Cardioprotection of voluntary exercise against breast cancer-induced cardiac injury via STAT3.

Exercise is an effective way to alleviate breast cancer-induced cardiac injury to a certain extent. However, whether voluntary exercise (VE) activates cardiac signal transducer and activator of transcription 3 (STAT3) and the underlying mechanisms remain unclear. This study investigated the role of STAT3-microRNA(miRNA)-targeted protein axis in VE against breast cancer-induced cardiac injury.VE for 4 weeks not only improved cardiac function of transgenic breast cancer female mice [mouse mammary tumor virus-polyomavirus middle T antigen (MMTV-PyMT +)] compared with littermate mice with no cancer (MMTV-PyMT -), but also increased myocardial STAT3 tyrosine 705 phosphorylation. Significantly more obvious cardiac fibrosis, smaller cardiomyocyte size, lower cell viability, and higher serum tumor necrosis factor (TNF)-α were shown in MMTV-PyMT + mice compared with MMTV-PyMT - mice, which were ameliorated by VE. However, VE did not influence the tumor growth. MiRNA sequencing identified that miR-181a-5p was upregulated and miR-130b-3p was downregulated in VE induced-cardioprotection. Myocardial injection of Adeno-associated virus serotype 9 driving STAT3 tyrosine 705 mutations abolished cardioprotective effects above. Myocardial STAT3 was identified as the transcription factor binding the promoters of pri-miR-181a (the precursor of miR-181a-5p) and HOX transcript antisense RNA (HOTAIR, sponged miR-130b-3p) in isolated cardiomyocytes. Furthermore, miR-181a-5p targeting PTEN and miR-130b-3p targeting Zinc finger and BTB domain containing protein 20 (Zbtb20) were proved in AC-16 cells. These findings indicated that VE protects against breast cancer-induced cardiac injury via activating STAT3 to promote miR-181a-5p targeting PTEN and to promote HOTAIR to sponge miR-130b-3p targeting Zbtb20, helping to develop new targets in exercise therapy for breast cancer-induced cardiac injury.

Wu L ，Li ZZ ，Yang H ，Cao LZ ，Wang XY ，Wang DL ，Chatterjee E ，Li YF ，Huang G ... -  《-》

Adipocyte-derived small extracellular vesicles exacerbate diabetic ischemic heart injury by promoting oxidative stress and mitochondrial-mediated cardiomyocyte apoptosis.

Diabetes increases ischemic heart injury via incompletely understood mechanisms. We recently reported that diabetic adipocytes-derived small extracellular vesicles (sEV) exacerbate myocardial reperfusion (MI/R) injury by promoting cardiomyocyte apoptosis. Combining in vitro mechanistic investigation and in vivo proof-concept demonstration, we determined the underlying molecular mechanism responsible for diabetic sEV-induced cardiomyocyte apoptosis after MI/R. Adult mice were fed a high-fat diet (HFD) for 12 weeks. sEV were isolated from plasma or epididymal adipose tissue. HFD significantly increased the number and size of plasma- and adipocyte-derived sEV. Intramyocardial injection of an equal number of diabetic plasma sEV in nondiabetic hearts significantly increased cardiac apoptosis and exacerbated MI/R-induced cardiac dysfunction. Diabetic plasma sEV significantly activated cardiac caspase 9 but not caspase 8, suggesting that diabetic sEV induces cardiac apoptosis via the mitochondrial pathway. These pathologic alterations were phenotyped by intramyocardial injection of sEV isolated from diabetic adipocytes or HGHL-challenged 3T3L1 adipocytes. To obtain direct evidence that diabetic sEV promotes cardiomyocyte apoptotic cell death, isolated neonatal rat ventricular cardiomyocytes (NRVMs) were treated with sEV and subjected to simulated ischemia/reperfusion (SI/R). Treatment of cardiomyocytes with sEV from diabetic plasma, diabetic adipocytes, or HGHL-challenged 3T3L1 adipocytes significantly enhanced SI/R-induced apoptosis and reduced cell viability. These pathologic effects were replicated by a miR-130b-3p (a molecule increased dramatically in diabetic sEV) mimic and blocked by a miRb-130b-3p inhibitor. Molecular studies identified PGC-1α (i.e. PGC-1α1/-a) as the direct downstream target of miR-130b-3p, whose downregulation causes mitochondrial dysfunction and apoptosis. Finally, treatment with diabetic adipocyte-derived sEV or a miR-130b-3p mimic significantly enhanced mitochondrial reactive oxygen species (ROS) production in SI/R cardiomyocytes. Conversely, treatment with a miR-130b-3p inhibitor or overexpression of PGC-1α extremely attenuated diabetic sEV-induced ROS production. We obtained the first evidence that diabetic sEV promotes oxidative stress and mitochondrial-mediated cardiomyocyte apoptotic cell death, exacerbating MI/R injury. These pathological phenotypes were mediated by miR-130b-3p-induced suppression of PGC-1α expression and subsequent mitochondrial ROS production. Targeting miR-130b-3p mediated cardiomyocyte apoptosis may be a novel strategy for attenuating diabetic exacerbation of MI/R injury.

