Circ_0004641 upregulates TRIM25/TRIM41 expression by sponging miR- 1192 to induce cardiac hypertrophy in vitro and in vivo

Circ_0004641 upregulates TRIM25/TRIM41 expression by sponging miR- 1192 to induce cardiac hypertrophy in vitro and in vivo | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Circ_0004641 upregulates TRIM25/TRIM41 expression by sponging miR- 1192 to induce cardiac hypertrophy in vitro and in vivo Liu Guang-cheng, Haipeng Zhang, Jingdai Zhang, Hao Qian, Liang Wang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4452125/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Aim To verify the cardiac hypertrophical role of circ_0004641 and its potential mechanism by targeting miR1192-TRIM25/TRIM41 axis. Methods Mice model was constructed by abdominal aortic coarctation (AAC) surgery and cell model was established from isolated neonatal mouse ventricular cardiomyocytes (NMVCs) by co-cultured with angiotensin II (Ang II). Differential expressed circRNAs were identified by Next-generation sequencing and target miRNAs and downstream mRNAs were predicted by bioinformational analysis. RT-qPCR and Western Blot were applied to validate myocardial-associated molecules on transcriptional and translational levels respectively. By transfecting siRNAs or co-culturing with mimics, expression of regulating-molecules was detected respectively. Dual luciferase reporter assay was performed to identify the interaction between circular RNA and miRNA. Results A total of 5 up-regulation and 25 down-regulation circRNAs were detected on hypertrophical myocardium by Next-generation sequencing. Among them, circ_0004641 was significantly increased both in in vivo and in vitro as ANP and β-MHC accumulated, while downstream target miRNA-1192 decreased and target mRNA (TRIM25/TRIM41) increased dramatically. Knock-down of circ_0004641 by transfecting siRNA shows a reverse effect on cardiac hypertrophy, along with contrary expressive trend of miR-1192 and TRIM25/TRIM41.Dual luciferase reporter assay identified the sponge-like interaction between circ_0004641 and miR-1192. By co-culturing NMVCs with miR-1192 mimics, its targets TRIM25/TRIM41 showed significant decrease. Moreover, NF-κB signaling pathway were identified to correlated by circ_0004641/miRNA-1192 axis as P65 protein present similar expressive trend with circ_0004641. Conclusion circ_0004641 may exert a stimulative role in cardiac hypertrophy by regulating miR-1192-TRIM25/TRIM41 axis and NF/κB p65 pathway is the underlying downstream pathway. Circular RNA CeRNA Cardiac hypertrophy Heart Failure TRIM Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Pathological cardiac hypertrophy is a maladaptive cardiac remodeling in variable clinical diseases involving Hypertension, cardiomyopathy and so on, which progressively deteriorate to Heart Failure(HF) 1 – 5 , remaining a major public healthcare problem with high morbidity and mortality in the worldwide 6 , 7 .There is an unmet need to detect underlying heart failure sensitively and to reverse myocardial enlargement and fibrosis effectively 5 . Several stimuli and signaling mechanisms have been postulated in pathological cardiac hypertrophy 8 – 10 , not just by pressure overloading but molecules on genome and transcriptional levels, such as impaired Ca2 + handling 11 , mitochondrial dysfunction 12 , 13 , oxidative stress 14 , 15 ,m6A methyletation 16 – 18 and so on. Researchers are devoting to resolve the knotty matter and breakthroughs are made via next-generation sequencing, which brings non-coding RNAs into researchers' fields of vision, shedding new lights on the cardiac complexity. With the advancement of transcriptional sequencing, non-coding RNAs(ncRNAs) are receiving increasing attention, including the newly emerging circRNA molecules entering researchers' horizons 19 – 21 . As an important transcription regulatory factor, circular RNA can act as a miRNA sponge, regulator of mRNA translation, or directly participate in gene transcription as a transcription factor 22 , 23 .Generally formulated by non-canonical back-splicing from parental gene, cirRNAs always exert as miRNA sponges 24 , 25 , thus, to modulate gene expression by acting as competing endogenous RNA(CeRNA). For example, circ_000203 can enhance the expression of fibrosis-associated genes in cardiac fibroblasts by depressing targeted miR-26b-5p and induce cardiac fibrosis 26 . While in contrast, circRNA HRCR can protect heart from pathological hypertrophy by targeting miR-223 27 . So far, many CeRNA networks has been identified as stimulating or inhibiting role in atherosclerosis (circ_0026218/ miR-338-3p 28 , circUSP36/miR-637 29 , circCHFR/miRNA-15b-5p 30 ), myocardial infarction(CDR1as/ miRNA-671-5p 31 , circNFIB/ miR-433 32 ), heart Failure induced by myocardial hypertrophy (circNfix/miR-145-5p 33 , circSlc8a1/miR-133a 34 , circmiRs/miR-132 35 )and so on. Those results contributing circRNA/miRNA axis as next molecular therapy for cardiac hypertrophy treatment, which are capturing massive attention. This study identified a novel circRNA(circ_0004641) in mice myocardium and constructed a brand-new CeRNA network (circ_0004641/miRNA-1192 axis). Loss of-and gain of function experiments demonstrated the essential role of circ_0004641/miR-1192 axis in regulation of myocardial hypertrophy both in vivo and in vitro by regulating downstream mRNA (TRIM25/TRIM41). Underlying function of NF-κB signaling pathway were also probed by regulation of CeRNA network. Results 1. Construction of cardiac hypertrophy models by AAC surgery We firstly established pressure overloading-induced myocardial hypertrophy model in C57B/L mice at 8 weeks after AAC surgery. Echocardiography and Staining was performed to evaluate the heart change concurrently. H.E. staining, Sirius red and WGA staining showed that the cross-sectional area of the myocardium increased on surgical group ( Fig. 1 A ) , along with increased Mass of hearts and enlarged ventricle (S1, Figure S1 A-D). Compared to sham group, LVPW, LVID, IVS, LV volume of surgical AAC mice were significantly increased both in cardiac systole and diastole period ( Fig. 1 B, C; S1, Figure S1 E-F) while EF and CO were sharply decreased ( Fig. 1 D, E ). Moreover, Western Blotting were applied to detect the cardiac biomarker and results illustrated that beta-myosin heavy chain(β-MHC) and atrial natriuretic peptides (ANP) were increasingly expressed, which refers to cardiac hypertrophy ( Fig. 1 F-G ) . 2. Identification of differential expressed circRNAs and target miRNAs prediction during cardiac hypertrophy After mice were euthanized, a total of 8 ventricular myocardium samples (each for 4 in sham and model) were subjected to circular RNA sequencing. We finally identified a total of 9813 circRNAs ( Supplementary 2 ; Table S1 ) in two groups. Based on the locations in host genes,the circRNAs were classified into three categories:95.85% exonic,3.44% intronic,0.71% intergenic circular molecules in sham group;96.19% exonic,3.01% intronic,0.79% intergenic molecules in model group ( Fig. 1 H; S1, Figure S2 A,2B) . Majority of the circRNAs ranged in length from 31 to 1970 bp (Fig. 1 I), and distributed across all chromosomes (S1, Figure S2 C) . Among them,5 molecules were identified to have increased expression, while 25 molecules (showed in valcanoplot and heatmap) had decreased expression compare to sham group ( Fig. 2 A, 2 B ) . Then targeted miRNAs were predicted by miRanda (Supplement 3) and Interaction diagram of circRNA-miRNA was also constructed by cytoscape 3.10 ( Fig. 2 E ) . For further insight into potential biological function, we annotated the parental genes of circRNAs by functional enrichment analysis. Gene ontology (GO) biological process enriched in transferase activity and ubiquitination ( Fig. 2 C ) . Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that functional circRNAs were mainly enriched in transcription factors, MAPK signaling pathway and Ubiquitin system ( Fig. 2 D ) . Herein, according to the P Value on expressive level, we specifically focus on highest expressed circ_0004641for further study (Table 1) . 3. Circ_0004641 was up-regulated both in vitro and in vivo To detect the expression of candidate circRNAs, we performed RT-qPCR validation on remaining myocardial tissue for sequencing. Results showed that only circ_0004641 ( Fig. 3 A ) had an significant expression increase ( Fig. 3 B ) . While circ_0009153 showed no significant difference; among the down-regulated genes, results showed a high expression on circ_0002423 and circ_0003046 and no significant expression on circ_0001213 and circ_0009685, which is contradicted to sequencing results (S1, Figure S2 D) . In addition, we duplicated the validation experiment on ventricular myocardium that not sent for sequencing. Results demonstrated the solely significant increase of circ_0004641, while the other molecules showed no difference or contradictory trend on expression (S1, Figure S2 E) . In order to support the results in tissues, hypertrophic cardiomyocytes were stimulated by Ang II and phalloidin staining demonstrated hypertrophy of myofilament and enlargement of cell size (S1, Figure S3 A, B) along with significant increase expression of β-MHC and ANP (S1, Figure S3 C-E) . Subsequently, qPCR validation on NMVCs showed that circ_0004641 ( Fig. 3 C ) and circ_0009153 has a match increase on expression, while circ_0002423 presenting a paradoxical trend of sequencing results and the others molecules showed no significant difference (S1, Figure S3 F). Hence, circ_0004641 were identified for further exploration for its highest expression and stable repeatability. Fluorescence in situ hybridization (FISH) identified its subcellular location in the nucleus ( Fig. 3 D ) . To explore its correlation with cardiac hypertrophy, knock-down experiments were applied by transfecting with siRNA. Results shows that inhibiting circ_0004641 significantly reverse the enlargement of cardiomyocytes both in control or Ang II group, along with the decreased transcriptional level of ANP and β-MHC ( Fig. 3 E; S1, Figure S3 G, H) . Western blot and quantitative analysis showed a sharp decrease of ANP and β-MHC after transfection ( Fig. 3 F-H ) . All above indicated a strong relation between circ_0004641 and cardiac hypertrophy. 