Non‐coding RNAs in cardiomyocyte proliferation and cardiac regeneration: Dissecting their therapeutic values

Abstract Cardiovascular diseases are associated with high incidence and mortality, contribute to disability and place a heavy economic burden on countries worldwide. Stimulating endogenous cardiomyocyte proliferation and regeneration has been considering as a key to repair the injured heart caused by ischaemia. Emerging evidence has proved that non‐coding RNAs participate in cardiac proliferation and regeneration. In this review, we focus on the observation and mechanism that microRNAs (or miRNAs), long non‐coding RNAs (or lncRNAs) and circular RNA (or circRNAs) regulate cardiomyocyte proliferation and regeneration to repair a damaged heart. Furthermore, we highlight the potential therapeutic role of some non‐coding RNAs used in stimulating CMs proliferation. Finally, perspective on the development of non‐coding RNAs therapy in cardiac regeneration is presented.

cells. 4,5 Although adult CMs can divide into two cells, these events naturally occur at a very low rate, which is not sufficient to restore the heart function after injury. Because of the dramatic decline in CMs cycle re-entry activity and the loss of regeneration potential in adult hearts, there has been increasing research interest to understand the cellular mechanism of CMs division. Numerous studies have reported that in CMs, signals from growth factors, internal signalling pathways, microRNAs and cell cycle regulators can promote the cell cycle re-entry in injured hearts. [6][7][8][9] Protein-coding RNAs account for less than 2% of all of the transcribed RNAs. 10 A large body of evidence has shown that non-coding RNAs play important roles in biological processes and diseases.
Based on the lengths, non-coding RNAs can be subdivided into 2 major groups: (a) small non-coding RNAs (<200 nucleotides) including rRNA, tRNA, microRNAs, PIWI-interacting RNAs and endogenous short interfering RNAs, etc and (b) long non-coding RNAs that have transcripts larger than 200 nucleotides in length and have no known protein-coding function. [11][12][13] In this review, we focus on non-coding RNAs' regulatory and therapeutic roles in CMs proliferation and cardiac regeneration ( Figure 1). Besides, the general mechanism of ncRNAs in CM proliferation and heart regeneration is depicted in Figure 2.

| miRNAs regulate CMs proliferation
In the cardiovascular system, miRNAs perform their physiological and pathological function in cardiac development, 14,15 diseases 16,17 and regeneration. 18, 19 Here, we discuss the roles of miRNAs on CMs proliferation and the associated mechanism, which are summarized in Table 1.

| miRNAs promote CMs proliferation miRNAs targeting Hippo-Yap signal pathway
Microscopy-based high-content screening functionally identified that hsa-miR-590 and hsa-miR-199a effectively increased both DNA synthesis and cytokinesis in neonatal mice and rat CMs. 20 After myocardial infarction, these miRNAs strongly stimulated cardiac regeneration and significantly recovered cardiac function. 20 The deepsequencing analysis revealed that Homer1, Hopx and Clic5 are targets of these miRNAs. 20 Their effects on stimulating CMs proliferation is F I G U R E 1 miRNAs, lncRNAs and circRNAs regulate cardiomyocyte proliferation to promote heart regeneration after injury. MicroRNAs, lncRNAs and circRNA are represented by red, blue and green boxes, respectively. The arrow indicates the promoting effect and the blunt end arrow indicates the inhibiting effect. (Created with BioRender.com) based on cumulative results on multiple targets. Hippo signal transduction pathway is considered a critical approach to regulate proliferation. 21 A recent study has shown that a series of miRNAs promote CMs proliferation, including hsa-miR-590 and hsa-miR-199a, by activating nuclear translocation of YAP and inducing the expression of YAP-responsive genes. 21,22 In addition, several miRNAs (including miR-199a-3p) also inhibit filamentous actin depolymerization by targeting Cofilin2 and activating YAP nuclear translocation. 22 The deletion of the miR-302-367 cluster using the Cre-LoxP system expressed during embryonic development confirmed that this cluster is essential for CMs proliferation. 23 Intracardiac injection of Gel-miR-302 stimulates both the wild-type and Myh6-MerCreMer:R26R-Confetti transgenic mice CMs to proliferate. 24 Furthermore, miR-302-367 cluster, hsa-miR-590 and hsa-miR-199a, exert their pro-proliferative effects on CMs by targeting components (Lats2, Mob1 and Mst1) of the Hippo signalling pathway. 23