Gan L ，Zhao J ，Yao P ，Christopher TA ，Lopez B ，Lau WB ，Koch W ，Gao E ，Ma X ，Wang Y ... -  《Redox Biology》

EGCG targeting STAT3 transcriptionally represses PLXNC1 to inhibit M2 polarization mediated by gastric cancer cell-derived exosomal miR-92b-5p.

M2-polarized tumor-associated macrophages (TAMs) predominate in tumor microenvironment (TME) and serve primary functions in tumor progression, including growth, angiogenesis, metastasis, immunosuppression, chemoresistance, and poor prognosis. The reversal of M2 polarization provides a new treatment strategy for cancer. Presently, the molecular mechanisms of M2 polarization have yet to be fully characterized, and there is a lack of effective therapeutic targets and drugs. Cancer cells initiate an immunosuppressive TME by recruiting macrophages and promoting M2 polarization through the secretion of inflammatory factors. Accordingly, blocking cancer cell-induced TAM M2 polarization may present a more effective strategy from the perspective of cancer cells. Hedyotis diffusa Willd (HDW) possesses immunomodulatory and antitumor properties, and is a precious and direct source of small molecule natural products with a dual function of inhibition of tumor growth and tumor cell-mediated M2 polarization. To identify a new target promoting gastric cancer (GC) cell growth and GC cell-mediated M2 polarization from mRNA profiles of GC cells treated with HDW injection (HDI) and to excavate a natural product from HDI that can regulate related mRNA and inhibit the aforementioned effects. RNA sequencing (RNA-seq) was used to analyze HDI-regulated differentially expressed mRNAs (HRmRNAs) in MKN45 cells. Weighted gene co-expression network analysis (WGCNA), univariate and multivariate Cox regression analysis, KM survival curves, and association analysis between HRmRNA and clinical characteristics/tumor infiltrating immune cells (TIICs) individually were utilized to screen out the target HRmRNA associated with prognosis and M2 macrophage infiltration in GC. shRNA lentiviral vectors were used for stably silencing, and transient overexpressing plasmids were constructed for overexpression. CCK8, EdU, colony formation, migration and invasion assays were used to validate the function of drugs and molecules in GC. HDI constituent analysis was performed using UHPLC-QE-MS. A network of HDI constituent-hub transcription factor (TF)-HRmRNA was constructed based on RNA-Seq, network pharmacology and TFs prediction. Exosome isolation and identification were performed using ultracentrifugation, NTA, TEM and western blot. Apoptosis and macrophage phenotypes were determined by flow cytometric analysis. Small RNA-Seq made exosomal miRNA identification. Small molecule interaction with targets were analyzed using molecular docking, SPR and CETSA. The direct relationship between transcription factors and promoters was verified using ChIP-QPCR and dual-luciferase reporter gene assay. A nude mice xenograft tumor model was established for vivo validation. HDI inhibited MKN45 cell proliferation, migration, invasion and promoted apoptosis. RNA-Seq identified 2583 HRmRNAs. PLXNC1 was screened out as the target HRmRNA associated with prognosis and M2 macrophage infiltration in GC. PLXNC1 promoted GC cell proliferation and facilitated TAMs M2 polarization by transferring GC cell-derived exosomal miR-92b-5p, inhibiting SOCS7-STAT3 interactions and subsequently activating STAT3 in macrophages. M2 TAMs induced by PLXNC1-mediated GC cell-derived exosomes promoted GC cell migration and invasion. PLXNC1 regulated exosomal miR-92b-5p through the MEK1/MSK1/CREB1 pathway. STAT3 could transcriptionally regulate PLXNC1 expression in GC cells. The network of HDI constituent-hub TF-HRmRNA showed epigallocatechin gallate (EGCG) from HDI targeted STAT3 to transcriptionally regulate PLXNC1 expression. EGCG as a natural product directly bound to STAT3 to diminish its nuclear localization, resulting in the transcriptional repression of PLXNC1 and the reversal of M2 polarization induced by PLXNC1-mediated GC cell-derived exosomes. PLXNC1 is a novel target exerting dual effects on GC cell proliferation and GC cell-mediated M2 polarization. EGCG derived from HDI inhibits GC cell proliferation and targets STAT3 to inhibit M2 polarization induced by PLXNC1-mediated exosomes derived from GC cells, which may be a multi-target therapeutic agent for GC cell proliferation and immune microenvironment.

