CDC-like kinase 3 deficiency aggravates hypoxia-induced cardiomyocyte apoptosis through AKT signaling pathway.

Hypoxia-induced cardiomyocyte apoptosis is one major pathological change of acute myocardial infarction (AMI), but the underlying mechanism remains unexplored. CDC-like kinase 3 (CLK3) plays crucial roles in cell proliferation, migration and invasion, and nucleotide metabolism, however, the role of CLK3 in AMI, especially hypoxia-induced apoptosis, is largely unknown. The expression of CLK3 was elevated in mouse myocardial infarction (MI) models and neonatal rat ventricular myocytes (NRVMs) under hypoxia. Furthermore, CLK3 knockdown significantly promoted apoptosis and inhibited NRVM survival, while CLK3 overexpression promoted NRVM survival and inhibited apoptosis under hypoxic conditions. Mechanistically, CLK3 regulated the phosphorylation status of AKT, a key player in the regulation of apoptosis. Furthermore, overexpression of AKT rescued hypoxia-induced apoptosis in NRVMs caused by CLK3 deficiency. Taken together, CLK3 deficiency promotes hypoxia-induced cardiomyocyte apoptosis through AKT signaling pathway.

Imp7 siRNA nanoparticles protect against mechanical ventilation-associated liver injury by inhibiting HMGB1 production and NETs formation.

Mechanical ventilation (MV) has the potential to induce extra-pulmonary organ damage by adversely affecting the lungs and promoting the secretion of inflammatory cytokines. High-mobility group box 1 protein (HMGB1) is a pro-inflammatory mediator in ventilator-induced lung injury (VILI), but its effect on MV-associated liver injury and the mechanisms are poorly understood. In the present study, mice were subjected to high-volume MV (20 ml/kg) to induce VILI. MV-induced HMGB1 prompted neutrophil extracellular traps (NETs) formation and PANoptosis within the liver. Inhibiting NETs formation by DNase I or PAD4 inhibitor, or by HMGB1 neutralizing ameliorated the liver injury. HMGB1 activated neutrophils to form NETs through TLR4/MyD88/TRAF6 pathway. Importantly, Importin7 siRNA nanoparticles inhibited HMGB1 release and protected against MV-associated liver injury. These data provide evidence of MV-induced HMGB1 prompted NETs formation and PANoptosis in the liver via the TLR4/MyD88/TRAF6 pathway. HMGB1 is a potential therapeutic target for MV-associated liver injury.

Systemic cytokines inhibition with Imp7 siRNA nanoparticle ameliorates gut injury in a mouse model of ventilator-induced lung injury.

Mechanical ventilation (MV) may negatively affect the lungs and cause the release of inflammatory mediators, resulting in extra-pulmonary organ dysfunction. Studies have revealed systemically elevated levels of proinflammatory cytokines in animal models of ventilator-induced lung injury (VILI); however, whether these cytokines have an effect on gut injury and the mechanisms involved remain unknown. In this study, VILI was generated in mice with high tidal volume mechanical ventilation (20 ml/kg). Tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and IL-6 concentrations in serum and gut measured by ELISA showed significant elevation in the VILI mice. Significant increases in gut injury and PANoptosis were observed in the VILI mice, which were positively correlated with the serum levels of TNF-α, IL-1ß, and IL-6. The VILI mice displayed intestinal barrier defects, decreased expressions of occludin and zonula occludin-1 (ZO-1), and increased expression of claudin-2 and the activation of myosin light chain (MLC). Importantly, intratracheal administration of Imp7 siRNA nanoparticle effectively inhibited cytokines production and protected mice from VILI-induced gut injury. These data provide evidence of systemic cytokines contributing to gut injury following VILI and highlight the possibility of targeting cytokines inhibition via Imp7 siRNA nanoparticle as a potential therapeutic intervention for alleviating gut injury following VILI.

miR-301a-PTEN-AKT Signaling Induces Cardiomyocyte Proliferation and Promotes Cardiac Repair Post-MI.