miRNAs regulate the cell cycle to promote CMs proliferation
The regulation of cyclin, cyclin-dependent kinase and regulators highly expressed in the foetal stage, significantly stimulate adult CMs to re-enter cell cycle. 25 The majority of these proteins are targets of miRNAs. miR-1825 is one of the miRNAs screened by the above approach 20 and a master regulator of miR-199a. 26 Transfecting with miR-1825 mimics markedly increases the proliferation of adult mice CMs. 26 MiR-1825 has been reported to reduce the mitochondrial numbers and destroy their function by direct inhibition of NDUFA10 and cell cycle genes. 26 miR-204 stimulates human CM progenitor cells to proliferate and differentiate. 27 Transgenic mice with highly cardiac expression of miR-204 exhibit a thicker ventricular wall, which is associated with CMs proliferation rather than hypertrophy during heart development by directly targeting Jarid2, 28 while Jarid2 binds to the promoter of cyclin D1 and represses its expression. 29 MiR-210, up-regulated in many cardiac diseases, exerts its beneficial effects against ischaemic injury when injected into the myocardium. 30 Transfection adult rat CMs with miR-210 significantly increases the amount of CMs and inhibits apoptosis as well. 31 Overexpression of miR-210 in transgenic mice results in recovery against injury and also promotes CMs proliferation and angiogenesis. 31 In silico analysis indicates that APC (adenomatous polyposis coli)-cell cycle inhibitor is involved in the canonical Wnt signalling pathway, which is a target of miR-210. 31 miR-294 is highly expressed during embryonic cardiac development and rapidly declines after maturation, which has been found to promote both neonatal rat ventricular myocytes (NRVMs) and feline adult CMs to enter the cell cycle. 32 In another study, miR-294 F I G U R E 2 General mechanisms of ncRNAs in CM proliferation and heart regeneration. miRNAs recognize their targets mRNAs by "seed region" resulting in translation inhibition and protein degradation. CircRNAs and lncRNAs could both act as a sponge to compete with endogenous miRNAs. LncRNAs function as signal, decoy, scaffold, guide and enhancer depends on their specific subcellular location. CircRNAs exert their biological function as regulators of splicing and transcription and modifiers of parental gene expression.

Exercise-induced miRNAs regulate CMs proliferation
Exercise induces physiological cardiac growth evidenced by increased proliferation markers and protection of the heart against pathological remodelling. miR-17-3p is induced by exercise and protects the heart against ventricular remodelling. 36 Inhibition of miR-17-3p can attenuate CMs hypertrophy and inhibit their proliferation. 36 Besides, miR-17-3p directly targets tissue inhibitor of metalloproteinase-3 (TIMP3) to induce CMs proliferation via EGFR/ JNK/SP-1 signalling 37 and indirectly regulates PTEN to promote CMs hypertrophy. 36 The expression level of miR-222 is also up-regulated in exercise models. 38 Overexpression of miR-222 is sufficient to induce neonatal CMs physiological growth, cellular hypertrophy and proliferation by reducing the expression of p27, negatively regulating the cell cycle and transcription factor HIPK1 39 and inhibiting apoptosis. 38 Cardiac-specific expression of miR-222 protects against cardiac remodelling and dysfunction after ischaemic injury. 38 The negative function of miR-222 was further demonstrated by multiisotope imaging mass spectrometry (MIMS) to identify newly formed CMs in the exercise model, which could be completely blocked by inhibition of miR-222. 40 Other mechanisms miR-17-92 cluster, known as OncomiR-1, is required for CMs proliferation in the embryonic and postnatal mouse hearts. 41 Overexpression of miR-17-92 induces CMs proliferation in embryonic, postnatal and adult heart and protects the adult heart from myocardial infarction through targeting Pten. 41 MiR-19a/19b, family members of miR-17-92 cluster, are highly expressed in heart failure patients. 42 Overexpression of miR-19a/19b promotes CMs proliferation, reduces apoptosis and blocks inflammation through targeting Pten, Bim1 and SOCX1/3. MiR-19a/19b protect the adult heart in two distinctive phases after myocardial infarction: early-phase and longterm protection. 42 Furthermore, miR-25 also belongs to an oncogene named MCM7, it promotes CMs growth and migration by targeting Bim. 43 miR-31-5p is up-regulated in P10 CMs compared to P0, but it promotes NRCMs proliferation through targeting RhoBTB1, 44 a subfamily of the Rho small GTPases. 45 This up-regulation of miR-31-5p is probably a compensatory mechanism of the CMs in response to exiting the cell cycle.
Unbiased miRNA-sequencing indicated that miR-486 was enriched in striated muscle and was up-regulated in neonatal patients with hypoplastic left heart syndrome which was confirmed by sheep dilated right ventricle. 46 The ventricle of neonatal mice treated with miR-486 mimics exhibited increased growth of the ventricles without changes in wall thickness and CMs proliferation. 46 Previously, iTRAQ-based mass spectrometry proteomics studies indicated that Stat1 was one of the most up-regulated proteins after miR-486 mimic treatment. 46

