Molecular Epidemiological Study Of The Role And Mechanism Of Circular RNA Hsa＿circ＿0000118 And Long Non-coding RNA H19 In Atrial Fibrillation

Atrial fibrillation（AF）is a frequently occurring arrhythmia in clinical practice.The harm of atrial fibrillation is mainly reflected in its complications,including stroke,heart failure and so on.Its harm not only lies in the health of patients,but also brings heavy economic burden to patients and their families.With the acceleration of the aging process in China,the prevalence of atrial fibrillation is also increasing year by year,which has become a serious public health problem in China.Meanwhile,the mechanism of atrial fibrillation is not very clear.Recent studies suggest that both genetic and epigenetic factors may significantly contribute to the development of AF.Non-coding RNAs（nc RNA）have been found to be involved in the development of many cardiovascular diseases and may be used as a biomarker for such diseases.However,the relationship between non-coding RNA and AF is still not well understood.Based on this background,we conducted a study in which we screened long non-coding RNAs（lnc RNA）and circular RNAs（circ RNA）with prognostic value for AF using high-throughput transcriptome sequencing and online literature databases.We also explored the role and mechanism of circ RNA hsa＿circ＿0000118 with prognostic value for AF in atrial remodeling.Part Ⅰ The role of peripheral blood circular RNA hsa＿circ＿0000118 and long non-coding RNA H19 in the prognosis of atrial fibrillationObjectivesWe aimed to identify circ RNAs and lnc RNAs associated with atrial fibrillation prognosis by conducting a cohort study.We screened them using high-throughput transcriptome sequencing and online literature databases,and assessed their predictive value for prognosis.MethodsIn our study,we first screened circ RNAs and lnc RNAs that may be associated with AF from the heart atrium tissue high-throughput transcriptome sequencing data of 7 atrial fibrillation patients and 7 controls,and verified their stability in plasma.We then conducted a retrospective cohort study of 192 newly diagnosed AF patients,using the Cox proportional hazard regression model to analyze the association between plasma expression of circ RNAs and lnc RNAs and two prognostic outcomes of all-cause mortality and stroke,and drawing Kaplan-Meier curves.Our study aimed to investigate whether circ RNA and lnc RNA with prognostic predictive value could improve the accuracy of CHA₂DS₂-VASc score of predicting stroke and all-cause mortality in atrial fibrillation patients.Results1.In our study,we screened 15 circ RNAs and 14 lnc RNAs from high-throughput transcriptome sequencing and online literature database at last,and verified the plasma stability of the above 29 candidate nc RNAs in plasma samples of 29 AF patients.Finally,we selected the lnc RNAs:NDUFV2P1,RPL18AP3,H19,GAS5 and UCA1,and circ RNA:hsa＿circ＿0000118 with plasma stability for subsequent retrospective cohort study.2.We conducted a retrospective cohort study of 192 newly diagnosed non-valvular atrial fibrillation patients and found that high expression of hsa＿circ＿0000118 in plasma was associated with a higher risk of all-cause mortality after adjusting for confounding factors using a multivariate Cox proportional hazard regression model.Additionally,high levels of lnc RNA H19 in plasma were independently associated with stroke outcome.Kaplan-Meier curves demonstrated that high levels of hsa＿circ＿0000118 and H19 in plasma were significantly associated with all-cause mortality and stroke,respectively.3.We compared the discrimination,reclassification,calibration and clinical net benefit of the CHA₂DS₂-VASc score model before and after incorporating hsa＿circ＿0000118 and H19.The results showed that after adding the relative level of H19 in the analysis of stroke model,the c-statistic of CHA₂DS₂-VASc score was 0.74（95%CI:0.62-0.79）,the c-statistic increment was 0.04（95%CI:0.01-0.07;P=0.02）,and the c-statistic was significantly increased;after adding the relative level of hsa＿circ＿0000118 in the analysis of all-cause mortality,the c-statistic of CHA₂DS₂-VASc score was 0.68（95%CI:0.62-0.79）,the C statistic increment was 0.03（95%CI:0.01-0.07;P=0.02）,and