Yi J ，Ye Z ，Xu H ，Zhang H ，Cao H ，Li X ，Wang T ，Dong C ，Du Y ，Dong S ，Zhou W ... -  《-》

CircCHSY1 protects hearts against ischemia/reperfusion injury by enhancing heme oxygenase 1 expression via miR-24-3p.

Circular RNAs are important players involving in a variety of physiological and pathological processes. However, their functions and mechanisms during myocardial ischemic injury and protection remain largely unknown. We recently found significant alterations of many circRNAs including circCHSY1 following myocardial ischemia/reperfusion (I/R) injury, whereas their exact functions are unclear. Here, we investigated roles of circCHSY1 in the acute myocardial I/R injury and the potential mechanisms involved. The expression of circCHSY1 was detected in cardiomyocytes from mouse, rat and human embryonic stem cells (hESC-CMs). It was further upregulated in mouse I/R (30 min/24 h) hearts, oxygen glucose deprivation/reperfused (OGD/R, 6 h/2 h) primary neonatal rat ventricular cardiomyocytes (NRCMs) and OGD/R (48 h/2 h) hESC-CMs. Adenovirus-mediated circCHSY1-overexpression significantly decreased infarct size and lactate dehydrogenase (LDH) release in mouse I/R hearts. Consistently, circCHSY1 overexpression reduced the LDH release in the OGD/R NRCMs and hESC-CMs, improved cell viability, and preserved mitochondrial function in the OGD/R NRCMs, whereas there were no significant differences in cell viability and LDH release between the OGD/R NRCMs with and without siRNA-mediated circCHSY1 knockdown. Mechanistically, circCHSY1 was detected to bind with miR-24-3p analyzed by dual luciferase assay and RNA pull-down assays. CircCHSY1 overexpression-mediated protective effects on cells and mitochondria in OGD/R NRCMs were reversed by the miR-24-3p mimic. Further, dual luciferase assay showed that miR-24-3p directly bound to heme oxygenase 1 (HO1) via its 3'UTR. The protein level of HO1 was downregulated by miR-24-3p mimic in OGD/R NRCMs but enhanced by the circCHSY1 overexpression in vitro and in vivo. Functionally, the HO1 knockdown by adenovirus in vivo and by siRNA in vitro eliminated cardioprotective effects of circCHSY1 overexpression. CircCHSY1 is upregulated following myocardial I/R injury. The higher level of circCHSY1 protects I/R hearts and cardiomyocytes. The protection of circCHSY1 is mediated through enhancement of the HO1 level, resulting in preserving mitochondrial homeostasis via targeting miR-24-3p in cardiomyocytes. These findings suggest circCHSY1 as a protective factor.

Tan J ，Min J ，Jiang Y ，Liu S ，Ke M ，Wang Z ，Yang HT ... -  《-》

Erratum: Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs.

《Jove-Journal of Visualized Experiments》