| miRNAs inhibit CMs proliferation
The role of miR-1-2/miR-133a-1 and miR-1-1/miR-133a-2 The cardiac-and skeletal muscle-specific miRNA genes miR-1-1 and miR-1-2 are specifically expressed in ventricle during cardiogenesis and activated during differentiation. 47 Transgenic mice with β-myosin heavy chain (MHC) promoter highly express miR-1 at E9.0 resulting in the thinner ventricular wall and less CMs proliferation via targeting Hand2, 47 which is required for the expansion of the embryonic cardiac ventricles. 48 Targeted deletion of miR-1-2 without affecting the resident gene Mib1 showed ventricular septal defect at E15.5 and some (~15%) mice survived to 2-3 months would suddenly die due to electrophysiologic defects as a consequence of direct inhibition of Irx5 by miR-1-2. 49 Besides, most adult miR-1-2 mutants have a thicker ventricular wall due to the increased proliferation of CMs. The effect on ventricular wall is consistent with overexpressed miR-1 in the heart. 49 miR-133a-1 and miR-133a-2 have identical sequences and are highly expressed in the heart. 50 The two miRNAs are transcribed as bicistronic transcripts with miR-1-2 and miR-1-1, respectively, in skeletal and cardiac muscle. 50 Mice deleting single gene were normal, whereas double knockout mice died during late embryonic or neonatal stage due to ventricular septal defect, along with enhancement of CMs proliferation, apoptosis and aberrant expression of smooth muscle genes in the heart. 50 Cyclin-D2, a positive regulator of cell cycle, is a target of miR-133a-1 and miR-133a-2. 50 Microarray data indicated that miR-133 was reduced during regeneration after resection of the ventricular apex in zebrafish. 51 Transgenic overexpression of miR-133 by heating shock single time after resection in short-term or daily in the long-term inhibited cardiac regeneration due to decreased CMs proliferation. 51 However, the deletion of miR-133 significantly increased the proliferation index of CMs and restored the myocardium through persistent inhibition of miR-133. 51 Besides the regulators of the cell cycle, pharmacological inhibition and EGFP sensor interaction studies indicated that cx43, a component of the cell junction, was a miR-133 target. 51 Another study on sheep showed miR-133 expression in heart enhanced while it's direct target gene IGF1R's expression decreased with age. 52 The expression profile of other targets of miR-133 such as CCND2, SRF, PGAM1 and GJA1(Cx43) did not show the reverse tendency of miR-133; hence, the regulatory effect of miR-133 could be through an indirect signalling pathway. 52 Cell cycle regulators miR-195 is one of the miR-15 families with 6 miRNAs sharing a similar seed region and is the most up-regulated miRNA in P1 and P10 mice. 53 Transgenic mice overexpressed miR-195 during embryonic would partly die on the consequences of the large apical ventricular septal defect and ventricular hypoplasia. 53 Though the survival parts had a normal cardiac function, they showed fewer proliferating CMs and depressed cardiac function. 54 However, neonatal mice receiving LNA (locked nucleic acid)-modified miR-15b and miR-16 showed more CMs mitosis re-entry and progression without cytokinesis. 53 Furthermore, LNA-modified miRNA injection from neonatal to adult improved heart function and stimulated CMs proliferation after MI injury. 54 RISC-seq confirmed that the miR-15 family negatively regulated cell cycle by directly targeting Chek1 (checkpoint kinase 1), a conserved target between humans and mice, and was required for 53,55 Global gene profiling of injured mouse and zebrafish hearts has revealed that miR-26a is down-regulated in the injured zebrafish hearts while keeping constant in the injured mouse hearts. 56  H9c2 and NRVMs to proliferate through targeting CCND2, a positive regulator of cell cycle. 57 In addition, CCND2 was also a target of an anti-proliferating miRNA let-7i-5p. 58 Another study found that miR-29a was up-regulated in purified adult rats CMs compared with neonatal and postnatal as well as miR-30a and miR-141 families. 59 Anti-miR of miR-29a, miR-30a and miR-141 enhanced the cell cycle re-entry of NRVMs and the predicted targets were Ccna2 and CDK6. 59 miR-34a is a regulator of age-associated physiology and prevents heart from regenerating in MI injury and its negative effect on cell proliferation is by direct targeting Bcl2, Cyclin D1 and Sirt1. 60 RNA-sequencing analysis of P1, P7 and P28 mice cardiac ventricles indicated that miR-128 expression was increased upon