Adult hearts are hard to recover after cardiac injury due to the limited proliferative ability of cardiomyocytes. Emerging evidence indicates the induction of cell cycle reentry of cardiomyocytes by special treatment or stimulation, which offers adult heart regenerative potential. Herein, a microRNA (miRNA) screening in cardiomyocytes identified miR-301a enriched specially in the neonatal cardiomyocytes from rats and mice. Overexpression of miR-301a in primary neonatal cardiomyocytes and H9C2 cells induced G1/S transition of the cell cycle, promoted cellular proliferation, and protected cardiomyocytes against hypoxia-induced apoptosis. Adeno-associated virus (AAV)9-mediated cardiac delivery of miR-301a to the mice model with myocardial infarction (MI) dramatically promoted cardiac repair post-MI in vivo. Phosphatase and tensin homolog (PTEN)/phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway was confirmed to mediate miR-301a-induced cell proliferation in cardiomyocytes. Loss of function of PTEN mimicked the miR-301a-induced phenotype, while gain of function of PTEN attenuated the miR-301a-induced cell proliferation in cardiomyocytes. Application of RG7440, a small molecule inhibitor of AKT, blocked the function of miR-301a in cardiomyocytes. The current study revealed a miRNA signaling in inducing the cell cycle reentry of cardiomyocytes in the injured heart, and it demonstrated the miR-301a/PTEN/AKT signaling as a potential therapeutic target to reconstitute lost cardiomyocytes in mammals.

Starvation stress attenuates the miRNA-target interaction in suppressing breast cancer cell proliferation.

BACKGROUND: Emerging evidence has demonstrated the limited access to metabolic substrates as an effective approach to block cancer cell growth. The mechanisms remain unclear. Our previous work has revealed that miR-221/222 plays important role in regulating breast cancer development and progression through interaction with target gene p27. RESULTS: Herein, we determined the miRNA-mRNA interaction in breast cancer cells under induced stress status of starvation. Starvation stimulation attenuated the miR-221/222-p27 interaction in MDA-MB-231 cells, thereby increased p27 expression and suppressed cell proliferation. Through overexpression or knockdown of miR-221/222, we found that starvation-induced stress attenuated the negative regulation of p27 expression by miR-221/222. Similar patterns for miRNA-target mRNA interaction were observed between miR-17-5p and CyclinD1, and between mR-155 and Socs1. Expression of Ago2, one of the key components of RNA-induced silencing complex (RISC), was decreased under starvation-induced stress status, which took responsibility for the impaired miRNA-target interaction since addition of exogenous Ago2 into MDA-MB-231 cells restored the miR-221/222-p27 interaction in starvation condition. CONCLUSIONS: We demonstrated the attenuated interaction between miR-221/222 and p27 by starvation-induced stress in MDA-MB-231 breast cancer cells. The findings add a new page to the general knowledge of negative regulation of gene expression by miRNAs, also demonstrate a novel mechanism through which limited access to nutrients suppresses cancer cell proliferation. These insights provide a basis for development of novel therapeutic options for breast cancer.

MiR-301a promotes embryonic stem cell differentiation to cardiomyocytes.

BACKGROUND: Cardiovascular disease is the leading cause of death worldwide. Tissue repair after pathological injury in the heart remains a major challenge due to the limited regenerative ability of cardiomyocytes in adults. Stem cell-derived cardiomyocytes provide a promising source for the cell transplantation-based treatment of injured hearts. AIM: To explore the function and mechanisms of miR-301a in regulating cardiomyocyte differentiation of mouse embryonic stem (mES) cells, and provide experimental evidence for applying miR-301a to the cardiomyocyte differentiation induction from stem cells. METHODS: mES cells with or without overexpression of miR-301a were applied for all functional assays. The hanging drop technique was applied to form embryoid bodies from mES cells. Cardiac markers including GATA-4, TBX5, MEF2C, and α-actinin were used to determine cardiomyocyte differentiation from mES cells. RESULTS: High expression of miR-301a was detected in the heart from late embryonic to neonatal mice. Overexpression of miR-301a in mES cells significantly induced the expression of cardiac transcription factors, thereby promoting cardiomyocyte differentiation and beating cardiomyocyte clone formation. PTEN is a target gene of miR-301a in cardiomyocytes. PTEN-regulated PI3K-AKT-mTOR-Stat3 signaling showed involvement in regulating miR-301a-promoted cardiomyocyte differentiation from mES cells. CONCLUSION: MiR-301a is capable of promoting embryonic stem cell differentiation to cardiomyocytes.

