Loss of circIGF1R Suppresses Cardiomyocytes Proliferation by Sponging miR-362-5p.

Circular RNAs (circRNAs) are generally formed by the back-splicing of precursor mRNA. Increasing evidence implicates the important role of circRNAs in cardiovascular diseases. However, the role of circ-insulin-like growth factor 1 receptor (circIGF1R) in cardiomyocyte (CM) proliferation remains unclear. Here, we investigated the potential role of the circIGF1R in the proliferation of CMs. We found that circIGF1R expression in heart tissues and primary CMs from adult mice was significantly lower than that in neonatal mice at postnatal 1 day (p1). Increased circIGF1R expression was detected in the injured neonatal heart at 0.5 and 1 days post-resection. circIGF1R knockdown significantly decreased the proliferation of primary CMs. Combined prediction software, luciferase reporter gene analysis, and quantitative real time-PCR (qPCR) revealed that circIGF1R interacted with miR-362-5p. A significant increase in miR-362-5p expression was detected in the adult heart compared with that in the neonatal heart. Further, heart injury significantly decreased the expression of miR-362-5p in neonatal mice. Treatment with miR-362-5p mimics significantly suppressed the proliferation of primary CMs, whereas knockdown of miR-362-5p promoted the CMs proliferation. Meanwhile, miR-362-5p silencing can rescue the proliferation inhibition of CMs induced by circIGF1R knockdown. Target prediction and qPCR validation revealed that miR-362-5p significantly inhibited the expression of Phf3 in primary CMs. In addition, decreased Phf3 expression was detected in adult hearts compared with neonatal hearts. Consistently, increased Phf3 expression was detected in injured neonatal hearts compared with that in sham hearts. Knockdown of Phf3 markedly repressed CMs proliferation. Taken together, these findings suggest that circIGF1R might contribute to cardiomyocyte proliferation by promoting Pfh3 expression by sponging miR-362-5p and provide an important experimental basis for the regulation of heart regeneration.

Mettl3-mediated m6A modification of Fgf16 restricts cardiomyocyte proliferation during heart regeneration.

Cardiovascular disease is the leading cause of death worldwide due to the inability of adult heart to regenerate after injury. N6-methyladenosine (m6A) methylation catalyzed by the enzyme methyltransferase-like 3 (Mettl3) plays an important role in various physiological and pathological bioprocesses. However, the role of m6A in heart regeneration remains largely unclear. To study m6A function in heart regeneration, we modulated Mettl3 expression in vitro and in vivo. Knockdown of Mettl3 significantly increased the proliferation of cardiomyocytes and accelerated heart regeneration following heart injury in neonatal and adult mice. However, Mettl3 overexpression decreased cardiomyocyte proliferation and suppressed heart regeneration in postnatal mice. Conjoint analysis of methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA-seq identified Fgf16 as a downstream target of Mettl3-mediated m6A modification during postnatal heart regeneration. RIP-qPCR and luciferase reporter assays revealed that Mettl3 negatively regulates Fgf16 mRNA expression in an m6A-Ythdf2-dependent manner. The silencing of Fgf16 suppressed the proliferation of cardiomyocytes. However, the overexpression of ΔFgf16, in which the m6A consensus sequence was mutated, significantly increased cardiomyocyte proliferation and accelerated heart regeneration in postnatal mice compared with wild-type Fgf16. Our data demonstrate that Mettl3 post-transcriptionally reduces Fgf16 mRNA levels through an m6A-Ythdf2-dependen pathway, thereby controlling cardiomyocyte proliferation and heart regeneration.

Cardiovascular diseases are one of the world's biggest killers. Even for patients who survive a heart attack, recovery can be difficult. This is because ­ unlike some amphibians and fish ­ humans lack the ability to produce enough new heart muscle cells to replace damaged tissue after a heart injury. In other words, the human heart cannot repair itself. Molecules known as messenger RNA (mRNA) carry the 'instructions' from the DNA inside the cell nucleus to its protein-making machinery in the cytoplasm of the cell. These messenger molecules can also be altered by different enzymes that attach or remove chemical groups. These modifications can change the stability of the mRNA, or even 'silence' it altogether by stopping it from interacting with the protein-making machinery, thus halting production of the protein it encodes. For example, a protein called Mettl3 can attach a methyl group to a specific part of the mRNA, causing a reversible mRNA modification known as m⁶A. This type of alteration has been shown to play a role in many conditions, including heart disease, but it has been unclear whether m⁶A could also be important for the regeneration of heart tissue. To find out more, Jiang, Liu, Chen et al. studied heart injury in mice of various ages. Newborn mice can regenerate their heart muscle for a short time, but adult mice lack this ability, which makes them a useful model to study heart disease. Analyses of the proteins and mRNAs in mouse heart cells confirmed that both Mettl3 and m⁶A-modified mRNAs were present. The amount of each also increased with age. Next, experiments in genetically manipulated mice revealed that removing Mettl3 greatly improved tissue repair after heart injury in both newborn and adult mice. In contrast, mouse hearts that produced abnormally high quantities of Mettl3 were unable to regenerate ­ even if the mice were young. Moreover, a detailed analysis of gene activity revealed that Mettl3 was suppressing heart regeneration by decreasing the production of a growth-promoting protein called FGF16. These results reveal a key biological mechanism controlling the heart's ability to repair itself after injury. In the future, Jiang et al. hope that Mettl3 can be harnessed for new, effective therapies to promote heart regeneration in patients suffering from heart disease.

FoxO3 restricts liver regeneration by suppressing the proliferation of hepatocytes.

Upon injury, the liver is capable of substantial regeneration from the original tissue until an appropriate functional size. The underlying mechanisms controlling the liver regeneration processes are not well elucidated. Previous studies have proposed that the transcription factor FoxO3 is involved in various liver diseases, but its exact role in the regulation of liver regeneration remains largely unclear. To directly test the detailed role of FoxO3 in liver regeneration, both a constitutive Albumin-Cre driver line and adeno-associated virus serotype 8 (AAV8)-Tbg-Cre (AAV-Cre)-injected adult FoxO3fl/fl mice were subjected to 70% partial hepatectomy (PH). Our data demonstrate that FoxO3 deletion accelerates liver regeneration primarily by limiting polyploidization and promoting the proliferation of hepatocytes during liver regeneration. RNA-seq analysis indicates that FoxO3 deficiency greatly alters the expression of gene sets associated with cell proliferation and apoptosis during liver regeneration. Chromatin immunoprecipitation-PCR (ChIP-PCR) and luciferase reporter assays reveal that FoxO3 promotes the expression of Nox4 but suppresses the expression of Nr4a1 in hepatocytes. AAV8 virus-mediated overexpression of Nox4 and knockdown of Nr4a1 significantly suppressed hepatocyte proliferation and liver regeneration in FoxO3-deficient mice. We demonstrate that FoxO3 negatively controls hepatocyte proliferation through Nox4 upregulation and Nr4a1 downregulation, thereby ensuring appropriate functional regeneration of the liver. Our findings provide novel mechanistic insight into the therapeutic mechanisms of FoxO3 in liver damage and repair.