4. miRNA-1192 was downstream target of circ_0004641 and negatively associated with myocardial hypertrophy Downstream predicted miR-1192 of circ_0004641 were synchronously detected and significant decrease was observed in ventricular myocardium and hypertrophic cardiomyocytes ( Fig. 4 A, B ) . After transfection of siRNA, qPCR results demonstrated significant increase of miR-1192, along with down regulation of ANP and BNP ( Fig. 4 C; S1, Figure S3 G, H) , indicating the underlying regulation between two molecules. To clarify the interaction between circ_0004641 and miR-1192, we conducted dual luciferase reporter assays. Under 293T engineered cell system, we observed a strong interaction between the WT-circ_0004641-3’UTR and miR-1192-mimics, while the mutant-circ-3’UTR did not react with miR-1192 ( Fig. 4 D, E ) . Thus, we verified that circ_0004641 and miR-1192 Interacted like sponge adsorption as we hypothesized. To explore the functional role of miR-1192, we over-expressed miR-1192 by mimics in NMVCs, and results showed miR-1192 plays an inhibitory role in cardiac hypertrophy, as transcriptional expression of ANP and β-MHC drop dramatically both in control and Ang II group ( Fig. 4 F-H ) . 5. TRIM25 and TRIM41 act as the downstream mRNA targeted by CeRNA network Through miRDB website, we totally identified 14 targeted downstream mRNAs regulated by miRNA-1192(both molecules consists of ubiquitin system as bioinformatic analysis predicted), including the TRIM protein family (TRIM6, TRIM21, TRIM25, TRIM32, TRIM33, TRIM41, TRIM71), the RNF protein family (RNF13, RNF138, RNF139, RNF169, RNF170), Dtx31 and RAD18.RT-qPCR validation were conducted both on mice myocardium and NMVCs, and resluts shows that TRIM25 and TRIM41 has stable expressive trend with a significant increase ( Fig. 5 A; Fig. 5 B ) . Other mRNAs were also detected but had contradictory or non-stable expression level. (S1, Figure S4 A-L; S1, Figure S5A). Repeatable experiments are applied on mice myocardium that not used for sequencing, and only TRIM25 and TRIM41 matched previous results ( S1-Figure S5B ), indicating that TRIM215/TRIM41 are two critical molecules regulated by circ_0004641/miRNA-1192 axis in cardiac hypertrophy. To verify, tranfection of siRNA-circ_0004641 were down in NMVCs, and results demonstrated TRIM25 and TRIM41 was down-regulated significantly both in trancriptional and translational level ( Fig. 5 C-E; S1, Figure S5C, D) , which is contrary to the expressive trend before transfection. Moreover, we co-cultured NMVCs with miR-1192 mimics and, as supposed, the expression of TRIM25/TRIM41 decreased sharply both in control and Ang II group ( Fig. 5 F-H ) . To this extent, we can conclude that over-expression of circ_0004641 can competitively adsorb miR-1192 molecules, thereby suppressing the inhibitory role of miR-1192 on the expression of TRIM25/TRIM41 in cardiac hypertrophy. 6. NF-κB may act as underlying key pathway in regulation of cardiac hypertrophy We also detected underlying downstream pathways invovling in cardiac hypertrophy and western blot and quantitative analysis suggested that, compared to the control group, there was an increased expression of P65 and phosphorylated P65(p-P65) protein in mouse myocardial ventricular tissue ( Fig. 6 A, 6 B ) . Repeated results are presented in cell model with same expressive trend ( Fig. 6 C, 6 D ). These results are consistent with previous studies on NF-κB pathway in myocardial hypertrophy 37 , 38 . By knocking down the expression of circ_0046461, we found that, under the premise that Ang-II group expression was higher than the control group, both two group was observed a significant decrease in expression of P65 and p-P65 after siRNA transfection ( Fig. 6 E, F ) , which indicates circ_0004641/miR-1192 axis plays an important role in the activation of the NF-κB signaling pathway in cardiac hypertrophy, but the underlying mechanism still needs in-depth research. In addition, we also detect the TGFβ1/smad signaling pathway and phosphorylated Smad protein, which present the same expressive trend of NF-κB( S1,Figure S6A-E ). To conclude, we identified a novel_circ_0004641 by sequencing and constructed a brand-new regulatory network via circ_0004641/miR-1192-Trim25/TRIM41 axis in hypertrophic mice myocardium and NF-κB pathway may function as the downstream object of TRIM to cardiac hypertrophy. Discussion CircRNAs, which are dominant sequencings without protein-encoding function, have been recently recognized as dominant regulatory roles since its discovery 22 , 39 . Endogenous circRNAs have been reported to engage in various biological process, such as cell development 40 , tumor proliferation and inhibition 41 – 43 and cardiovascular diseases 44 – 48 .Thus, circRNAs have been found to play an important role in the cardiovascular field, involving almost whole process 49 – 51 from cardiac embryonic development to physiological and pathological regulation. Identification of these key molecules facilitates the subsequent comprehensive research on pathological mechanism for myocardial hypertrophy, as a growing number of CeRNA networks are being uncovered 27 , 35 , 52 , 53 . In our study, a novel circ_0004641 (which would be named as circRftn1 from its parental gene as canonical rules 24 , 36 ) and CeRNA network(circ_0004641/miR-1192) was characterized in cardiac hypertrophy. For first time, we verified the interaction of circ_0004641 and miR-1192 and established the sponge-like effect in the regulation on cardiac hypertrophy. Our study also identified the downstream mRNA encoding TRIM25/41 protein regulated by circ_0004641/ miRNA-1192 axis. TRIM (Tripartite motif) protein family 54 , 55 , known as E3 ligase, were prominent molecules that participate in cardiac hypertrophy by activating downstream signaling pathway involving in pathological cardiac myotrophy(such as TRIM8 56 , TRIM32 57 , TRIM44 58 , TRIM63 59 and so on). Our study found the underlying regulation between CeRNA network and TRIM25/TRIM41 for the first time. Previous studies revealed the unique function of TRIM 25 60 and TRIM41 61,62 ,as activator, targeting downstream NF-κB pathway at the K63 tyrosine site, thereby enhancing or depressing the tumor proliferation and metastasis. While potential mechanism of TRIM25/TRIM41 on cardiac hypertrophy remains unknown. Our study identified that circ_0004641/miRNA-1192 axis promote the expression of TRIM 25 and TRIM41 on pathological cardiomyotrophy, and firstly speculated that TRIM25/TRIM41 would enhance downstream NF-κB pathway by activating P65 protein and promote myocardial hypertrophy associated transcriptional process. There are still some lackage in our study: 1) Firstly, the miRNAs and mRNAs we verified were predicted by the miRanda and miRDB. Therefore, to ensure the accuracy of the study, we conducted repeated validation experiments in both vivo and vitro levels; 2) Secondly, the relation between TRIM protein and NF-κB signaling pathway still requires validation by Co-Immunoprecipitation (CO-IP) or Immunoprecipitation-Mass Spectrometry (IP-Mass).For further study, amino-acid binding cite for ubiquitination and underlying regulatory mechanism require comprehensive verification. Conclusion To conclude, we revealed a brand-new CeRNA network axis (circ_0004641/ miRNA1192-TRIM41/TRIM25) in myocardial hypertrophy. Mechanically, up-regulated circ_0004641 sponged miR-1192 by depressing its inhibitory role on TRIM25/TRIM41, which contribute to the progression of myocardial hypertrophy. Underlying myocardial hypertrophic regulation by TRIM25/41 protein via NF-κB pathway need in-depth functional verification. Materials and Methods AAC animal model establishment and Evaluation The SPF C57BL/6 mice (male, 6–8 weeks, 20–22 g) were purchased from the Experimental Animal Tech of Weitonglihua (Beijing, China). Abdominal aortic coarctation (AAC) is a widely-used method 63 , 64 and the procedure was described in Supplement 4 . Echocardiography in mice was performed at 8 weeks post-AAC operation using the Vevo 2100 Imaging system (visual sonics,Canada). Mice were anesthetized with isoflurane (2.5% for induction, 1.0% for maintenance) and left ventricular parameters during diastole and systole periods were detected from the M-modes. Statistical analysis was applied by Prisma 9 software on cardiac indexes in Supplement 4 . Histological analysis and RNA fluscence in situ hybridization (FISH) Mice were euthanized and hearts were excised and fixed in 4% paraformaldehyde and routinely dehydrated and paraffin-embedded. The wax blocks were cut into sections with a thickness of 4–5 µm. Subsequently, cross heart sections were stained with hematoxylin-eosin (HE, Servicebio, China) and Sirius Red staining (sigma, USA) to assess the size and morphologic alterations of the heart. FITC-conjugated wheat germ agglutinin (WGA, sigma, L4895, 1:500, USA) staining was performed to demarcate cardiomyocyte boundaries and 4ʹ,6-diamidino-2-phenylindole (DAPI, Servicebio, China) was used to label the nuclei. Images were captured using Leica microscope (Leica, Wetzlar, Germany) and processed with CaseView 2.4. Cardiomyocytes were fixed with paraformaldehyde (4%), followed by treatment with 0.5% Triton. FISH was performed using a FISH Tag™ RNA Multicolor Kit (Invitrogen, USA) and DAPI was used to stain the cell nucleus. The localization of circRNA was observed by using Leica SP5 Spectral scanning laser confocal microscope (Leica, Wetzlar, Germany). Neonatal mice ventricle cell separating and culture Neonatal mouse ventricle cardiomyocytes (NMVCs) were isolated from the hearts of neonatal C57BL/6 mice (0–3 days after birth). Ventricle tissues were sectioned into 1 mm 3 pieces and isolated by using 0.1% collagenase II (Gibico, China). NMVCs were cultured in Dulbecco’s Modified Eagle’s medium/Nutrient Mixture F-12 (DMEM/F12; Thermo Fisher Scientific, USA) containing 10% fetal bovine serum (Hyclone, USA) and maintained at 37°C with 5% CO2. NMVCs were incubated with 10 − 7 M Angiotensin II (Ang-II,Selleck,USA) for 48h to induce the hypertrophic phenotype. SiRNA targeting mmu_circ_0004641 (5nm, RiboBio, China) was transfected into NMVCs by co-culture with Ang II or DMSO for 48h.The sequences of siRNA of circ_0004641 is as follows: CATGGCCAATGGTGCAGGA. Overexpression mimics targeting mmu_miR-1192(50pmol, Sangon Biotech, China) was co-cultured with NMVCs by Ang II or DMSO for 48h.The sequences of miR-1192 mimics is as follows: AAACAAACAAACAGACCAAAUU. RNA extraction, library preparation, and CircRNA sequencing Total RNA was isolated from the left ventricular tissues of mice with TRIzol™ reagent (Invitrogen, United States). