| Objective: Atrial fibrillation(AF)is one of the most common arrhythmia in the world and the incidence of AF rises with the age increases.AF could induce palpitation and heart failure and increase the risks of stroke and death.People investigated the mechanism of AF in the past years to relieve the symptoms,improve the function of heart and make the risks of stroke and death lower.However,the results of drug therapy,catheter ablation and MAZE procedure were far from satisfactory,which was owing to the non-understanding of the mechanism of AF.Metabonomic study found that the expression of metabolismrelated proteins changed in AF patients,which indicted that energy metabolism played a role in the development of AF.Mitochondrial transcription factor A(TFAM)is encoded in nucleus and transported into mitochondria to take actions.The human TFAM gene is located at 10q21 and the TFAM protein is a 25 kD molecule which belongs to high mobility protein family.TFAM is composed of 2 high mobility groups and a C-tail.The high mobility groups could connect to DNA and the C-tail could interact with mitochondrial transcription factor B1 and mitochondrial transcription factor B2 or interact with the specific sequence in the upstream of promotor to activate the transcription.The effect of TFAM on mitochondrial DNA leads to the regulation of expression of mitochondrial genome,which has an effect on ATP synthesis.As reported,the abnormal content and function of TFAM took part in the development of many diseases,such as cancer,neurodegeneration diseases and cardiovascular diseases.However,the expression level and function of TFAM in AF tissues have not been reported.Long noncoding RNAs,which consists of more than 200 nucleotides,play an important role in the development of various diseases,although they do not encode proteins.As reported,long noncoding RNAs were involved in cancer,Alzheimer’s disease,cardiomyopathy,fibrosis and so on.Recent studies showed that the abnormal expression of long noncoding RNAs in AF patients.We selected the differential expressed long noncoding RNAs through the microarray database and found that the fold change of LINC01018 was about 0.25 in the AF tissues compared to sinus rhythm.However,the expression,function and mechanism of LINC01018 in AF tissues and cardiomyocytes require further investigation.The purpose of this study is to explore the expression of LINC01018 in AF tissues and sinus rhythm tissues and investigate the function of LINC01018 in cardiomyocytes.In addition,we want to demonstrate that LINC01018 has an effect on energy synthesis of cardiomyocytes by the regulation of TFAM and explain the regulatory mechanism between LINC01018 and TFAM.Methods:1.From 2017.6 to 2019.5,left atrial appendage(LAA)tissues were collected from 40 AF patients and 20 sinus rhythm patients who were treated by cardiac surgery in the First Affiliated Hospital of China Medical University.2.The expression of TFAM in AF and SR tissues was detected by RT-qPCR and Western Blot.The location and expression of TFAM was tested by immunohistochemistry and average optical density was calculated.The relationship between the expression of TFAM in AF tissues and clinical characteristics was analyzed by chi-square test and the correlation was analyzed by Pearson correlation.3.The ATP content in AF and SR tissues was tested by ATP assay kit and the expression of proteins related to ATP synthesis were tested by RT-qPCR 和 Western Blot.4.We selected the differential expressed long noncoding RNAs between AF and SR tissues through the microarray database and predicted the microRNA by bioinformatics software.5.The expression of LINC01018 and miR-199a-3p in AF and SR tissues were measured by RT-qPCR.6.We used the primary cultured cardiomyocytes and constructed the model of rapid pacing cardiomyocytes.7.The expression of LINC01018 and miR-199a-3p in rapid pacing and non-pacing cardiomyocytes were measured by RT-qPCR.Luciferase assay was used to prove whether LINC01018 can directly bind to mi R-199a-3p.The expression of TFAM in rapid pacing and non-pacing cardiomyocytes were measured by RT-qPCR and Western Blot.The ATP content in rapid pacing and non-pacing cardiomyocytes were measured by ATP assay kit.The expression of molecules related to ATP synthesis in rapid pacing and non-pacing cardiomyocytes were measured by RT-qPCR and Western Blot.8.Transfection efficiency was detected by RT-qPCR after LINC01018 transfection.RT-qPCR and Western Blot were used to test the effect of LINC01018 on the expression of miR-199a-3p and TFAM.ATP assay kit was used to test the effect of LINC01018 on the ATP content.Western Blot was used to test the effect of LINC01018 