the c-statistic was marginally significantly increased（P=0.10）.ConclusionsOur study found that high levels of circ RNA hsa＿circ＿0000118 in plasma are an independent risk factor for all-cause mortality prognosis outcome of atrial fibrillation.Adding it to the CHA₂DS₂-VASc score model can improve the predictive value of the score for all-cause mortality.Additionally,high levels of lnc RNA H19 in plasma are an independent risk factor for stroke prognosis outcome of atrial fibrillation.Adding it to the CHA₂DS₂-VASc score model can improve the predictive value of the score for stroke.Part Ⅱ The mechanism study of mmu＿circ＿0010297,the conserved mm9 circ RNA of hsa＿circ＿0000118,promoting fibrosis of mouse cardiac fibroblast via mmu-mi R-34a-5p/Smad4 axis.ObjectivesWe aim to investigate the effects of mmu＿circ＿0010297,the homologous circ RNA of hsa＿circ＿0000118 in mice,on the proliferation,migration,differentiation to myofibroblasts,and collagen expression of cardiac fibroblasts.Additionally,we aim to reveal the role of mmu＿circ＿0010297 in the process of structural remodeling in atrial fibrillation.MethodsGain-of-function cell models were created by transfecting p LC5-ci R-C10297 vectors,which overexpressed mmu＿circ＿0010297,into mouse cardiac fibroblasts.Additionally,the expression of mmu＿circ＿0010297 in mouse cardiac fibroblasts was knocked down using two independent si RNAs.The gain-and loss-of-function of mmu＿circ＿0010297 were evaluated using Cell Counting Kit-8 and cell migration assays in mouse cardiac fibroblasts in vitro.QRT-PCR was used to measure the expression of Acta2,Vim,Col1a1,and Col3a1.Western blot was used to detect whether the expression of Col1a1 and Col3a1 proteins changed with the expression level of mmu＿circ＿0010297 in mouse cardiac myofibroblasts.RNA fluorescence in situ hybridization（FISH）was used to investigate the subcellular localization of mmu＿circ＿0010297 in mouse cardiac myofibroblasts.Possible mi RNAs associated with mmu＿circ＿0010297 were screened by integrating the prediction results of three online target mi RNA databases and the results of previous studies.Whether there is mi RNA that conforms to the reverse regulation trend in the cardiac fibroblasts overexpressed or interfered with mmu＿circ＿0010297 was observed.After determining the possible target mi RNA,RNA binding protein immunoprecipitation（RIP）assay and Dual-Luciferase Reporter experiment were performed to verify whether mmu＿circ＿0010297 targets and binds mmu-mi R-34a-5p.A recovery experiment was also carried out to verify whether the promotion effect of mmu＿circ＿0010297 on the expression of downstream target gene Smad4 can be reversed by overexpressing mmu-mi R-34a-5p.Results1.Our study found that the overexpression of mmu＿circ＿0010297 promotes the migration of mouse cardiac myofibroblasts,while knockdown of mmu＿circ＿0010297attenuates their migration ability.Additionally,the expression levels of fibrosis genes Col1a1and Col3a1 were significantly up-regulated in mouse cardiac myofibroblasts overexpressing mmu＿circ＿0010297,while they were significantly down-regulated in mouse cardiac myofibroblasts in which mmu＿circ＿0010297 was knocked down.The Western blot experiment results confirmed the positive regulation relationship between the expression levels of Col1a1 and Col3a1 and the expression level of mmu＿circ＿0010297.2.Mmu＿circ＿0010297 was distributed in both the cytoplasm and nucleus of mouse cardiac fibroblasts.3.Using a combination of prediction results from three mi RNA databases（mi Randa,mi RDB,and RNA22）and previous studies,we identified ten mi RNAs that may bind to mmu＿circ＿0010297.These mi RNAs include mmu-mi R-6946-3p,mmu-mi R-7032-3p,mmu-mi R-6387,mmu-mi R-6987-5p,mmu-mi R-546,mmu-mi R-7675-3p,mmu-mi R-686,mmu-mi R-1951,mmu-mi R-7215-5p,and mmu-mi R-34a-5p.After overexpression or knockdown of mmu＿circ＿0010297,we observed that the expression levels of mmu-mi R-34a-5p and mmu-mi R-6387 in mouse cardiac fibroblasts were in accordance with the inverse regulation relationship.4.The RIP assay results showed that the concentrations of mmu＿circ＿0010297 and mmu-mi R-34a-5p were significantly higher in the precipitates of the Ago2 group compared to the precipitates of the Ig G