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, United States) before library preparation. Qualified RNA samples were treated with Ribo-ZeroTM Gold Kits (Epicenter Technologies, United States) to remove ribosomal RNA (rRNA). Sequencing libraries were generated using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, United States) following the manufacturer’s recommendations. Sequencing process was on Illumina HiSeq 4000 platform, and 125 bp/150 bp paired-end reads were generated. CircRNA identification and Target prediction To ensure the quality and reliability of sequencing data, the original data was filtered (See Supplement 2). Hisat2 software was used to accurately align clean reads with the reference genome, and obtain localization information. Find_ Circle algorithm and CIRI algorithm (Supplement 2) were used for identifying candidate circRNAs, and TMM algorithm was applied to standardize readcount data before conducting differential analysis. Fold change and corrected significance level are identified for screening (p_adj = 1.0 as threshold). Visualization by volcano plots and heatmap are down to infer differential circRNAs. Gene Ontology and KEGG enrichment analyses were performed on the sets of parental genes with differential expression of circRNA in each group. Based on miRanda software and miRDB software ( http://mirdb.org/miRDB ), miRNA binding sites targeted by the identified circRNAs and downstream mRNAs were predicted respectively. Then, interaction between circRNA and miRNA was constructed by cytoscape 3.10.( http://www.cytoscape.org/ ) Western Blot analysis Total protein was isolated and quantified using a ImageQuant LAS 4000 system (Cytiva,USA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel (10%) was prepared and 40 µg protein was loaded for electrophoresis; the protein was then transferred onto a polyvinylidene fluoride (PVDF) membrane. Next, the blots were blocked in 5% skimmed milk at room temperature for 2 h and incubated with the primary antibody β-MHC (1:1,000; ab172967, Abcam, UK), ANP (1:2,000; 27426-1-AP, Proteintech,USA),TRIM25(1:1000;Proteintech,12573-1-AP), TRIM41(1:1000, Proteintech, 18468-1-AP),p-NFκB (1:1,000; 3033, CST, USA), NFκB (1:1,000; 8242, CST, USA), TGFβ1 (1:1,000; ab179695, Abcam, UK), p-SMAD1/5 (1:1,000; 9516, CST, USA), SMAD1/5 (1:1,000; ab75273, Abcam, UK), p-SMAD2/3 (1:1,000; 8828, CST, USA), SMAD2/3 (1:1,000; 8685, CST, USA), GAPDH (1:10,000; ab181602, Abcam, UK)overnight, followed by incubation with the secondary antibody (1:5,000; 5220 − 0336, KPL, USA) for a further 2 h. sigma, L4895, 1:500. The data were analyzed using Image J software. Quantitative real-time PCR Total RNA was isolated from the left ventricular tissues of mice with TRIzolTM reagent (Invitrogen, United States). Then, reverse transcription was accomplished using a reverse transcription kit (TOYOBO, FSQ-101, Japan). Next, qRT–PCR was performed to detect gene expression using the ChamQ Universal SYBR qPCR Master Mix Kit (Vazyme, Q711-03, China) on a CFX96 detection system (BioRad, United States). The relative expression of circRNA was calculated based on the cycle threshold values according to the 2 −ΔΔCt method. GADPH was used as a reference gene for normalization. The primers used are listed in Supplementary 4 Dual-Luciferase Reporter Assay The recombinant luciferase reporter plasmids containing the potential circ_0004641 binding site sequences were constructed. Human embryonic kidney (HEK) 293 cells (3 × 10 5 cells per well in 12-well plate) were co-transfected with 200 ng of recombinant luciferase reporter plasmid, 20 ng of pRL-TK as an internal control (Promega, Madison, WI), 200 ng of pDsRed2-N1 or 200 ng of pDsRed2-miR-192, 50 nM circ_0004641 mimic or 50 nM mutant circ_0004641 mimic, respectively. Activities of firefly luciferase (FL) and Renilla luciferase (RL) were measured 24 hour after transfection, and the relative ratio of the FL/RL was used to indicate the circ_0004641-mediated knockdown of target genes. Statistical analysis Data are presented as the mean ± standard error of the mean (SEM). Unpaired Student’s t test (two-tailed) was used for comparisons between groups or one-way ANOVA test was performed to analyze multiple groups followed by Bonferroni post hoc tests. All data were analyzed using by GraphPad Prism 9 and P value < 0.05 was considered significant.Graphical Abstract was portrayed by Figdraw2.0. Abbreviations AAC Abdominal aortic coarctation Ang II Angiotensin II CircRNA Circular RNA miRNA microRNA TRIM Tripartite motif CeRNA Network Competitive endogenous RNA Network Declarations Author contributions All authors contributed to the study conception and formulation. Methods design and model sonstruction were excecuted by Guangcheng Liu and Haipeng Zhang. Material preparation and experiments were performed by Guangcheng Liu, Haipeng Zhang and Jingdai Zhang. Data collection,correction and analysis were performed by Hao Qian, Liang Wang and Lianfeng Chen. The first draft of the manuscript was written by Guangcheng Liu. Zhujun Shen drafted the work critically for important intellectual content. All authors commented on previous versions of the manuscript and we confirmed that all authors in the list contributed to the article and approved the finally submitted version. Funding This research was funded by the National Natural Science Foundation of China (NSFC) projects:82170372. Institutional Review Board Statement The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of Peking Union Medical College Hospital (PUMCH,China).Written informed consent was obtained from the owners for the participation of their animals in this study. Informed Consent Statement: Not applicable for studies not involving humans. Data availability statement The datasets generated during during the current study are available in the Mendeley Database repository, doi: 10.17632/hy5pmx39g5.1. Conflict of interest The authors declare no potential conflict of interest in this research. References Mishra S, Kass DA. Cellular and molecular pathobiology of heart failure with preserved ejection fraction. Nat Rev Cardiol . Jun 2021;18(6):400-423. doi:10.1038/s41569-020-00480-6 Nakamura M, Sadoshima J. Cardiomyopathy in obesity, insulin resistance and diabetes. J Physiol . Jul 2020;598(14):2977-2993. doi:10.1113/jp276747 Packer M. Differential Pathophysiological Mechanisms in Heart Failure With a Reduced or Preserved Ejection Fraction in Diabetes. JACC Heart Fail . Aug 2021;9(8):535-549. doi:10.1016/j.jchf.2021.05.019 Murphy SP, Ibrahim NE, Januzzi JL, Jr. Heart Failure With Reduced Ejection Fraction: A Review. Jama . 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Adv Exp Med Biol . 2020;1229:231-245. doi:10.1007/978-981-15-1671-9_13 Altesha MA, Ni T, Khan A, Liu K, Zheng X. Circular RNA in cardiovascular disease. J Cell Physiol . May 2019;234(5):5588-5600. doi:10.1002/jcp.27384 Wang L, Feng J, Feng X, et al. Exercise-induced circular RNA circUtrn is required for cardiac physiological hypertrophy and prevents myocardial ischemia-reperfusion injury. Cardiovasc Res . Oct 28 2023;doi:10.1093/cvr/cvad161 Li Y, Zhang J, Huo C, et al. Dynamic Organization of lncRNA and Circular RNA Regulators Collectively Controlled Cardiac Differentiation in Humans. EBioMedicine . Oct 2017;24:137-146. doi:10.1016/j.ebiom.2017.09.015 Du WW, Yang W, Chen Y, et al. Foxo3 circular RNA promotes cardiac senescence by modulating multiple factors associated with stress and senescence responses. Eur Heart J . May 7 2017;38(18):1402-1412. doi:10.1093/eurheartj/ehw001 Yang F, Qin Y, Lv J, et al. Silencing long non-coding RNA Kcnq1ot1 alleviates pyroptosis and fibrosis in diabetic cardiomyopathy. Cell Death Dis . Sep 24 2018;9(10):1000. doi:10.1038/s41419-018-1029-4 Wang K, Liu F, Zhou LY, et al. The long noncoding RNA CHRF regulates cardiac hypertrophy by targeting miR-489. Circ Res . Apr 25 2014;114(9):1377-88. doi:10.1161/circresaha.114.302476 Borlepawar A, Frey N, Rangrez AY. A systematic view on E3 ligase Ring TRIMmers with a focus on cardiac function and disease. Trends Cardiovasc Med . Jan 2019;29(1):1-8. doi:10.1016/j.tcm.2018.05.007 Lu K, Pan Y, Huang Z, Liang H, Ding ZY, Zhang B. TRIM proteins in hepatocellular carcinoma. J Biomed Sci . Sep 13 2022;29(1):69. doi:10.1186/s12929-022-00854-7 Chen L, Huang J, Ji YX, et al. Tripartite Motif 8 Contributes to Pathological Cardiac Hypertrophy Through Enhancing Transforming Growth Factor β-Activated Kinase 1-Dependent Signaling Pathways. Hypertension . Feb 2017;69(2):249-258. doi:10.1161/hypertensionaha.116.07741 Chen L, Huang J, Ji Y, et al. Tripartite motif 32 prevents pathological cardiac hypertrophy. Clin Sci (Lond) . May 1 2016;130(10):813-28. doi:10.1042/cs20150619 Jiang XY, Guan FF, Ma JX, et al. Cardiac-specific Trim44 knockout in rat attenuates isoproterenol-induced cardiac remodeling via inhibition of AKT/mTOR pathway. Dis Model Mech . May 1 2023;16(5)doi:10.1242/dmm.049444 Chen SN, Czernuszewicz G, Tan Y, et al. Human molecular genetic and functional studies identify TRIM63, encoding Muscle RING Finger Protein 1, as a novel gene for human hypertrophic cardiomyopathy. Circ Res . Sep 14 2012;111(7):907-19. doi:10.1161/circresaha.112.270207 Gack MU, Shin YC, Joo CH, et al. TRIM25 RING-finger E3 ubiquitin ligase is essential for RIG-I-mediated antiviral activity. Nature . Apr 19 2007;446(7138):916-920. doi:10.1038/nature05732 Yu Z, Li X, Yang M, et al. TRIM41 is required to innate antiviral response by polyubiquitinating BCL10 and recruiting NEMO. Signal Transduct Target Ther . Feb 28 2021;6(1):90. doi:10.1038/s41392-021-00477-8 Liu Y, Liu K, Huang Y, et al. TRIM25 Promotes TNF-α-Induced NF-κB Activation through Potentiating the K63-Linked Ubiquitination of TRAF2. J Immunol . Mar 15 2020;204(6):1499-1507. doi:10.4049/jimmunol.1900482 Tarnavski O. Mouse surgical models in cardiovascular research. Methods Mol Biol . 