YY1 mediates TGF-ß1-induced EMT and pro-fibrogenesis in alveolar epithelial cells.

Pulmonary fibrosis is a chronic, progressive lung disease associated with lung damage and scarring. The pathological mechanism causing pulmonary fibrosis remains unknown. Emerging evidence suggests prominent roles of epithelial-mesenchymal transition (EMT) of alveolar epithelial cells (AECs) in myofibroblast formation and progressive pulmonary fibrosis. Our previous work has demonstrated the regulation of YY1 in idiopathic pulmonary fibrosis and pathogenesis of fibroid lung. However, the specific function of YY1 in AECs during the pathogenesis of pulmonary fibrosis is yet to be determined. Herein, we found the higher level of YY1 in primary fibroblasts than that in primary epithelial cells from the lung of mouse. A549 and BEAS-2B cells, serving as models for type II alveolar pulmonary epithelium in vitro, were used to determine the function of YY1 during EMT of AECs. TGF-ß-induced activation of the pro-fibrotic program was applied to determine the role YY1 may play in pro-fibrogenesis of type II alveolar epithelial cells. Upregulation of YY1 was associated with EMT and pro-fibrotic phenotype induced by TGF-ß treatment. Targeted knockdown of YY1 abrogated the EMT induction by TGF-ß treatment. Enforced expression of YY1 can partly mimic the TGF-ß-induced pro-fibrotic change in either A549 cell line or primary alveolar epithelial cells, indicating the induction of YY1 expression may mediate the TGF-ß-induced EMT and pro-fibrosis. In addition, the translocation of NF-κB p65 from the cytoplasm to the nucleus was demonstrated in A549 cells after TGF-ß treatment and/or YY1 overexpression, suggesting that NF-κB-YY1 signaling pathway regulates pulmonary fibrotic progression in lung epithelial cells. These findings will shed light on the better understanding of mechanisms regulating pro-fibrogenesis in AECs and pathogenesis of lung fibrosis.

Neonatal Heart-Enriched miR-708 Promotes Proliferation and Stress Resistance of Cardiomyocytes in Rodents.

Adult heart has limited potential for regeneration after pathological injury due to the limited cell proliferation of cardiomyocytes and the quiescent status of progenitor cells. As such, induction of cell-cycle reentry of cardiomyocytes is one of the key strategies for regeneration of damaged heart. In this study, a subset of miRNAs including miR-708 were identified to be much more abundant in the embryonic and neonatal cardiomyocytes than that in adult rodents. Overexpression of miR-708 promoted cellular proliferation of H9C2 cells or primary cardiomyocytes from neonatal rats or mice in vitro. Lipid nanoparticle delivery of miR-708 promoted myocardial regeneration and heart function recovery in vivo. In addition, miR-708 protected cardiomyocytes against stress-induced apoptosis under hypoxia or isoproterenol treatments. miR-708 inhibited the expression of MAPK14, which has been demonstrated arresting the cell cycle in cardiomyocytes. The cell proliferation-promoting function of miR-708 was dependent at least partly on the expression of MAPK14. These findings strengthen the potential of applying miRNAs to reconstitute lost cardiomyocytes in injured hearts, and may provide a novel miRNA candidate for promoting heart regeneration.

Small RNA zippers lock miRNA molecules and block miRNA function in mammalian cells.

