LC3B Activation Research Articles

LncRNA TINCR knockdown inhibits colon cancer cells via regulation of autophagy.

The present study aimed to evaluate the effects of long noncoding (lnc)RNA TINCR ubiquitin domain containing (TINCR) on the development of colon cancer, and the specific underlying mechanisms. The present study used adjacent healthy and cancer tissues obtained from patients with colon cancer and measured lncRNA TINCR expression using reverse transcription-quantitative (RT-q) PCR and in situ hybridization assays. Moreover, associations between lncRNA TINCR and clinicopathology and prognosis were also investigated. In addition, the gene and protein expression levels of lncRNA TINCR, mTOR, LC 3B, P62, and Beclin1 were measured using RT-qPCR and western blotting assays. Cell proliferation, apoptosis, invasion, and migration were measured using MTT, Edu staining, flow cytometry, TUNEL, Transwell, and wound-healing assays, and cell ultrastructure and LC 3B activation were measured using transmission electron microscopy and cellular immunofluorescence. Results of the present study demonstrated that lncRNA TINCR expression was significantly upregulated in colon cancer tissues, and the overall survival of the low-expression group was significantly increased, compared with that of the high-expression groups. In addition, the results of the present study demonstrated that lncRNA TINCR was associated with clinicopathology in patients with colon cancer. Moreover, following lncRNA TINCR knockdown using transfection with small interfering RNA-TINCR, results of the present study demonstrated that cell proliferation was significantly reduced, while cell apoptosis was significantly increased. In addition, cell invasion and migration were significantly reduced, and autophagy was increased in HT-29 and SW620 cell lines. However, following treatment with an mTOR agonist (an autophagy inhibitor), biological activities were significantly increased in HT-29 and SW-620 cell lines. Collectively, these results demonstrated that lncRNA TINCR may induce colon cancer development through the regulation of autophagy.

Non‐acidotic hypercapnia limits the loss of diaphragm force at single fiber level in mechanically ventilated rats for 5 days

The diaphragm loss of function resulting from Mechanical Ventilation (MV) in Intensive Care Unit (ICU) patients has been termed “Ventilator Induced Diaphragmatic Dysfunction” (VIDD) and is characterized by a rapid and progressive loss of diaphragm muscle mass and function. Hypercapnia is a frequent condition in mechanically ventilated ICU patients and accepted as “permissive hypercapnia” in ARDS. There are numerous negative effects of this gas imbalance on different tissues and their functions are well known, whereas the positive effects are still poorly understood. Acute hypercapnic exposure has shown benefit to in vivo diaphragm function in porcine and rat ICU models, however our understanding of the effects of longer‐term ventilation and the downstream signaling are less known. Here, we hypothesize that normoxic non acidotic hypercapnia would augment diaphragm muscle function during development of VIDD. Adult female Sprague‐Dawley rats were deeply sedated, pharmacologically paralyzed, hydrated, nourished and controlled mechanical ventilated (CMV) for 5 days in normoxic‐normocapnic or normoxic, hypercapnic conditions. Hypercapnic conditions maintained EtCO2 between 55 and 70 mmHg for the duration of the experiment ensuring normal arterial blood pH (7.37 – 7.43), thus they were non‐acidotic hypercapnic conditions. This unique experimental rat model is more representative of the prolonged nature and effects of VIDD and thus provides clinically relevant assessment of hypercapnic treatment efficacy. Diaphragm muscles were dissected and prepared for contractile function assessment and downstream signalling assessed at the gene and protein level.Our results show that: 1) cross sectional area (CSA) was decreased by 40% following normocapnic CMV when compared with CTR (p< 0.001), which was unaltered by hypercapnia treatment; 2) Specific force (SF; force normalised to CSA) was decreased to 58% of CTR following normocapnic CMV (p< 0.001). Non‐acidotic hypercapnia increased SF by 15% when compared with normocapnic rats (p< 0.05); 3) Muscle E3 ligases Murf1 and Atrogin‐1 protein expression is unchanged following 5 days of CMV, irrespective of C02 conditions. LC3B is a known marker for autophagy, with LC3B II:I ratio signifying activation. Following 5 days normocapnic CMV LC3B II:I ratio (p< 0.05) was reduced by 70% in diaphragm muscle. Non‐acidotic hypercapnic conditions failed to alter the activation of LC3B or these muscle degradation pathways; 4) Non‐acidotic hypercapnic conditions however increased TNFα and Interleukin 1β transcript expression in the diaphragm when compared with normocapnic CMV and CTR groups; 5) Non‐acidotic hypercapnic conditions increased stability of respiratory peak pressure, peripheral oxygenation and perfusion, hemodynamic conditions, thus promoting the benefits of non‐acidotic hypercapnic conditions.Our results suggest that non‐acidotic hypercapnic conditions have beneficial effects on single fibre diaphragm function irrespective of significant increase in muscle inflammation in non‐septic animals. This, combined with the positive respiratory and hemodynamic effects, encourage further study of the therapeutic potential of CO2 in ICU contexts.