2009;573:115-37. doi:10.1007/978-1-60761-247-6_7 Wang L, Wang P, Xu S, et al. The cross-talk between PARylation and SUMOylation in C/EBPβ at K134 site participates in pathological cardiac hypertrophy. Int J Biol Sci . 2022;18(2):783-799. doi:10.7150/ijbs.65211 Table 1 Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files GraphicalAbstract.tif Supplement1FigureS1S7.pdf Supplement2.doc Supplement3.doc Supplement4.doc Table1.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 29 Jul, 2024 Reviews received at journal 29 Jul, 2024 Reviewers agreed at journal 28 Jul, 2024 Reviews received at journal 19 Jul, 2024 Reviewers agreed at journal 19 Jul, 2024 Reviewers invited by journal 08 Jul, 2024 Editor assigned by journal 08 Jul, 2024 Editor invited by journal 23 May, 2024 Submission checks completed at journal 23 May, 2024 First submitted to journal 21 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4452125","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":310031032,"identity":"9a735f9e-7814-4e12-960e-b52cb21cd6fb","order_by":0,"name":"Liu Guang-cheng","email":"","orcid":"","institution":"Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Liu","middleName":"","lastName":"Guang-cheng","suffix":""},{"id":310031033,"identity":"e9596789-73ea-45a9-9320-760c928ff747","order_by":1,"name":"Haipeng Zhang","email":"","orcid":"","institution":"Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Haipeng","middleName":"","lastName":"Zhang","suffix":""},{"id":310031034,"identity":"a84341f0-4b5e-4831-ab70-a5b86d68c0aa","order_by":2,"name":"Jingdai Zhang","email":"","orcid":"","institution":"Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Jingdai","middleName":"","lastName":"Zhang","suffix":""},{"id":310031039,"identity":"5bf1bb1a-6818-4ced-b85a-601a712d03e0","order_by":3,"name":"Hao Qian","email":"","orcid":"","institution":"Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Qian","suffix":""},{"id":310031040,"identity":"f1af33ab-b7ab-438d-8f10-3431ae9b6d69","order_by":4,"name":"Liang Wang","email":"","orcid":"","institution":"Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Wang","suffix":""},{"id":310031042,"identity":"dd86e2ab-90f3-454d-9272-7f9fe0062c07","order_by":5,"name":"Lianfeng Chen","email":"","orcid":"","institution":"Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Lianfeng","middleName":"","lastName":"Chen","suffix":""},{"id":310031043,"identity":"d87d47a8-39b9-4da9-a255-db3b3643f576","order_by":6,"name":"Zhujun Shen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIiWNgGAWjYDACCQYGAwYGGzkQg+GDAZBBpJY0YxCDcUYBkEGMFiA4lAhiMPN8OJxIUIf87B6DYt4dBxLkZzcfe2xjwJzAwH746AZ8WhjnnDEw5j1zJ8/gzrF04xwDtjwGnrS0G/i0MEvkALW0PSs2kMgxk84x4ClmkOAxw6uFDaLlcOL8GfnfpC0MJBIbCGnhgWlpuJHDJs1gYEBYi4REWoHh3LY0Y4MbaWaSPQYJxmyE/CI/I3mbwds2Gzkg45nEjz//5fjZDx/DqwXkHQNULgHlIMD8gAhFo2AUjIJRMJIBAMvJRHz9UXkiAAAAAElFTkSuQmCC","orcid":"","institution":"Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College","correspondingAuthor":true,"prefix":"","firstName":"Zhujun","middleName":"","lastName":"Shen","suffix":""}],"badges":[],"createdAt":"2024-05-21 04:29:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4452125/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4452125/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57942772,"identity":"bd6f127e-7e17-40e1-9ae3-04ea204116c7","added_by":"auto","created_at":"2024-06-07 19:02:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":890846,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConstruction cardiac hypertrophy mouse model of AAC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eRepresentative histopathology evaluation of cardiac hypertrophy:HE staining;Sirius Red staining and WGA staining; \u003cstrong\u003e(B)\u003c/strong\u003eRepresentative M-mode image of model and sham mice;\u003cstrong\u003e(C)(D)(E)\u003c/strong\u003eEchocardiography evaluation on cardiac parameters：Left ventricular posterior wall thickness, end-diastolic/systolic (LVPW;d / LVPW;s)；Left ventricular ejection fraction (LVEF),ejection fraction = (stroke volume/diastolic volume) x 100%;Cardiac output(CO) = stroke volume x heart rate,stroke volume = diastolic volume – systolic volume; \u003cstrong\u003e(F)(G)\u003c/strong\u003eWestern Blotting showed beta-myosin heavy chain(β-MHC) and atrial natriuretic peptide(ANP) overexpression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H)\u003c/strong\u003eClass distribution of circRNAs in sham and model groups by next-generation sequencings;\u003cstrong\u003e(I)\u003c/strong\u003e Length distribution of circRNAs;\u003c/p\u003e\n\u003cp\u003eData are shown as mean ±SEM, *indicates P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001 ， N =8, 5 in (C)-(E) ,(G)respectively.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4452125/v1/fda5238e24ce618958f3c0aa.png"},{"id":57942920,"identity":"1ee468a1-f50a-4e1d-baa2-8b73acbe1b5a","added_by":"auto","created_at":"2024-06-07 19:02:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":949477,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution profiles of circRNAs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eThe Volcanoplot of DE circRNAs in sham and model mouse,shown are top5 up-regulation and top10 down-regulation molecules;\u003cstrong\u003e(B)\u003c/strong\u003eThe heatmap of DE circRNAs in sham and model mouseClass distribution of circRNAs in sham and model groups;\u003cstrong\u003e(C)\u003c/strong\u003eGO analysis of the DE parent genes of circRNAs;\u003cstrong\u003e(D) \u003c/strong\u003eKEGG pathway enrichment analysis of the DE parent genes of circRNAs;\u003cstrong\u003e(E)\u003c/strong\u003eNetwork of circRNA and miRNA, circRNA in yellow and miRNA in black；green represents down regulation and red represents up regulation.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4452125/v1/54051b7265e3ab2618f7f29c.png"},{"id":57942761,"identity":"5396fe41-aa42-4d66-8777-0cdda590805f","added_by":"auto","created_at":"2024-06-07 19:01:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":813013,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCirc_0004641 was up regulated in vivo and was correlated with cardiac hypertrophy.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eCircRNA was back-splicing from Rftn1 gene with 595 bp;\u003cstrong\u003e(B)(C) \u003c/strong\u003eCompared to the control group,circ_004641 has a significant rise both in mouse ventricular myocardium and Ang II-induced cardiomyocytes;\u003cstrong\u003e(D)\u003c/strong\u003e RNA FISH for circRNA_004641 in mouse myocardium. CircRNA_0004641 was shown in orange and nuclei were stained with DAPI. The scale bar is 100 μm; \u003cstrong\u003e(E)\u003c/strong\u003eRepresentative Phalloidin staining of NMVCs in both DMSO or Ang II coculture with or without siRNA transfection; \u003cstrong\u003e(F)-(H)\u003c/strong\u003e Western blot assay of cardiac specific peptide and quantitative analysis shows the transfection of siRNA significantly reverse the expression of ANP and β-NHC.\u003c/p\u003e\n\u003cp\u003eData are shown as mean ±SEM, *indicates P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001 ，****P \u0026lt; 0.0001 N =4, 4,3 in (B),(C),(F)-(G) respectively.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4452125/v1/8996d64b0065cf65b52861ba.png"},{"id":57942966,"identity":"6ac7d756-2a9c-4cf8-b167-89bab65c5efc","added_by":"auto","created_at":"2024-06-07 19:02:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":333735,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-1192 was the downstream target of circRNA and was correlated with cardiac hypertrophy.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)(B)\u003c/strong\u003emiR-1192 was down regulated both in hypertrophic myocardium and cardiomyocytes;\u003cstrong\u003e(C) \u003c/strong\u003eBy transfected with siRNA-circ_0004641,miR-1192 has a significant up-regulation in cardiomyocytes(both in control group and Ang II group; \u003cstrong\u003e(D)(E)\u003c/strong\u003eDual luciferase reporter assay illustrates that only miRNA-1192 and circ_0004641 reaction has strong reduction on luciferase expression;\u003cstrong\u003e(F)-(G)\u003c/strong\u003e Western blot assay and quantitative analysis shows that by coculture with miR-1192 mimics, cardiac specific peptide of ANP and β-NHC significantly decrease both in control group and Ang II group.\u003c/p\u003e\n\u003cp\u003eData are shown as mean ±SEM, *indicates P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001 ，****P \u0026lt; 0.0001 N =4, 3,3 in (A), (B),(C)-(F) respectively.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4452125/v1/252d68274705991e4db4dd97.png"},{"id":57942779,"identity":"6becd5d1-2fd3-4453-b05e-d77b19cd88c7","added_by":"auto","created_at":"2024-06-07 19:02:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":366326,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTRIM25 and TRIM41 are downstream mRNA regulated by circ_0004641-miR-1192 axis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)(B)\u003c/strong\u003eTRIM25 and TRIM41 are up-regulated both in myocardium and NMVCs; \u003cstrong\u003e(C)-(E) \u003c/strong\u003eWestern blot showed that, after transfected with siRNA, TRIM25 and TRIM41 has a significant decrease in cardiomyocytes (both in control and Ang II group); \u003cstrong\u003e(F)-(H) \u003c/strong\u003eWestern blot and quantitative analysis illustrate that, after transfected with miR-1192 mimics, TRIM25 and TRIM41 has a significant decrease in cardiomyocytes (both in control and Ang II group).