F I G U R E 3
The mechanisms of lncRNAs in CM proliferation. The pink boxes indicate the lncRNAs that promotes the proliferation of CM, and the green boxes indicate the lncRNAs that inhibits proliferation. The round boxes represent the protein targets they regulate. The specific mechanisms are shown in Table 2 growth. 61 Cardiac-specific overexpression of miR-128 in early-stage led to the enlargement of the ventricle, reduced function and decreased regeneration after apex resection due to decreased proliferating of CMs. 61 Conditional deletion of miR-128 in neonatal mice showed similar heart size but smaller and more proliferating CMs. 61 Knockout of miR-128 in adult mouse hearts resulted in more proliferating and differential CMs under basal conditions as well as improved heart regeneration at injury conditions. 61

| LncRNAs regulate CMs proliferation
More recently, lncRNAs have come into the spotlight due to their roles in regulating gene expression and biological processes.
LncRNAs are generated by RNA polymerase Ⅱ, 5'-capped, spliced and 3'-polyadenylated (except some specific non-polyadenylated lncRNAs 63,64 )-but they are not translated into proteins and are expressed at a relatively low level. 65 Based on their gene location, they are divided into six subgroups: sense, sense intronic, antisense, bidirectional, enhancer or intergenic lncRNAs. 65 However, this standard of classification is not enough due to the abundant amount of lncR-NAs. 10 Therefore, based on their functions, nuclear-expressed lncR-NAs are divided into signal, decoy, guide or scaffold and enhancer lncRNAs. 66 The rest of the cytosol-expressed lncRNAs support the following functions: they can be integrated into a complex of ribonucleoprotein (RNP) and trafficking, act as a sponge to sequester miRNAs and combined with mRNAs to stabilizing or destabilizing them. 66 The specific definition of this classification has been reviewed in detail previously. 65,66 Here, we discuss the role of lncRNAs in regulating CMs proliferation and cardiac repair as well as their potential therapeutic role. The specific regulating mechanism of each lncRNA is depicted in Figure 3.