Retinal ganglion cell loss in an ex vivo mouse model of optic nerve cut is prevented by curcumin treatment

Retinal ganglion cell (RGC) loss is a pathologic feature common to several retinopathies associated to optic nerve damage, leading to visual loss and blindness. Although several scientific efforts have been spent to understand the molecular and cellular changes occurring in retinal degeneration, an effective therapy to counteract the retinal damage is still not available. Here we show that eyeballs, enucleated with the concomitant optic nerve cut (ONC), when kept in PBS for 24 h showed retinal and optic nerve degeneration. Examining retinas and optic nerves at different time points in a temporal window of 24 h, we found a thinning of some retinal layers especially RGC’s layer, observing a powerful RGC loss after 24 h correlated with an apoptotic, MAPKs and degradative pathways dysfunctions. Specifically, we detected a time-dependent increase of Caspase-3, -9 and pro-apoptotic marker levels, associated with a strong reduction of BRN3A and NeuN levels. Importantly, a powerful activation of JNK, c-Jun, and ERK signaling (MAPKs) were observed, correlated with a significant augmented SUMO-1 and UBC9 protein levels. The degradation signaling pathways was also altered, causing a significant decrease of ubiquitination level and an increased LC3B activation. Notably, it was also detected an augmented Tau protein level. Curcumin, a powerful antioxidant natural compound, prevented the alterations of apoptotic cascade, MAPKs, and SUMO-1 pathways and the degradation system, preserving the RGC survival and the retinal layer thickness. This ex vivo retinal degeneration model could be a useful method to study, in a short time window, the effect of neuroprotective tools like curcumin that could represent a potential treatment to contrast retinal cell death.

Cinnamaldehyde induces autophagy-mediated cell death through ER stress and epigenetic modification in gastric cancer cells

Previous reports suggested that cinnamaldehyde (CA), the bioactive ingredient in Cinnamomum cassia, can suppress tumor growth, migratory, and invasive abilities. However, the role and molecular mechanisms of CA in GC are not completely understood. In the present study, we found that CA-induced ER stress and cell death via the PERK–CHOP axis and Ca2+ release in GC cells. Inhibition of ER stress using specific–siRNA blocked CA-induced cell death. Interestingly, CA treatment resulted in autophagic cell death by inducing Beclin-1, ATG5, and LC3B expression and by inhibiting p62 expression whereas autophagy inhibition suppressed CA-induced cell death. We showed that CA induces the inhibition of G9a and the activation of LC3B. Moreover, CA inhibited G9a binding on Beclin-1 and LC3B promoter. Overall, these results suggested that CA regulates the PERK–CHOP signaling, and G9a inhibition activates autophagic cell death via ER stress in GC cells.

α-Synuclein mutation impairs processing of endomembrane compartments and promotes exocytosis and seeding of α-synuclein pathology.

Neuronal loss in Parkinson's disease (PD) is associated with impaired proteostasis and accumulation of α-syn microaggregates in dopaminergic neurons. These microaggregates promote seeding of α-synuclein (α-syn) pathology between synaptically linked neurons. However, the mechanism by which seeding is initiated is not clear. Using human pluripotent stem cell (hPSC) models of PD that allow comparison of SNCA mutant cells with isogenic controls, we find that SNCA mutant neurons accumulate α-syn deposits that cluster to multiple endomembrane compartments, specifically multivesicular bodies (MVBs) and lysosomes. We demonstrate that A53T and E46K α-syn variants bind and sequester LC3B monomers into detergent-insoluble microaggregates on the surface of late endosomes, increasing α-syn excretion via exosomes and promoting seeding of α-syn from SNCA mutant neurons to wild-type (WT) isogenic controls. Finally, we show that constitutive inactivation of LC3B promotes α-syn accumulation and seeding, while LC3B activation inhibits these events, offering mechanistic insight into the spread of synucleinopathy in PD.