\u003c/p\u003e\n\u003cp\u003eData are shown as mean ±SEM, *indicates P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001 N =4, 3 in (A), (B)-(H) respectively.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4452125/v1/2d0ff58ce1daed7968e0e204.png"},{"id":57942925,"identity":"cbc207bf-3121-421a-8bdc-511aafa27de2","added_by":"auto","created_at":"2024-06-07 19:02:12","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":511841,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNF-κB signaling pathway may act as the regulatory role by CeRNA network on cardiac hypertrophy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)(B)\u003c/strong\u003eCompared to the sham group，NF-κB signaling pathway has a significant increase in model mice after AAC surgery;\u003cstrong\u003e(C)(D)\u003c/strong\u003e Compared to the control group，NF-κB signaling pathway has a significant increase in Ang-II induced cardiomyocytes;\u003cstrong\u003e(E)(F) \u003c/strong\u003eAfter transfection of si-circ_004641, P65 and p-P65 has been significant suppressed both in DMSO- and Ang-II induced cardiomyocytes;\u003c/p\u003e\n\u003cp\u003eData are shown as mean ±SEM, *indicates P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001 ，****P \u0026lt; 0.0001 N =5,3,3 in (A)-(B),(C)-(D),(E)-(F)respectively.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4452125/v1/3da7d362ae5b72e3335e4ce4.png"},{"id":57944646,"identity":"801ba259-68f1-4bf1-8c19-57af9522eac6","added_by":"auto","created_at":"2024-06-07 19:10:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5210076,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4452125/v1/d1c99b0f-691b-4842-8187-ce6ae8c78db7.pdf"},{"id":57942929,"identity":"edf66859-5a52-42b6-ba95-48a8606d3093","added_by":"auto","created_at":"2024-06-07 19:02:19","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":79709946,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-4452125/v1/c76dfd03ec602691546238ff.tif"},{"id":57942775,"identity":"74cc8c9f-3022-48b3-86c9-fa37e799c28d","added_by":"auto","created_at":"2024-06-07 19:02:04","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4744155,"visible":true,"origin":"","legend":"","description":"","filename":"Supplement1FigureS1S7.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4452125/v1/99a9a3b0d24b4cbddfea662c.pdf"},{"id":57942778,"identity":"e4173944-2c06-47fd-b4ae-5d1674311081","added_by":"auto","created_at":"2024-06-07 19:02:05","extension":"doc","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1381408,"visible":true,"origin":"","legend":"","description":"","filename":"Supplement2.doc","url":"https://assets-eu.researchsquare.com/files/rs-4452125/v1/9d04c5f75d15780f79193e43.doc"},{"id":57942928,"identity":"f8e170e0-80b3-48c7-8119-4a372cc03788","added_by":"auto","created_at":"2024-06-07 19:02:14","extension":"doc","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":6527450,"visible":true,"origin":"","legend":"","description":"","filename":"Supplement3.doc","url":"https://assets-eu.researchsquare.com/files/rs-4452125/v1/254073f17e3d48f9205c2c00.doc"},{"id":57942777,"identity":"d5344f3f-98a6-4e07-aef6-5becea2ed2ab","added_by":"auto","created_at":"2024-06-07 19:02:05","extension":"doc","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":42322,"visible":true,"origin":"","legend":"","description":"","filename":"Supplement4.doc","url":"https://assets-eu.researchsquare.com/files/rs-4452125/v1/e2f7fa29ef11978a8b408ddc.doc"},{"id":57942781,"identity":"275352be-1ee1-413c-b9c0-fb7d50219d4a","added_by":"auto","created_at":"2024-06-07 19:02:07","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":89545,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4452125/v1/071a02f73eae1493f7b3746e.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Circ_0004641 upregulates TRIM25/TRIM41 expression by sponging miR- 1192 to induce cardiac hypertrophy in vitro and in vivo","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePathological cardiac hypertrophy is a maladaptive cardiac remodeling in variable clinical diseases involving Hypertension, cardiomyopathy and so on, which progressively deteriorate to Heart Failure(HF)\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, remaining a major public healthcare problem with high morbidity and mortality in the worldwide\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.There is an unmet need to detect underlying heart failure sensitively and to reverse myocardial enlargement and fibrosis effectively\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Several stimuli and signaling mechanisms have been postulated in pathological cardiac hypertrophy\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, not just by pressure overloading but molecules on genome and transcriptional levels, such as impaired Ca2\u0026thinsp;+\u0026thinsp;handling\u003csup\u003e11\u003c/sup\u003e, mitochondrial dysfunction\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, oxidative stress\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e,m6A methyletation\u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e and so on. Researchers are devoting to resolve the knotty matter and breakthroughs are made via next-generation sequencing, which brings non-coding RNAs into researchers' fields of vision, shedding new lights on the cardiac complexity.\u003c/p\u003e \u003cp\u003eWith the advancement of transcriptional sequencing, non-coding RNAs(ncRNAs) are receiving increasing attention, including the newly emerging circRNA molecules entering researchers' horizons\u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. As an important transcription regulatory factor, circular RNA can act as a miRNA sponge, regulator of mRNA translation, or directly participate in gene transcription as a transcription factor\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.Generally formulated by non-canonical back-splicing from parental gene, cirRNAs always exert as miRNA sponges\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, thus, to modulate gene expression by acting as competing endogenous RNA(CeRNA). For example, circ_000203 can enhance the expression of fibrosis-associated genes in cardiac fibroblasts by depressing targeted miR-26b-5p and induce cardiac fibrosis\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. While in contrast, circRNA HRCR can protect heart from pathological hypertrophy by targeting miR-223\u003csup\u003e27\u003c/sup\u003e. So far, many CeRNA networks has been identified as stimulating or inhibiting role in atherosclerosis (circ_0026218/ miR-338-3p\u003csup\u003e28\u003c/sup\u003e, circUSP36/miR-637\u003csup\u003e29\u003c/sup\u003e, circCHFR/miRNA-15b-5p\u003csup\u003e30\u003c/sup\u003e), myocardial infarction(CDR1as/ miRNA-671-5p\u003csup\u003e31\u003c/sup\u003e, circNFIB/ miR-433\u003csup\u003e32\u003c/sup\u003e), heart Failure induced by myocardial hypertrophy (circNfix/miR-145-5p\u003csup\u003e33\u003c/sup\u003e, circSlc8a1/miR-133a\u003csup\u003e34\u003c/sup\u003e, circmiRs/miR-132\u003csup\u003e35\u003c/sup\u003e)and so on. Those results contributing circRNA/miRNA axis as next molecular therapy for cardiac hypertrophy treatment, which are capturing massive attention.\u003c/p\u003e \u003cp\u003eThis study identified a novel circRNA(circ_0004641) in mice myocardium and constructed a brand-new CeRNA network (circ_0004641/miRNA-1192 axis). Loss of-and gain of function experiments demonstrated the essential role of circ_0004641/miR-1192 axis in regulation of myocardial hypertrophy both in vivo and in vitro by regulating downstream mRNA (TRIM25/TRIM41). Underlying function of NF-κB signaling pathway were also probed by regulation of CeRNA network.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e1. Construction of cardiac hypertrophy models by AAC surgery\u003c/h2\u003e \u003cp\u003eWe firstly established pressure overloading-induced myocardial hypertrophy model in C57B/L mice at 8 weeks after AAC surgery. Echocardiography and Staining was performed to evaluate the heart change concurrently. H.E. staining, Sirius red and WGA staining showed that the cross-sectional area of the myocardium increased on surgical group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e, along with increased Mass of hearts and enlarged ventricle \u003cb\u003e(S1, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-D).\u003c/b\u003e Compared to sham group, LVPW, LVID, IVS, LV volume of surgical AAC mice were significantly increased both in cardiac systole and diastole period \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C; S1, \u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eE-F)\u003c/b\u003e while EF and CO were sharply decreased \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E\u003cb\u003e).