TA B L E 2 (Continued)
human heart compared with adults. 67 Adenovirus-or AAV-mediated overexpression of ECRAR was found to promote postnatal and adult CMs to re-enter cell cycle without leading to hypertrophy at baseline or subjected MI injury. 67 Besides, ECRAR knockdown in neonatal rat using shRNA showed decreased proliferation of CMs which was further confirmed in human AC16 cells. 67  Sirt-1 antisense lncRNA is highly expressed in the embryonic stage and rapidly decreases after birth. 68 Adenovirus-mediated gain-of-function promotes NRVMs to undergo mitosis, karyokinesis and sarcomere disassembly. 68 LNA-mediated loss-of-function approach in neonatal and AAV9-mediated overexpression of this lncRNA in adult mice, both indicated that this lncRNA was required and sufficient to induce CMs proliferation. 68 Elevated expression of this lncRNA also enhances survival rate, improves cardiac function, reduces infarct area and inhibits fibrosis after MI. 68 Complementary with 3'UTR of Sirt1 mRNA, Sirt1 antisense lncRNA interacts with Sirt1 mRNA and augments its stability and pro-proliferating ability. 68 NR-045363 is an antisense lncRNA to human CDK6, which is reported to be down-regulated in the embryo but up-regulated in adults 7 days after apex resection and correlates with CMs proliferation. 69 AAV-mediated overexpression improves cardiac function and reduces scar size due to increased CMs proliferation. 69  Microarray analysis has shown that CAREL is up-regulated with growth and development. 73 Transgenic mice with cardiac-specific overexpression of CAREL impede CMs proliferation and cardiac regeneration after apex resection which has been confirmed in the intracardiac injection of CAREL adenovirus. 73 However, CAREL deletion mediated by adenovirus reduces the scar size, improves cardiac function and enhances CMs cell cycle re-entry after MI injury. 73 CAREL, expressed in the cytoplasm of CMs, acts as an endogenous competitor of miR-296 and promotes CMs proliferation by directly targeting Trp53inp1 or Itm2a. 73 RNA-seq data from human foetal and adult heart revealed that the expression of AZIN2-sv and CRRL were correlated with cell cycle-related protein-coding genes and increased with age. 74,75 Adenovirus-mediated gene regulation of these lncRNA revealed that they are negative regulators of CMs proliferation. 74

| CircRNAs regulate CMs proliferation
CircRNAs (circular RNAs) are circularized by connecting the 3' end to 5' end to provide stability compared with non-circular RNAs and thus play an important role in the regulatory pathway. 76,77 Superenhancer associated circRNA circNfix is highly expressed in adult hearts and also highly expressed in the cytoplasm of CMs. 78 SiRNA-mediated knockdown and adenovirus-mediated overexpression revealed that circNfix was a negative regulator of CMs proliferation. 78 The knockdown of circNfix by AAV9 packaging shRNA significantly facilitates adult CMs proliferation and dedifferentiation marked by increased RUNX1. 78 CircNfix exerted its anti-regenerating effect through two independent pathways-inhibiting CMs proliferation and angiogenesis. 78 First, transcription factor Meis1 binds to the superenhancer of CircNfix and promotes transcription and cyclizing of Nfix. 78 CircNfix then combines with Ybx1-a positive regulator of CyclinA2/B1-and Nedd4l, to the consequence of ubiquitination and degradation of Ybx1. 78 Second, circNfix acts as a sponge to absorb miR-214 which directly targets Gsk3β and promotes the expression of β-catenin to inhibit angiogenesis. 78

TA B L E 3 (Continued)
Highly expressed in foetal and neonatal heart, CircHipk3 can facilitate cardiomyogenesis and angiogenesis. 79 CircHipk3 could increase the stability of Notch1 intracellular domain (N1CID) by acetylation and prevent its degradation to stimulate CMs proliferation. 79 CircHipk3 also acts as a sponge for miR-133a to increase the expression level of connective tissue growth factor (CTGF), then activates endothelial cells. 79 The summary of LncRNA and CircRNA in CMs proliferation are listed in Table 2.

| RNA-BA S ED THER APEUTI C S TR ATEG IE S
Given the significance of non-coding RNA in regulation of CMs proliferation, the therapeutic potential of non-coding RNAs has aroused extensive research interests. The first question is how to direct the non-coding RNAs into the specific tissue or cell to play their biological roles? During the last two decades, viral particles were found to be effective tools to package plasmid containing therapeutic noncoding RNAs. Besides, many oligonucleotides were also designed and modified to enhance their affinity and stability and strengthen the curative effect.