MTMR14 protects against cerebral stroke through suppressing PTEN-regulated autophagy

The phosphoinositide phosphatase, myotubularinrelated protein 14 (MTMR14), plays a critical role in the regulating autophagy. However, its functional contribution to neuronal autophagy is still unclear. In the present study, we attempted to explore the effects of MTMR14 on ischemic stroke progression, as well as the underlying molecular mechanisms. Oxygen-glucose deprivation/reoxygenation (OGDR)-induced primary cortical neurons and pheochromocytoma (PC12) cells, and middle cerebral artery occlusion (MCAO)-operated mice were used to establish cerebral ischemia/reperfusion (I/R) injury in vitro and in vivo, respectively. OGDR treatment markedly decreased the expression of MTMR14 expression from mRNA and protein levels in the cultured primary neurons and PC12 cells. Functional analysis showed that OGDR-reduced cell viability was further accelerated by MTMR14 knockdown. On the contrary, MTMR14 over-expression significantly rescued the cell survival in OGDR-exposed cells. Moreover, autophagic markers including LC3BII and Beclin 1 were highly up-regulated in OGDR-incubated neurons and PC12 cells, while being further exacerbated by MTMR14 deletion. However, promoting MTMR14 dramatically alleviated LC3BII and Beclin 1 expression levels stimulated by OGDR. Importantly, we found that MTMR14-regulated autophagy was through its interactions with phosphatase and tensin homolog (PTEN). MTMR14 negatively modulated PTEN protein expression levels in OGDR-exposed cells. In vivo, MCAO-operated mice exhibited significantly reduced expression of MTMR14 in the ischemic penumbra tissues. After MCAO operation, MTMR14 over-expression effectively reduced infarct volume and neurological deficits scores, along with decreased activation of LC3B in neurons. Consistently, MCAO-increased PTEN, LC3BII and Beclin 1 were repressed by MTMR14 in mice. An interaction between MTMR14 and PTEN in response to MCAO was confirmed in vivo. Together, these results indicated the neuroprotective effects of MTMR14 on modulating PTEN-dependent excessive autophagy during cerebral I/R injury. Thus, targeting MTMR14 may provide feasible therapy for ischemic stroke onset and progression.

Naringin Induces Lysosomal Permeabilization and Autophagy Cell Death in AGS Gastric Cancer Cells.

Autophagy is a process of active programmed cell death, where a dying cell induces autophagosomes and subsequently regulated by degradative machinery. The aim of this study was to investigate the mechanism behind induction of autophagic cell death by Naringin flavonoid in AGS cancer cells. Growth inhibition of AGS cells showed downregulation of PI3K/Akt/mTOR signaling by Naringin treatment. Transmission electron microscopy observation showed swollen mitochondria and lysosome near peri-nuclear zone fused with autophagic vacuoles. Rapamycin pre-treatment with Naringin showed significant decrease in mTOR phosphorylation and increase in LC3B activation in AGS cells. Decrease in mTOR phosphorylation is associated with lysosomal function activation was observed by time-dependent treatment of Naringin. Induction of lysosomal membrane permeabilization (LMP) was observed by LAMP1 activation leading lysosomal cell death by releasing Cathepsin D from lysosomal lumen to cytosol. Naringin treated AGS cells showed up-regulating BH3 domain Bad, down-regulating Bcl-xL, and Bad phosphorylation and significant mitochondrial fluorescence intensity expression. Significant localization of mitochondria and LC3B activation was examined by person coefficient correlation. Activation of ERK1/2-p38 MAPKs and production of intracellular ROS has been observed over Naringin treatment. It has also been elucidated that pre-treatment with NAC inhibited mitochondria-LC3B colocalization, where ROS acted as upstream of ERK1/2-p38 MAPKs activation. Lysosomal cell death involvement has been evaluated by BAF A1 pre-treatment, inhibiting LAMP1, Cathepsin D, ROS, and blocking autophagolysosome in AGS cell death. Taken together, these findings show that, Naringin induced autophagy cell death involves LMP mediated lysosomal damage and BH3 protein Bad activation in AGS cancer cells.