\u003c/b\u003e Moreover, Western Blotting were applied to detect the cardiac biomarker and results illustrated that beta-myosin heavy chain(β-MHC) and atrial natriuretic peptides (ANP) were increasingly expressed, which refers to cardiac hypertrophy \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-G\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2. Identification of differential expressed circRNAs and target miRNAs prediction during cardiac hypertrophy\u003c/h2\u003e \u003cp\u003eAfter mice were euthanized, a total of 8 ventricular myocardium samples (each for 4 in sham and model) were subjected to circular RNA sequencing. We finally identified a total of 9813 circRNAs \u003cb\u003e(\u003c/b\u003e\u003cb\u003eSupplementary 2\u003c/b\u003e;\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e)\u003c/b\u003e in two groups. Based on the locations in host genes,the circRNAs were classified into three categories:95.85% exonic,3.44% intronic,0.71% intergenic circular molecules in sham group;96.19% exonic,3.01% intronic,0.79% intergenic molecules in model group\u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH; S1,\u003cb\u003eFigure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA,2B)\u003c/b\u003e. Majority of the circRNAs ranged in length from 31 to 1970 bp (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI), and distributed across all chromosomes \u003cb\u003e(S1, Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC)\u003c/b\u003e. Among them,5 molecules were identified to have increased expression, while 25 molecules (showed in valcanoplot and heatmap) had decreased expression compare to sham group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA,\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eThen targeted miRNAs were predicted by \u003cb\u003emiRanda (Supplement 3)\u003c/b\u003e and Interaction diagram of circRNA-miRNA was also constructed by cytoscape 3.10 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e. For further insight into potential biological function, we annotated the parental genes of circRNAs by functional enrichment analysis. Gene ontology (GO) biological process enriched in transferase activity and ubiquitination \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that functional circRNAs were mainly enriched in transcription factors, MAPK signaling pathway and Ubiquitin system \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. Herein, according to the P Value on expressive level, we specifically focus on highest expressed circ_0004641for further study \u003cb\u003e(Table\u0026nbsp;1)\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e3. Circ_0004641 was up-regulated both in vitro and in vivo\u003c/h2\u003e \u003cp\u003eTo detect the expression of candidate circRNAs, we performed RT-qPCR validation on remaining myocardial tissue for sequencing. Results showed that only circ_0004641\u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e had an significant expression increase \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. While circ_0009153 showed no significant difference; among the down-regulated genes, results showed a high expression on circ_0002423 and circ_0003046 and no significant expression on circ_0001213 and circ_0009685, which is contradicted to sequencing results \u003cb\u003e(S1, Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eD)\u003c/b\u003e. In addition, we duplicated the validation experiment on ventricular myocardium that not sent for sequencing. Results demonstrated the solely significant increase of circ_0004641, while the other molecules showed no difference or contradictory trend on expression \u003cb\u003e(S1, Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eE)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eIn order to support the results in tissues, hypertrophic cardiomyocytes were stimulated by Ang II and phalloidin staining demonstrated hypertrophy of myofilament and enlargement of cell size \u003cb\u003e(S1, Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA, B)\u003c/b\u003e along with significant increase expression of β-MHC and ANP \u003cb\u003e(S1, Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eC-E)\u003c/b\u003e. Subsequently, qPCR validation on NMVCs showed that circ_0004641\u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e and circ_0009153 has a match increase on expression, while circ_0002423 presenting a paradoxical trend of sequencing results and the others molecules showed no significant difference \u003cb\u003e(S1, Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eF).\u003c/b\u003e Hence, circ_0004641 were identified for further exploration for its highest expression and stable repeatability. Fluorescence in situ hybridization (FISH) identified its subcellular location in the nucleus \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eTo explore its correlation with cardiac hypertrophy, knock-down experiments were applied by transfecting with siRNA. Results shows that inhibiting circ_0004641 significantly reverse the\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eenlargement of cardiomyocytes both in control or Ang II group, along with the decreased transcriptional level of ANP and β-MHC \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE; S1, \u003cb\u003eFigure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eG, H)\u003c/b\u003e. Western blot and quantitative analysis showed a sharp decrease of ANP and β-MHC after transfection \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-H\u003cb\u003e)\u003c/b\u003e. All above indicated a strong relation between circ_0004641 and cardiac hypertrophy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e4. miRNA-1192 was downstream target of circ_0004641 and negatively associated with myocardial hypertrophy\u003c/h2\u003e \u003cp\u003eDownstream predicted miR-1192 of circ_0004641 were synchronously detected and significant decrease was observed in ventricular myocardium and hypertrophic cardiomyocytes \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B\u003cb\u003e)\u003c/b\u003e. After transfection of siRNA, qPCR results demonstrated significant increase of miR-1192, along with down regulation of ANP and BNP \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC; S1, \u003cb\u003eFigure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eG, H)\u003c/b\u003e, indicating the underlying regulation between two molecules. To clarify the interaction between circ_0004641 and miR-1192, we conducted dual luciferase reporter assays. Under 293T engineered cell system, we observed a strong interaction between the WT-circ_0004641-3\u0026rsquo;UTR and miR-1192-mimics, while the mutant-circ-3\u0026rsquo;UTR did not react with miR-1192\u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, E\u003cb\u003e)\u003c/b\u003e. Thus, we verified that circ_0004641 and miR-1192 Interacted like sponge adsorption as we hypothesized.\u003c/p\u003e \u003cp\u003eTo explore the functional role of miR-1192, we over-expressed miR-1192 by mimics in NMVCs, and results showed miR-1192 plays an inhibitory role in cardiac hypertrophy, as transcriptional expression of ANP and β-MHC drop dramatically both in control and Ang II group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF-H\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e5. TRIM25 and TRIM41 act as the downstream mRNA targeted by CeRNA network\u003c/h2\u003e \u003cp\u003eThrough \u003cb\u003emiRDB\u003c/b\u003e website, we totally identified 14 targeted downstream mRNAs regulated by miRNA-1192(both molecules consists of ubiquitin system as bioinformatic analysis predicted), including the TRIM protein family (TRIM6, TRIM21, TRIM25, TRIM32, TRIM33, TRIM41, TRIM71), the RNF protein family (RNF13, RNF138, RNF139, RNF169, RNF170), Dtx31 and RAD18.RT-qPCR validation were conducted both on mice myocardium and NMVCs, and resluts shows that TRIM25 and TRIM41 has stable expressive trend with a significant increase \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Other mRNAs were also detected but had contradictory or non-stable expression level. \u003cb\u003e(S1, Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA-L; S1, Figure S5A).\u003c/b\u003e Repeatable experiments are applied on mice myocardium that not used for sequencing, and only TRIM25 and TRIM41 matched previous results (\u003cb\u003eS1-Figure S5B\u003c/b\u003e), indicating that TRIM215/TRIM41 are two critical molecules regulated by circ_0004641/miRNA-1192 axis in cardiac hypertrophy.\u003c/p\u003e \u003cp\u003eTo verify, tranfection of siRNA-circ_0004641 were down in NMVCs, and results demonstrated TRIM25 and TRIM41 was down-regulated significantly both in trancriptional and translational level \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-E; S1, \u003cb\u003eFigure S5C, D)\u003c/b\u003e, which is contrary to the expressive trend before transfection. Moreover, we co-cultured NMVCs with miR-1192 mimics and, as supposed, the expression of TRIM25/TRIM41 decreased sharply both in control and Ang II group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF-H\u003cb\u003e)\u003c/b\u003e. To this extent, we can conclude that over-expression of circ_0004641 can competitively adsorb miR-1192 molecules, thereby suppressing the inhibitory role of miR-1192 on the expression of TRIM25/TRIM41 in cardiac hypertrophy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e6. NF-κB may act as underlying key pathway in regulation of cardiac hypertrophy\u003c/h2\u003e \u003cp\u003eWe also detected underlying downstream pathways invovling in cardiac hypertrophy and western blot and quantitative analysis suggested that, compared to the control group, there was an increased expression of P65 and phosphorylated P65(p-P65) protein in mouse myocardial ventricular tissue \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA,\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Repeated results are presented in cell model with same expressive trend \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC,\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD\u003cb\u003e).\u003c/b\u003e These results are consistent with previous studies on NF-κB pathway in myocardial hypertrophy\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. By knocking down the expression of circ_0046461, we found that, under the premise that Ang-II group expression was higher than the control group, both two group was observed a significant decrease in expression of P65 and p-P65 after siRNA transfection \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, F\u003cb\u003e)\u003c/b\u003e, which indicates circ_0004641/miR-1192 axis plays an important role in the activation of the NF-κB signaling pathway in cardiac hypertrophy, but the underlying mechanism still needs in-depth research. In addition, we also detect the TGFβ1/smad signaling pathway and phosphorylated Smad protein, which present the same expressive trend of NF-κB(\u003cb\u003eS1,Figure S6A-E\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eTo conclude, we identified a novel_circ_0004641 by sequencing and constructed a brand-new regulatory network via circ_0004641/miR-1192-Trim25/TRIM41 axis in hypertrophic mice myocardium and NF-κB pathway may function as the downstream object of TRIM to cardiac hypertrophy.