control group.In addition,the Dual-Luciferase Reporter experiments revealed that the wild-type dual-luciferase reporter construct,which contained the wild-type sequence near the binding site of mmu＿circ＿0010297 and mmu-mi R-34a-5p,was significantly inhibited by the overexpression of mmu-mi R-34a-5p.However,the luciferase activity of the mutant group did not significantly change.5.The recovery experiment demonstrated that the promoting effect of overexpressing mmu＿circ＿0010297 on the downstream target gene Smad4 could be counteracted by overexpressing mmu-mi R-34a-5p.ConclusionsOur study found that mmu＿circ＿0010297,the mouse homologous circ RNA of hsa＿circ＿0000118,promoted the migration and expression of fibrosis markers gene Col1a1and Col3a1 in mouse cardiac fibroblasts.Furthermore,mmu＿circ＿0010297 was found to regulate the downstream target gene Smad4 by acting as an endogenous sponge of mmu-mi R-34a-5p.Part Ⅲ Mmu＿circ＿0010297 regulates ion channel in mouse cardiomyocytes through the FUS/Kcnn3 pathway,and participates in the mechanism of atrial electrical remodeling.ObjectivesExplore whether mmu＿circ＿0010297 affects apoptosis of cardiomyocytes and the expression of ion channel proteins in cardiomyocytes,then reveal its role in the process of atrial fibrillation electrical remodeling.MethodsInvestigate the influence of mmu＿circ＿0010297 on the action potential of mouse cardiomyocytes by patch clamp whole-cell recording technique.Observe whether the expression of apoptosis,transient outward potassium channels(I_to),fast sodium channels(I_NA),and Small-conductance calcium-activated potassium channels(I_SK)in mouse cardiomyocytes is affected by mmu＿circ＿0010297.Record the current intensity of the calcium-activated potassium current through patch clamp whole-cell recording technique to explore the effect of mmu＿circ＿0010297 on I_SK.Apply FISH experiment to explore the subcellular localization of mmu＿circ＿0010297 in mouse cardiomyocytes.Integrate the prediction results of circ RNA targeting binding RNA binding protein（RBP）databases to obtain the RBP that may bind to mmu＿circ＿0010297,and predict whether Kcnn3 gene and candidate target RBP exist target binding relationship.Then carry out RIP experiment to verify whether it is targeted binding.Verify whether mmu＿circ＿0010297 regulates the expression of Kcnn3 through RBP FUS by recovery experiment.Results1.The apoptosis of mouse cardiomyocytes was not affected by mmu＿circ＿0010297.2.Overexpression of mmu＿circ＿0010297 will shorten the action potential duration of mouse cardiomyocytes,while knockdown of mmu＿circ＿0010297 will prolong the action potential duration of mouse cardiomyocytes.3.These results suggest that overexpression of mmu＿circ＿0010297 can significantly up-regulate the expression level of Kcnn3 in mouse cardiomyocytes,while knockdown of mmu＿circ＿0010297 can significantly down-regulate the expression level of Kcnn3.Furthermore,the patch clamp whole-cell recording technique recording the current intensity of I_SK showed that the current intensity of I_SK was significantly increased after overexpression of mmu＿circ＿0010297,and the current intensity of I_SK was significantly decreased after knockdown of mmu＿circ＿0010297.4.The results showed that mmu＿circ＿0010297 was distributed in both the cytoplasm and nucleus of mouse cardiomyocytes.5.Combining the prediction results of cat RAPID,RBPDB and RBPmap,three possible RBPs that may bind to circ RNA were obtained:FUS,KHSRP and KHDRBS3.Meanwhile,the target RBP prediction results of Kcnn3 showed that there were binding sites between FUS and KHSRP.The RIP experiment results showed that mmu＿circ＿0010297 was significantly enriched in the precipitation of FUS group compared with the precipitation in the Ig G control group.6.The rescue experiment results showed that the promotion effect of mmu＿circ＿0010297 on the expression of downstream target gene Kcnn3 could be reversed by knocking down FUS.ConclusionsMmu＿circ＿0010297,the mouse homologous circ RNA of hsa＿circ＿0000118,may enhance the I_SK current intensity of mouse cardiomyocytes and shorten the action potential duration,leading to electrical remodeling.Mmu＿circ＿0010297 can bind to RBP FUS,thus regulating the downstream target gene Kcnn3.