MicroRNAs (miRNAs) loss-of-function phenotypes are mainly induced by chemically modified antisense oligonucleotides. Here we develop an alternative inhibitor for miRNAs, termed 'small RNA zipper'. It is designed to connect miRNA molecules end to end, forming a DNA-RNA duplex through a complementary interaction with high affinity, high specificity and high stability. Two miRNAs, miR-221 and miR-17, are tested in human breast cancer cell lines, demonstrating the 70∼90% knockdown of miRNA levels by 30-50 nM small RNA zippers. The miR-221 zipper shows capability in rescuing the expression of target genes of miR-221 and reversing the oncogenic function of miR-221 in breast cancer cells. In addition, we demonstrate that the miR-221 zipper attenuates doxorubicin resistance with higher efficiency than anti-miR-221 in human breast cancer cells. Taken together, small RNA zippers are a miRNA inhibitor, which can be used to induce miRNA loss-of-function phenotypes and validate miRNA target genes.

Neonatal Heart-Enriched miR-708 Promotes Differentiation of Cardiac Progenitor Cells in Rats.

Cardiovascular disease is becoming the leading cause of death throughout the world. However, adult hearts have limited potential for regeneration after pathological injury, partly due to the quiescent status of stem/progenitor cells. Reactivation of cardiac stem/progenitor cells to create more myocyte progeny is one of the key steps in the regeneration of a damaged heart. In this study, miR-708 was identified to be enriched in the neonatal cardiomyocytes of rats, but this has not yet been proven in adult humans. A lower level of miR-708 in c-kit(+) stem/progenitor cells was detected compared to non-progenitors. Overexpression of miR-708 induced cardiomyocyte differentiation of cardiac stem/progenitor cells. This finding strengthened the potential of applying miRNAs in the regeneration of injured hearts, and this indicates that miR-708 could be a novel candidate for treatment of heart diseases.

LRP6 acts as a scaffold protein in cardiac gap junction assembly.

Low-density lipoprotein receptor-related protein 6 (LRP6) is a Wnt co-receptor in the canonical Wnt/ß-catenin signalling. Here, we report the scaffold function of LRP6 in gap junction formation of cardiomyocytes. Cardiac LRP6 is spatially restricted to intercalated discs and binds to gap junction protein connexin 43 (Cx43). A deficiency in LRP6 disrupts Cx43 gap junction formation and thereby impairs the cell-to-cell coupling, which is independent of Wnt/ß-catenin signalling. The defect in Cx43 gap junction resulting from LRP6 reduction is attributable to the defective traffic of de novo Cx43 proteins from the endoplasmic reticulum to the Golgi apparatus, leading to the lysosomal degradation of Cx43 proteins. Accordingly, the hearts of conditional cardiac-specific Lrp6-knockout mice consistently exhibit overt reduction of Cx43 gap junction plaques without any abnormality in Wnt signalling and are predisposed to lethal arrhythmias. These findings uncover a distinct role of LRP6 as a platform for intracellular protein trafficking.

miR-134 Modulates the Proliferation of Human Cardiomyocyte Progenitor Cells by Targeting Meis2.

Cardiomyocyte progenitor cells play essential roles in early heart development, which requires highly controlled cellular organization. microRNAs (miRs) are involved in various cell behaviors by post-transcriptional regulation of target genes. However, the roles of miRNAs in human cardiomyocyte progenitor cells (hCMPCs) remain to be elucidated. Our previous study showed that miR-134 was significantly downregulated in heart tissue suffering from congenital heart disease, underlying the potential role of miR-134 in cardiogenesis. In the present work, we showed that the upregulation of miR-134 reduced the proliferation of hCMPCs, as determined by EdU assay and Ki-67 immunostaining, while the inhibition of miR-134 exhibited an opposite effect. Both up- and downregulation of miR-134 expression altered the transcriptional level of cell-cycle genes. We identified Meis2 as the target of miR-134 in the regulation of hCMPC proliferation through bioinformatic prediction, luciferase reporter assay and western blot. The over-expression of Meis2 mitigated the effect of miR-134 on hCMPC proliferation. Moreover, miR-134 did not change the degree of hCMPC differentiation into cardiomyocytes in our model, suggesting that miR-134 is not required in this process. These findings reveal an essential role for miR-134 in cardiomyocyte progenitor cell biology and provide new insights into the physiology and pathology of cardiogenesis.