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCircRNAs, which are dominant sequencings without protein-encoding function, have been recently recognized as dominant regulatory roles since its discovery\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Endogenous circRNAs have been reported to engage in various biological process, such as cell development\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, tumor proliferation and inhibition\u003csup\u003e\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e and cardiovascular diseases\u003csup\u003e\u003cspan additionalcitationids=\"CR45 CR46 CR47\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e.Thus, circRNAs have been found to play an important role in the cardiovascular field, involving almost whole process\u003csup\u003e\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e from cardiac embryonic development to physiological and pathological regulation. Identification of these key molecules facilitates the subsequent comprehensive research on pathological mechanism for myocardial hypertrophy, as a growing number of CeRNA networks are being uncovered\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn our study, a novel circ_0004641 (which would be named as circRftn1 from its parental gene as canonical rules\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e) and CeRNA network(circ_0004641/miR-1192) was characterized in cardiac hypertrophy. For first time, we verified the interaction of circ_0004641 and miR-1192 and established the sponge-like effect in the regulation on cardiac hypertrophy. Our study also identified the downstream mRNA encoding TRIM25/41 protein regulated by circ_0004641/ miRNA-1192 axis. TRIM (Tripartite motif) protein family\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, known as E3 ligase, were prominent molecules that participate in cardiac hypertrophy by activating downstream signaling pathway involving in pathological cardiac myotrophy(such as TRIM8\u003csup\u003e56\u003c/sup\u003e, TRIM32\u003csup\u003e57\u003c/sup\u003e, TRIM44\u003csup\u003e58\u003c/sup\u003e, TRIM63\u003csup\u003e59\u003c/sup\u003e and so on). Our study found the underlying regulation between CeRNA network and TRIM25/TRIM41 for the first time. Previous studies revealed the unique function of TRIM 25\u003csup\u003e60\u003c/sup\u003e and TRIM41\u003csup\u003e61,62\u003c/sup\u003e,as activator, targeting downstream NF-κB pathway at the K63 tyrosine site, thereby enhancing or depressing the tumor proliferation and metastasis. While potential mechanism of TRIM25/TRIM41 on cardiac hypertrophy remains unknown. Our study identified that circ_0004641/miRNA-1192 axis promote the expression of TRIM 25 and TRIM41 on pathological cardiomyotrophy, and firstly speculated that TRIM25/TRIM41 would enhance downstream NF-κB pathway by activating P65 protein and\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003epromote myocardial hypertrophy associated transcriptional process.\u003c/p\u003e \u003cp\u003eThere are still some lackage in our study: 1) Firstly, the miRNAs and mRNAs we verified were predicted by the miRanda and miRDB. Therefore, to ensure the accuracy of the study, we conducted repeated validation experiments in both vivo and vitro levels; 2) Secondly, the relation between TRIM protein and NF-κB signaling pathway still requires validation by Co-Immunoprecipitation (CO-IP) or Immunoprecipitation-Mass Spectrometry (IP-Mass).For further study, amino-acid binding cite for ubiquitination and underlying regulatory mechanism require comprehensive verification.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTo conclude, we revealed a brand-new CeRNA network axis (circ_0004641/ miRNA1192-TRIM41/TRIM25) in myocardial hypertrophy. Mechanically, up-regulated circ_0004641 sponged miR-1192 by depressing its inhibitory role on TRIM25/TRIM41, which contribute to the progression of myocardial hypertrophy. Underlying myocardial hypertrophic regulation by TRIM25/41 protein via NF-κB pathway need in-depth functional verification.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAAC animal model establishment and Evaluation\u003c/h2\u003e \u003cp\u003eThe SPF C57BL/6 mice (male, 6\u0026ndash;8 weeks, 20\u0026ndash;22 g) were purchased from the Experimental Animal Tech of Weitonglihua (Beijing, China). Abdominal aortic coarctation (AAC) is a widely-used method\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e and the procedure was described in \u003cb\u003eSupplement 4\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eEchocardiography in mice was performed at 8 weeks post-AAC operation using the Vevo 2100 Imaging system (visual sonics,Canada). Mice were anesthetized with isoflurane (2.5% for induction, 1.0% for maintenance) and left ventricular parameters during diastole and systole periods were detected from the M-modes. Statistical analysis was applied by Prisma 9 software on cardiac indexes in \u003cb\u003eSupplement 4\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHistological analysis and RNA fluscence in situ hybridization (FISH)\u003c/h2\u003e \u003cp\u003eMice were euthanized and hearts were excised and fixed in 4% paraformaldehyde and routinely dehydrated and paraffin-embedded. The wax blocks were cut into sections with a thickness of 4\u0026ndash;5 \u0026micro;m. Subsequently, cross heart sections were stained with hematoxylin-eosin (HE, Servicebio, China) and Sirius Red staining (sigma, USA) to assess the size and morphologic alterations of the heart. FITC-conjugated wheat germ agglutinin (WGA, sigma, L4895, 1:500, USA) staining was performed to demarcate cardiomyocyte boundaries and 4ʹ,6-diamidino-2-phenylindole (DAPI, Servicebio, China) was used to label the nuclei. Images were captured using Leica microscope (Leica, Wetzlar, Germany) and processed with CaseView 2.4.\u003c/p\u003e \u003cp\u003e Cardiomyocytes were fixed with paraformaldehyde (4%), followed by treatment with 0.5% Triton. FISH was performed using a FISH Tag\u0026trade; RNA Multicolor Kit (Invitrogen, USA) and DAPI was used to stain the cell nucleus. The localization of circRNA was observed by using Leica SP5 Spectral scanning laser confocal microscope (Leica, Wetzlar, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eNeonatal mice ventricle cell separating and culture\u003c/h2\u003e \u003cp\u003eNeonatal mouse ventricle cardiomyocytes (NMVCs) were isolated from the hearts of neonatal C57BL/6 mice (0\u0026ndash;3 days after birth). Ventricle tissues were sectioned into 1 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e pieces and isolated by using 0.1% collagenase II (Gibico, China). NMVCs were cultured in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s medium/Nutrient Mixture F-12 (DMEM/F12; Thermo Fisher Scientific, USA) containing 10% fetal bovine serum (Hyclone, USA) and maintained at 37\u0026deg;C with 5% CO2.\u003c/p\u003e \u003cp\u003eNMVCs were incubated with 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003eM Angiotensin II (Ang-II,Selleck,USA) for 48h to induce the hypertrophic phenotype. SiRNA targeting mmu_circ_0004641 (5nm, RiboBio, China) was transfected into NMVCs by co-culture with Ang II or DMSO for 48h.The sequences of siRNA of circ_0004641 is as follows: CATGGCCAATGGTGCAGGA. Overexpression mimics targeting mmu_miR-1192(50pmol, Sangon Biotech, China) was co-cultured with NMVCs by Ang II or DMSO for 48h.The sequences of miR-1192 mimics is as follows: AAACAAACAAACAGACCAAAUU.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction, library preparation, and CircRNA sequencing\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated from the left ventricular tissues of mice with TRIzol\u0026trade; reagent (Invitrogen, United States). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, United States) before library preparation. Qualified RNA samples were treated with Ribo-ZeroTM Gold Kits (Epicenter Technologies, United States) to remove ribosomal RNA (rRNA). Sequencing libraries were generated using the NEBNext\u0026reg; Ultra\u0026trade; RNA Library Prep Kit for Illumina\u0026reg; (NEB, United States) following the manufacturer\u0026rsquo;s recommendations. Sequencing process was on Illumina HiSeq 4000 platform, and 125 bp/150 bp paired-end reads were generated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCircRNA identification and Target prediction\u003c/h2\u003e \u003cp\u003eTo ensure the quality and reliability of sequencing data, the original data was filtered (See Supplement 2). Hisat2 software was used to accurately align clean reads with the reference genome, and obtain localization information. Find_ Circle algorithm and CIRI algorithm (Supplement 2) were used for identifying candidate circRNAs, and TMM algorithm was applied to standardize readcount data before conducting differential analysis. Fold change and corrected significance level are identified for screening (p_adj\u0026thinsp;\u0026lt;\u0026thinsp;=\u0026thinsp;0.05 and log2FoldChange\u0026thinsp;\u0026gt;\u0026thinsp;=\u0026thinsp;1.0 as threshold). Visualization by volcano plots and heatmap are down to infer differential circRNAs. Gene Ontology and KEGG enrichment analyses were performed on the sets of parental genes with differential expression of circRNA in each group. Based on miRanda software and miRDB software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://mirdb.org/miRDB\u003c/span\u003e\u003cspan address=\"http://mirdb.org/miRDB\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ), miRNA binding sites targeted by the identified circRNAs and downstream mRNAs were predicted respectively. Then, interaction between circRNA and miRNA was constructed by cytoscape 3.10.