed by the microscope.Secondly,western blot was utilized to detect the expressions of HCN4 in the three groups,and the corresponding results demonstrated that the expression of HCN4 in the LncRNA TCONS＿00017953 up-regulation group significantly up-regulated compared with the LncRNA TCONS＿00017953 down-regulation group(0.879±0.064 vs0.257±0.072,P=0.023,n=2),that the expression of HCN4 in the LncRNA TCONS＿00017953 up-regulation group also significantly up-regulated compared with the blank transfection group(0.879±0.064 vs 0.212±0.048,P=0.026,n=2).Although the expression of HCN4 in the LncRNA TCONS＿00017953 down-regulation group was lower than the blank transfection group,but there was no significant difference(0.212±0.048 vs 0.257±0.072,P=0.824,n=2).Thirdly,according to the results of the whole-cell patch-clamp experiments,at-150 m V,the I_f current density was measured to be-5.1±1.9p A/p F and-10.6±2.7 p A/p F in the blank transfection group and the LncRNA TCONS＿00017953 up-regulation group.Compared with the blank transfection group,the current in the LncRNA TCONS＿00017953 up-regulation group was significantly higher(10.6±2.7 vs 5.1±1.9 P=0.014,n=3).Conclusion:Our study provided evidence that:First,the expression of LncRNA TCONS＿00017953 and HCN4 in the patients with permanent AF was higher than patients with SR.Second,the LncRNA TCONS＿00017953 could regulate the expression of HCN4 in vitro experiment.So we could deduce that LncRNA TCONS＿00017953 might induce AF through regulating HCN4 expression. |
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