on the expression of molecules related to ATP synthesis.9.Transfection efficiency was detected by RT-qPCR after miR-199a-3p transfection.RT-qPCR and Western Blot were used to test the effect of mi R-199a-3p on the expression TFAM.ATP assay kit was used to test the effect of mi R-199a-3p on the ATP content.Western Blot was used to test the effect of miR-199a-3p on the expression of molecules related to ATP synthesis.10.Transfection efficiency was detected by RT-qPCR after TFAM transfection.ATP assay kit was used to test the effect of TFAM on the ATP content.Western Blot was used to test the effect of TFAM on the expression of molecules related to ATP synthesis.11.The expression of TFAM and molecules related to ATP synthesis and ATP content were tested after cotransfection of LINC01018 overexpression plasmid and miR-199a-3p mimic in rapid pacing cardiomyocytes.12.The expression of TFAM and molecules related to ATP synthesis and ATP content were tested after co-transfection of si-LINC01018 and mi R-199a-3p inhibitor in non-pacing cardiomyocytes.Results: 1.The ATP content was decreased in AF tissues compared to SR tissues.The ATP content was decreased in rapid pacing cardiomyocytes compared to non-pacing cardiomyocytes.2.The electron microscope showed that the morphology of mitochondria in SR tissue was uniform and the cristae were clear and regularly distributed in the mitochondria.However,in AF tissue,the mitochondria became swollen,the morphology was irregular and the cristae were disappeared.3.The expression of MT-ND1 and MTCO1 were decreased in AF tissues compared to SR tissues.However,there was no difference in the expression of NDUFS1 and COX6 C between AF and SR tissues.The expression of MT-ND1 and MT-CO1 were decreased in rapid pacing cardiomyocytes compared to non-pacing cardiomyocytes.However,there was no difference in the expression of NDUFS1 and COX6 C between rapid pacing and non-pacing cardiomyocytes.4.The expression of TFAM was decreased in AF tissues compared to SR tissues and there was a negative correlation between the expression of TFAM and age,history and left atrial diameter,separately.5.The expression of TFAM were decreased in rapid pacing cardiomyocytes compared to non-pacing cardiomyocytes.The overexpression of TFAM could increase the ATP content and the expression of MT-ND1 and MT-CO1.Downregulation of TFAM could decrease the ATP content and the expression of MT-ND1 and MT-CO1.6.The expression of miR-199a-3p were increased in AF tissues compared to SR tissues.7.The expression of miR-199a-3p were increased in rapid pacing cardiomyocytes compared to non-pacing cardiomyocytes.Downregulation of miR-199a-3p could increase the expression of TFAM.Downregulation of miR-199a-3p could increase the ATP content and the expression of MT-ND1 and MT-CO1.Upregulation of miR-199a-3p could decrease the expression of TFAM.Upregulation of miR-199a-3p could decrease the ATP content and the expression of MT-ND1 and MT-CO1.8.The expression of LINC01018 were decreased in AF tissues compared to SR tissues.9.The expression of LINC01018 were decreased in rapid pacing cardiomyocytes compared to non-pacing cardiomyocytes.The overexpression of LINC01018 could decrease the expression of miR-199a-3p.The overexpression of LINC01018 could increase the expression of TFAM,MTND1 and MT-CO1.The overexpression of LINC01018 could increase the ATP content.Downregulation of LINC01018 could increase the expression of miR-199a-3p.Downregulation of LINC01018 could decrease the expression of TFAM,MT-ND1 and MT-CO1.Downregulation of LINC01018 could decrease the ATP content.10.The downregulation of miR-199a-3p could partially restore the decrease of ATP induced by transfection of si-LINC01018.11.Luciferase assay showed that LINC01018 could bind to miR-199a-3p directly and LINC01018 could regulate the expression of TFAM by sponging miR-199a-3p.Conclusion: 1.The ATP content was decreased in AF tissues and rapid pacing cardiomyocytes.2.The expression of TFAM was decreased in AF tissues and there was a negative correlation between the AOD of TFAM and age,history and left atrial diameter,separately.3.The expression of TFAM was decreased in rapid pacing cardiomyocytes and upregulation of TFAM could increase the ATP content in rapid pacing cardiomyocytes.4.The expression of LINC01018 was decreased in AF tissues and rapid pacing cardiomyocytes.Upregulation of LINC01018 could increase the ATP content in rapid pacing cardiomyocytes.5.LINC01018 could play a role in the synthesis of ATP in cardiomyocytes through functioning as a competing endogenous RNA to regulate TFAM expression by sponging miR-199a-3p. |
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