gain‐of‐function increased CM proliferation. Overexpression of CDK6 also blocked the inhibitory effect of miR‐449a‐5p on CM proliferation, indicating that CDK6 was a functional target of miR‐449a‐5p in CM proliferation. In conclusion, miR‐449a‐5p inhibited CM proliferation by targeting CDK6,

Induction of cardiomyocyte (CM) proliferation is a promising approach for cardiac regeneration following myocardial injury. MicroRNAs (miRs) have been reported to regulate CM proliferation. In particular, miR‐449a‐5p has been identified to be associated with CM proliferation in previous high throughput functional screening data. However, whether miR‐449a‐5p regulates CM proliferation has not been thoroughly investigated. This study aimed to explore whether miR‐449a‐5p modulates CM proliferation and to identify the molecular mechanism via which miR‐449a‐5p regulates CM proliferation. The current study demonstrated that miR‐449a‐5p expression levels were significantly increased during heart development. Furthermore, the results suggested that miR‐449a‐5p mimic inhibited CM prolifera‐ tion in vitro as determined via immunofluorescence for ki67 and histone H3 phosphorylated at serine 10 (pH3), as well as the numbers of CMs. However, miR‐449a‐5p knockdown promoted CM proliferation. CDK6 was identified as a direct target gene of miR‐449a‐5p, and CDK6 mRNA and protein expression was suppressed by miR‐449a‐5p. Moreover, CDK6 gain‐of‐function increased CM proliferation. Overexpression of CDK6 also blocked the inhibitory effect of miR‐449a‐5p on CM proliferation, indicating that CDK6 was a functional target of miR‐449a‐5p in CM proliferation. In conclusion, miR‐449a‐5p inhibited CM proliferation by targeting CDK6, which provides a potential molecular target for preventing myocardial injury. Introduction Cardiac disease is the most prevalent cause of mortality worldwide (1). Damage to the mammalian heart leads to cardiomyocyte (CM) loss, cardiac fibrosis and hypertrophy followed by cardiac dysfunction, which eventually leads to heart failure (2). Adult mammalian CMs become fully differ‐ entiated, exit the cell cycle and are unable to regenerate CMs following cardiac diseases (3). Therefore, the induction of CM proliferation presents a promising strategy to promote cardiac regeneration (4,5). MicroRNAs (miRNAs/miRs) are a group of ~22‐nucleo‐ tide, highly conserved, non‐coding RNAs that regulate gene expression via post‐transcriptional modification in numerous biological and pathological processes, such as cell proliferation and apoptosis (6). Previous studies have revealed that miRNA is a critical regulator in cardiac conditions, including in cardiac hypertrophy, myocardial infarction and heart failure (7,8). To date, multiple miRNAs have been identified that control fetal CM proliferation and postnatal CM cell cycle reentry (6,9,10). A high‐throughput functional screening identified that ~204 miRNAs promoted neonatal CM proliferation, whereas 331 miRNAs decreased CM proliferation (11). The miRNA clusters miR‐17‐92 (12) and miR‐302‐367 (9) are reported to be involved in CM proliferation in mouse embryonic and post‐ natal hearts. Moreover, miR‐590‐3p and miR‐199a‐3p increase cyclin A (CCNA), cyclin E (CCNE) and cyclin B (CCNB) expression levels in proliferating CMs (11). It has also been shown that miR‐204‐dependent regulation of Jumonji And AT‐Rich Interaction Domain Containing 2 (Jarid2) in CM is essential during embryo development and cardiogenesis (13). miR‐449a‐5p has been reported to be a tumor suppressor in numerous types of cancer including gastric cancer, liver cancer and neuroblastoma (14‐16). Previous studies have demonstrated that miR‐449a‐5p inhibits cell cycle progres‐ sion, proliferation, growth and invasion in cancer cells by targeting different target genes (17). A previous study also revealed that miR‐449a‐5p regulated hypoxia/reoxygenation injury‐induced CM mortality (18). Furthermore, the members of the miR‐34/449 family share high sequence homology (19). miR‐34a has been shown to inhibit CM proliferation in miR‐449a‐5p suppresses CDK6 expression to inhibit cardiomyocyte proliferation BING LI1, ZHI WANG2, FAN YANG3, JING HUANG3, XINGWEI HU3, SHIYAN DENG3, MAOBO TIAN3 and XIAOYUN SI4 1School of Medicine, Guizhou University, Guiyang, Guizhou 550025; 2Department of Emergency Medicine, Qingdao Municipal Hospital (Group), Qingdao, Shandong 266011; 3Department of Cardiology, Guizhou Provincial People's Hospital, Guiyang, Guizhou 550002; 4Department of Cardiology, The Affiliated Hospital of Guizhou Medical University, Guiyang, Guizhou 550004, P.R. China Received May 8, 2020; Accepted September 2, 2020 DOI: 10.3892/mmr.2020. 11652 Correspondence to: Dr Xiaoyun Si, Department of Cardiology, The Affiliated Hospital of Guizhou Medical University, 28 Guiyi Road, Guiyang, Guizhou 550004, P.R. China E‐mail: sixiaoyun1@qq.com