| Adenoviral-based gene delivery
Adenoviruses are double-stranded DNA viruses that packaged in a high-affinity protein capsid. Due to their high transfection efficiency and robust transgene expression, they are extensively used in scientific research. However, the characteristic of transient expression limits their application in the treatment of diseases. Adenovirus vectors containing shAZIN2-sv injected into the myocardium has been reported to preserve adult rats' cardiac function, reduce infarct area and promote angiogenesis from 14 to 60 days after MI injury. 74 Besides, adenoviral proteins elicit hosts' immune response and this is one of the major hurdles limiting its therapeutic application.

| Adeno-associated virus-based gene delivery
Adeno-associated viruses have a single strand DNA genome that does not integrate into the host genome. Currently, more than 10 serotypes are known and used in gene therapy. Another AAV's feature is the serotypes with different organotropism. AAV serotype 9 showed the best transgene expression and distribution when systemically delivering different AAV serotype to mice. 80 AAV9 has been widely used in rodents while AAV6 has been used to cure cardiac injury in pigs. 81 Though AAV9-packaged transgenes are highly expressed in cardiac, other organs such as the liver and lungs have comparable expression due to systemically delivery.
The episomal circular form of recombinant AAV results in long time expression ranging from days to months. AAV 9-mediated cardiac-targeted delivery of miR-19a/19b soon after MI injury provides long-term protection lasting from 7 days to 2-3 months after injection. 42 Intracardiac injection AAV9-anti-miR-99/100 or AAV9anti-Let-7a/c in the border of the infarcted area provides prolonged cardiac protection for up to 90 days after MI injury. 62 Non-coding RNAs that inhibit CMs proliferation can also be used as treatment through their shRNA packaged by AAV particles. Injection of AAV9-shcircNfix into the peri-infarct area of adult mice resulted in significant improvement in the ejection fraction post-MI. 78 In a previous study, adult mice subjected to MI surgery were injected with AAV9 vectors expressing hsa-miR-590 and hsa-miR-199a into peri-infarcted area. This resulted in improved cardiac function from 12 days to 1-2 months after injection. 20 However, in mammals, this long-term expression of transgene may be cause detrimental effects. Delivery of hsa-miR-199a through an AAV serotype 6 vector 1-month post-MI, treated pigs showed marked improvements in both global and regional contractility, increased muscle mass and reduced scar size. 81 It is worth noting that in pigs treated with the miR-199a, although 30% showed a continuous improvement in cardiac morphology and function with a recovery period lasting for two months, 70% sudden died 7-8 weeks after MI. 81 The electrocardiogram results revealed that the deaths were caused by accelerated heart rate which led to ventricular fibrillation. 81

| Oligonucleotides-based gene therapy
Although virus vectors are a powerful tool for expressing non-coding RNAs, several issues such as the short duration of expression, risk of infecting other unintended organs and possibility of triggering an immune response limit their clinical application. Because the dedifferentiation of CMs is a prerequisite step determining the ability of CMs to repair cardiac injuries, prolonged expression of pro-proliferating miRNAs may result in adverse effects.

| MiRNA mimics and lipid formulation
Synthetic oligonucleotides might be a more promising alternative.
This is because they produce prolonged proliferative effect without long-term potential adverse effects. 84 Up-regulation of some miR-NAs that improve CMs proliferation using miRNA mimics maybe an alternative approach to treat cardiovascular disease. MiRNA mimics are double-strand, chemically modified oligonucleotides that do not undergo natural miRNA biogenesis but have the same biology function such as inhibiting the expression of target genes. 85,86 Furthermore, 2' -O-methoxyethy of ribose, 5' Cholesterol and phosphorothioates backbone modified miRNA agonist called agomir was found to significantly improve the nuclease resistance and affinity of mimics. Mice injected with miR-17-3p agomir through tail vein were protected from adverse remodelling after cardiac I/R injury. 36 Oligonucleotides are much smaller than biomacromolecules such as proteins or ribonucleic acids, but they are bigger than some small molecules (<500 Da) which can passively diffuse across cellular membranes. Mimics or agomirs are larger than 14kDa and have numerous charges. Therefore, to enhance their cellular uptake, they should be packaged into some nanoparticles. Five kinds of lipid formulations were used to deliver pro-proliferation miR-199a-3p mimics. RNAiMAX was found to be the most effective (transfection efficiency > 80%) and less toxic formulation. 84 The expression level of miRNA significantly increased 3 days after intracardiac injections and the inhibition effect on their targets could maintain for 8-12 days. 84 Overexpression of miR302-367 cluster in adult mice heart reduced the scar formation following MI injury but it did not improve the heart function. 23  Lipid nanoparticle delivery of miR-708 using RNALancerII injected via tail vein protected against cardiac injury induced by isoproterenol. 87 The expression level of miR-708 was up-regulated for 16 days which is sufficient to inhibit hypertrophy and reduce fibrosis for 5-10 days following ISO treatment. 87