Fundc1 is necessary for proper body axis formation during embryogenesis in zebrafish

FUN14 domain-containing protein 1 (FUNDC1) is a mitochondrial outer membrane protein which is responsible for hypoxia-induced mitophagy in mammalian cells. Knockdown of fundc1 is known to cause severe defects in the body axis of a rare minnow. To understand the role of Fundc1 in embryogenesis, we used zebrafish in this study. We used bioimaging to locate zebrafish Fundc1 (DrFundc1) with MitoTracker, a marker of mitochondria, and/or CellLight Lysosomes-GFP, a label of lysosomes, in the transfected ovary cells of grass carp. The use of Western blotting detected DrFundc1 as a component of mitochondrial proteins with endogenous COX IV, LC3B, and FUNDC1 in transgenic human embryonic kidney 293 T cells. DrFundc1 induced LC3B activation. The ectopic expression of Drfundc1 caused cell death and apoptosis as well as impairing cell proliferation in the 293 T cell line, as detected by Trypan blue, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and incorporation of BrdU. DrFundc1 up-regulated expression of both autophagy- and apoptosis-related genes, including ATG5, ATG7, LC3B, BECLIN1, and BAX in transgenic 293 T cells. A knockdown of Drfundc1 using short hairpin RNA (shRNA) led to midline bifurcation with two notochords and two spinal cords in zebrafish embryos. Co-injection of Drfundc1 mRNA repaired defects resulting from shRNA. Knockdown of Drfundc1 resulted in up- or down-regulation of genes related to autophagy and apoptosis, as well as decreased expression of neural genes such as cyclinD1, pax2a, opl, and neuroD1. In summary, DrFundc1 is a mitochondrial protein which is involved in mitophagy and is critical for typical body axis development in zebrafish.

19 Autophagy related metals in IDH1 mutant glioma: Chloroquine and TMZ as a potential novel strategy to treat IDH1 mt gliomas

Glioblastoma is considered among the most aggressive cancers, dismal prognosis and overall survival is only 14 months, 80% of primary low grade gliomas and seccondary GBMs that progress from low grade to grade II or III WHO classification have isocitrate dehydrogenase, (IDH1) or IDH2, mutations [1]. IDH1 mutant glioma is characterized by impaired glycolysis activity resulting in an abnormal production of 2-hydroxyglutarate (2-HG) [2] resulting in an undifferentiated phenotype with permanent hyper-methylation status and enhanced proliferation and invasion[3]. Interestingly, the IDH1 mutant phenotype of U87MG glioma cells shows resistance to autophagy induced cell death even in starving and low oxygen conditions [4]. Recent evidence has demonstrated increased autophagy activity on IDH1 mt cytotoxic activity when cells are exposed to autophagy inhibitors such as chloroquine [5]. To this date it remains elusive whether increased autophagy phenotype is the result of IDH1 mutation or if it either represents a secondary co-existing condition in the setting of the IDH1 mutation. Autophagy is also a mechanism of detoxification induced by chronic heavy metal exposure in both normal and cancer cells. Thus, we hypothesized that autophagy activity in IDH1 mt glioma is partially induced by chronic heavy metal exposure, leading to increased cell survival and abnormal DNA repair. Our approach included characterization and quantification of metal content on IDH1 mt glioma cell lines and tissues, in addition to correlation analyses of the celullar metallome with autophagy markers, ROS and DNA repair of IDH1 mt glioma cells allowed us to explore targets responsible for cell survival and DNA repair response. Furthermore, we evaluated the potential therapeutic value of Chloroquine (CQ) and bafylomicin for IDH1 mutant gliomas targeting autophagy pathway in combination with TMZ and radiation. We demonstrated that 2-HG induces autophagy activity via LC3B activation, and autophagy inhibiton by beclin gene silencing results in a reduction of 2-HG leading to cell starvation and apoptosis. Remarkably, we observed a positive correlation on at least six different metals with autophagy induced LC3B and beclin1 expression that significantly differed between the mutant and the wt genotype in glioma cell lines. ROS and DNA repair were also positively associated with at least 6 different metals and only seen in the IDH1 mt cell lines, then suggesting a possible explanation for the increase on autophagy, analysis of both LC3B and beclin 1 expression demonstrated a positive correlation with Mo98, Fe54, and Zn 66 on IDH1 mt cell lines and a positively correlation with Mo98 and V concentrations in relation to H2AX expression. Co, SeO Mo, V and Mg were positively correlated to ROS expression. TMZ and CQ induced autophagy pathway activation as measured by LC3B, Beclin, Atg expression. Silencing beclin in IDH1 mutant glioma cell lines induced apoptosis and reduction on 2-HG production after treatment with TMZ and radiation. Overall the results contained in this study 1) identify cellular metal content in relationship to mechanisms leading to increased autophagy on IDH1 mt glioma cells. 2) evaluate the combination of CQ and TMZ to potentially target and inhibit autophagy as a mechanism downstream the 2-HG production in IDH1 mt glioma cells.