Nucleoporin 35 regulates cardiomyocyte pH homeostasis by controlling Na+-H+ exchanger-1 expression.

The mammalian nuclear pore complex is comprised of ∼ 30 different nucleoporins (Nups). It governs the nuclear import of gene expression modulators and the export of mRNAs. In cardiomyocytes, Na(+)-H(+) exchanger-1 (NHE1) is an integral membrane protein that exclusively regulates intracellular pH (pHi) by exchanging one intracellular H(+) for one extracellular Na(+). However, the role of Nups in cardiac NHE1 expression remains unknown. We herein report that Nup35 regulates cardiomyocyte NHE1 expression by controlling the nucleo-cytoplasmic trafficking of nhe1 mRNA. The N-terminal domain of Nup35 determines nhe1 mRNA nuclear export by targeting the 5'-UTR (-412 to -213 nt) of nhe1 mRNA. Nup35 ablation weakens the resistance of cardiomyocytes to an acid challenge by depressing NHE1 expression. Moreover, we identify that Nup35 and NHE1 are simultaneously downregulated in ischemic cardiomyocytes both in vivo and in vitro. Enforced expression of Nup35 effectively counteracts the anoxia-induced intracellular acidification. We conclude that Nup35 selectively regulates cardiomyocyte pHi homeostasis by posttranscriptionally controlling NHE1 expression. This finding reveals a novel regulatory mechanism of cardiomyocyte pHi, and may provide insight into the therapeutic strategy for ischemic cardiac diseases.

miRNA-204 drives cardiomyocyte proliferation via targeting Jarid2.

OBJECTIVES: In mammals, the heart grows by hypertrophy but not proliferation of cardiomyocytes after birth. The paucity of cardiomyocyte proliferation limits cardiac regeneration in a variety of heart diseases. To explore the efficient strategies that drive cardiomyocyte proliferation, we employed in vitro and in vivo models to investigate the function of miRNA-204, which was demonstrated to regulate the proliferation and differentiation of human cardiac progenitor cells in our previous study. METHODS AND RESULTS: miRNA-204 overexpression markedly promoted cardiomyocyte proliferation in both neonatal and adult rat cardiomyocytes in vitro. Transgenic mice with the cardiac-specific overexpression of miRNA-204 exhibited excessive cardiomyocyte proliferation throughout the embryonic and adult stages, leading to a pronounced increase in ventricular mass. Accordingly, the cell cycle regulators, including Cyclin A, Cyclin B, Cyclin D2, Cyclin E, CDC2 and PCNA, were upregulated in miRNA-204 transgenic embryonic hearts. Furthermore, we demonstrated that miRNA-204 directly targeted Jarid2. Knockdown of Jarid2 mimicked the pro-proliferative effect of miRNA-204 overexpression on cultured rat cardiomyocytes, whereas enhanced expression of Jarid2 conferred the myocytes with substantial resistance to proliferation by miRNA-204 overexpression. CONCLUSION: Our findings identify a conserved role for miRNA-204 in regulating cardiomyocyte proliferation by targeting the Jarid2 signaling pathway.

miR-10a regulates proliferation of human cardiomyocyte progenitor cells by targeting GATA6.

microRNAs (miRNAs) play essential roles in cardiogenesis. The altered expression of miRNAs can result in cardiac malformations by inducing abnormalities in the behavior of cardiac cells. However, the role of miR-10a in the regulation of cardiomyocyte progenitor cells (CMPCs) remains undetermined. In the present study, we found that up- or down-regulation of miR-10a inhibited or promoted the proliferation of human CMPCs, respectively, without affecting their differentiation toward cardiomyocytes. miR-10a bound to GATA6 directly and reduced GATA6 expression. Over-expression of GATA6 greatly attenuated the miR-10a-mediated inhibitory effect on the proliferation of human CMPCs. Thus, our results indicate that miR-10a could effectively modulate the proliferation of human CMPCs by targeting GATA6. The finding provides novel insights into the potency of miR-10a during heart development.