(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.cytoscape.org/\u003c/span\u003e\u003cspan address=\"http://www.cytoscape.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blot analysis\u003c/h2\u003e \u003cp\u003eTotal protein was isolated and quantified using a ImageQuant LAS 4000 system (Cytiva,USA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel (10%) was prepared and 40 \u0026micro;g protein was loaded for electrophoresis; the protein was then transferred onto a polyvinylidene fluoride (PVDF) membrane. Next, the blots were blocked in 5% skimmed milk at room temperature for 2 h and incubated with the primary antibody β-MHC (1:1,000; ab172967, Abcam, UK), ANP (1:2,000; 27426-1-AP, Proteintech,USA),TRIM25(1:1000;Proteintech,12573-1-AP), TRIM41(1:1000, Proteintech, 18468-1-AP),p-NFκB (1:1,000; 3033, CST, USA), NFκB (1:1,000; 8242, CST, USA), TGFβ1 (1:1,000; ab179695, Abcam, UK), p-SMAD1/5 (1:1,000; 9516, CST, USA), SMAD1/5 (1:1,000; ab75273, Abcam, UK), p-SMAD2/3 (1:1,000; 8828, CST, USA), SMAD2/3 (1:1,000; 8685, CST, USA), GAPDH (1:10,000; ab181602, Abcam, UK)overnight, followed by incubation with the secondary antibody (1:5,000; 5220\u0026thinsp;\u0026minus;\u0026thinsp;0336, KPL, USA) for a further 2 h. sigma, L4895, 1:500. The data were analyzed using Image J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real-time PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated from the left ventricular tissues of mice with TRIzolTM reagent (Invitrogen, United States). Then, reverse transcription was accomplished using a reverse transcription kit (TOYOBO, FSQ-101, Japan). Next, qRT\u0026ndash;PCR was performed to detect gene expression using the ChamQ Universal SYBR qPCR Master Mix Kit (Vazyme, Q711-03, China) on a CFX96 detection system (BioRad, United States). The relative expression of circRNA was calculated based on the cycle threshold values according to the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method. GADPH was used as a reference gene for normalization. The primers used are listed in \u003cb\u003eSupplementary 4\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eDual-Luciferase Reporter Assay\u003c/h2\u003e \u003cp\u003eThe recombinant luciferase reporter plasmids containing the potential circ_0004641 binding site sequences were constructed. Human embryonic kidney (HEK) 293 cells (3 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well in 12-well plate) were co-transfected with 200 ng of recombinant luciferase reporter plasmid, 20 ng of pRL-TK as an internal control (Promega, Madison, WI), 200 ng of pDsRed2-N1 or 200 ng of pDsRed2-miR-192, 50 nM circ_0004641 mimic or 50 nM mutant circ_0004641 mimic, respectively. Activities of firefly luciferase (FL) and Renilla luciferase (RL) were measured 24 hour after transfection, and the relative ratio of the FL/RL was used to indicate the circ_0004641-mediated knockdown of target genes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Unpaired Student\u0026rsquo;s t test (two-tailed) was used for comparisons between groups or one-way ANOVA test was performed to analyze multiple groups followed by Bonferroni post hoc tests. All data were analyzed using by GraphPad Prism 9 and P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.Graphical Abstract was portrayed by Figdraw2.0.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eAAC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Abdominal aortic coarctation \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAng II \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Angiotensin II \u0026nbsp; \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCircRNA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Circular RNA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emiRNA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;microRNA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTRIM \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Tripartite motif\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCeRNA Network \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Competitive endogenous RNA Network\u003c/strong\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and formulation. Methods design and model sonstruction were excecuted by Guangcheng Liu and Haipeng Zhang. Material preparation and experiments were performed by Guangcheng Liu, Haipeng Zhang and Jingdai Zhang. Data collection,correction and analysis were performed by Hao Qian, Liang Wang and Lianfeng Chen. The first draft of the manuscript was written by Guangcheng Liu. Zhujun Shen drafted the work critically for important intellectual content. All authors commented on previous versions of the manuscript and we confirmed that all authors in the list contributed to the article and approved the finally submitted version.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the National Natural Science Foundation of China (NSFC) projects:82170372.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eInstitutional Review Board Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee\u0026nbsp;of\u0026nbsp;Peking Union Medical College Hospital (PUMCH,China).Written informed consent was obtained from the owners for the participation of their animals in this study.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eInformed Consent Statement:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable for studies not involving humans.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during during the current study are available in the Mendeley Database repository, doi: 10.17632/hy5pmx39g5.1.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no potential conflict of interest in this research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMishra S, Kass DA. 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The cross-talk between PARylation and SUMOylation in C/EBP\u0026beta; at K134 site participates in pathological cardiac hypertrophy. \u003cem\u003eInt J Biol Sci\u003c/em\u003e. 2022;18(2):783-799. doi:10.7150/ijbs.65211\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Circular RNA, CeRNA, Cardiac hypertrophy, Heart Failure, TRIM","lastPublishedDoi":"10.21203/rs.3.rs-4452125/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4452125/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eAim \u003c/strong\u003eTo verify the cardiac hypertrophical role of circ_0004641 and its potential mechanism by targeting miR1192-TRIM25/TRIM41 axis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e Mice model was constructed by abdominal aortic coarctation (AAC) surgery and cell model was established from isolated neonatal mouse ventricular cardiomyocytes (NMVCs) by co-cultured with angiotensin II (Ang II). Differential expressed circRNAs were identified by Next-generation sequencing and target miRNAs and downstream mRNAs were predicted by bioinformational analysis. RT-qPCR and Western Blot were applied to validate myocardial-associated molecules on transcriptional and translational levels respectively. By transfecting siRNAs or co-culturing with mimics, expression of regulating-molecules was detected respectively. Dual luciferase reporter assay was performed to identify the interaction between circular RNA and miRNA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e A total of 5 up-regulation and 25 down-regulation circRNAs were detected on hypertrophical myocardium by Next-generation sequencing. Among them, circ_0004641 was significantly increased both in in vivo and in vitro as ANP and β-MHC accumulated, while downstream target miRNA-1192 decreased and target mRNA (TRIM25/TRIM41) increased dramatically. Knock-down of circ_0004641 by transfecting siRNA shows a reverse effect on cardiac hypertrophy, along with contrary expressive trend of miR-1192 and TRIM25/TRIM41.Dual luciferase reporter assay identified the sponge-like interaction between circ_0004641 and miR-1192. By co-culturing NMVCs with miR-1192 mimics, its targets TRIM25/TRIM41 showed significant decrease. Moreover, NF-κB signaling pathway were identified to correlated by circ_0004641/miRNA-1192 axis as P65 protein present similar expressive trend with circ_0004641.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e circ_0004641 may exert a stimulative role in cardiac hypertrophy by regulating miR-1192-TRIM25/TRIM41 axis and NF/κB p65 pathway is the underlying downstream pathway.\u003c/p\u003e","manuscriptTitle":"Circ_0004641 upregulates TRIM25/TRIM41 expression by sponging miR- 1192 to induce cardiac hypertrophy in vitro and in vivo","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-07 19:01:15","doi":"10.21203/rs.3.rs-4452125/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-29T08:34:26+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-29T08:11:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"242844538215659176554870032658403477270","date":"2024-07-28T11:45:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-19T19:42:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"259506926511421246155951445397180360804","date":"2024-07-19T17:22:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-08T11:09:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-08T11:03:33+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-05-23T04:59:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-23T04:56:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-05-21T04:28:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e304d9a1-f46e-4fb1-86a7-512792e2acad","owner":[],"postedDate":"June 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-10-14T05:08:50+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-07 19:01:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4452125","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4452125","identity":"rs-4452125","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}