| Locked nucleic acid
Locked nucleic acid (LNA) means the ribose sugar is locked in a C3'endo conformation by the introduction of a 2'-O-, 4'-C-methylene bridge to form 2' sugar modification. 88 This modification improved the affinity of complementary RNA. A previous study injected LNA-miR-294-3p mimic formulated with RNALancerII into the heart once soon after MI injury. 32 They found that expression level of miR-294-3p significantly up-regulated two days after injection. This was accompanied by increased CMs proliferation but the protective effect only lasted for 2-3 weeks as the infarct size was not reduced 8 weeks after MI injury. 32 LNA can also be used to inhibit miRNAs. This is achieved by complementary base pairing to form a DNA-RNA hybrid that activates RNase H-dependent degradation of target RNA. 89 Injection of LNAbased anti-miR-34a through tail vain down-regulated the expression level of miR-34a for more than 7 days and improved adult heart function, delayed remodelling and reduced the formation of fibrosis scars 7 days after MI injury. 60

| Hydrogel-based delivery
Another approach to deliver cholesterol-modified miR-302 using shear-thinning, injectable hydrogels based on the guest-host interaction of modified hyaluronic acid (HA) by intracardiac injection after MI could improve heart function for 4 weeks. 24 A biocompatible injectable gel composed of gelatin and silicate was used to deliver viral particles. 26 The gel prevented the viral particles from being rapidly metabolized by the beating heart and allowed the slow release of the particles from the gel to enhance therapeutic effects. 26 Simultaneous injection of gel and AAV-miR-1825 resulted in significant reduction in scar size, promoted peri-infarct region adult CMs proliferation and improved overall cardiac function up to 28 days after MI. 26 The detail of miRNAs therapy is listed in Table 3.

| CON CLUS ION
The limited capacity of the adult CMs to regenerate after cardiac injury is the major obstacle for heart repair. Recently, non-coding RNAs are emerging as a promising player in boosting cardiac proliferation and regeneration in heart diseases. In this review, we discuss recent non-coding RNAs associated with cardiac proliferation and potential therapeutic potential. Due to CM proliferation is transient and rare, more precise and powerful tools, such as lineage tracing strategy, 5,24 would be useful for dynamically capturing the CMs proliferation events and better understanding the mechanism for the regeneration field. In addition, the barrier between basic research and clinical implication requires more effort to overcome. RNAbased gene therapy works well, however, there are many barriers and limitations, such as pharmacokinetics and pharmacodynamics, hamper the progress. New delivery system, such as extracellular vesicles 90 and exosomes, 91,92 and synthetic hydrogel, would improve RNA-based therapeutic potential. There are tremendous non-coding RNAs that haven't been annotated, therefore, the perspective on cardiac regeneration stimulated by non-coding RNAs, advances us deeper understanding the world of non-coding RNA and novel clinical therapeutic strategies for heart diseases.

ACK N OWLED G EM ENTS
This work is supported by National Natural Science Foundation of China (Nos. 81670257, 81970227 to J. Chen, and 82000244 to F. Gao,); Zhejiang Provincial NSF project (LZ20H020001 to J. Chen.) and China Postdoctoral Science Foundation (2020M671751 to F. Gao).

CO N FLI C T S O F I NTE R E S T
The authors declare no competing interests.