Hyperoxia‐mediated LC3B activation contributes to the impaired transdifferentiation of type II alveolar epithelial cells (AECIIs) to type I cells (AECIs)

Life-saving mechanical ventilation can also cause lung injury through the overproduction of reactive oxygen species (ROS), leading to bronchopulmonary dysplasia (BPD)-like symptoms in preterm infants. It is reported that the autophagic protein microtubule-associated protein-1 light chain (LC)-3B can confer protection against hyperoxia-induced DNA damage in lung alveolar epithelium. However, its role in the transdifferentiation of type II alveolar epithelial cells (AECIIs) to type I cells (AECIs) is unclear and requires further investigation. In this study, newborn Sprague-Dawley rats were exposed to 90% oxygen for up to 14days to mimic BPD in human infants, with neonatal pups exposed to room air (21% oxygen) as controls. Primary rat AECIIs were cultured under hyperoxic conditions for up to 24hours to further investigate the underlying mechanisms. This study found that hyperoxia promoted a significant and time-dependent increase of AECII marker surfactant protein (SP)-C in the lung. The increase of AECI marker T1α was repressed by hyperoxia during lung development. These results indicated an impaired AECII transdifferentiation. Pulmonary ROS concentration and expression of autophagic protein LC-3B were increased gradually in response to hyperoxia exposure. Furthermore, AECIIs produced more ROS when cultured under hyperoxic conditions in vitro. Both the LC3B expression and the conversion from LC3BI to LC3BII were enhanced in hyperoxic AECs. Interestingly, inhibition of LC3B either by ROS inhibitor N-acetyl-l-cysteine (NAC) or adenovirus-mediated LC3B shRNA could partly restore AECII transdifferentiation under hyperoxia condition. In summary, the current study reveals a novel role of activated LC3B induced by hyperoxia in AECII transdifferentiation.

Abstract 2910: The dual role of autophagy in cadmium induced prostate carcinogenesis

Abstract Cadmium chloride (Cd) and cadmium products cause both acute and chronic cytotoxicity and Cd exposure correlates with the risk of cancer, especially prostate cancer in humans. On the cellular and molecular level, Cd compounds may promote transformation of healthy cells to a malignant state by inducing expression of pro-survival signaling molecules. However, the precise molecular mechanism by which Cd affects cellular transformation in different tissue types, especially in prostate, is yet to be delineated. Autophagy is an intracellular lysosomal degradation process by which cells remove damaged organelles and maintain the integrity of intracellular components. In cancer cells, autophagy promotes cancer cell survival as well as cell death. It is unclear of how, and under what circumstances, these dual and opposite roles of autophagy affect both anti-oncogenic and oncogenic functions. It is possible that uncharacterized molecular switch that influences the anti-oncogenic function of autophagy in healthy cells to become oncogenic. In our studies, we found that exposure of normal prostate epithelial cells (RWPE-2) in culture to Cd (10μM) results in growth inhibition. By contrast, the growth of Cd-transformed prostate epithelial (CTPE) cells was not inhibited by the same Cd treatment. While analyzing the molecular link between Cd induced toxicity and generation of reactive oxygen species (ROS) generation in RWPE-2 and CTPE cells; we found there was no significant change in the ROS induction in both cell types. In addition, no changes in the expression of autophagy targeted genes (Atg family proteins) were seen in Cd treated in both non-transformed and transformed cells. However, microtubule-associated protein 1A/1B-light chain 3B (LC-3B), which is directly involved in autophagy initiation was upregulated in Cd-treated non-transformed cells, but not in Cd-treated transformed cells. LC-3B activation also correlated with growth inhibition in non-transformed cells, while impairing LC-3B activation in RWPE-2 cells causes resistance to CD induced growth inhibition. Our ongoing study will determine whether induction of autophagy via LC-3B is a key event that facilitates Cd-mediated conversion of non-transformed prostate epithelial cells to a malignant state. We strongly believe our studies will define a new concept of LC-3B mediated autophagy in Cd-induced prostate carcinogenesis. Citation Format: Trinath P. Das, Arokya MS John, Suman Suman, Murali Ankem, Chendil Damodaran. The dual role of autophagy in cadmium induced prostate carcinogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2910. doi:10.1158/1538-7445.AM2015-2910

The pro-apoptotic role of autophagy in breast cancer.

Background:Autophagy is a catabolic process that has a vital role in cancer progression and treatment. Current chemotherapeutic agents, which target autophagy, result in growth inhibition in many cancer types. In this study, we examined the role of autophagy in breast cancer (BCa) patients as well as BCa cell lines.Methods:Tissue microarray was used to detect the expression of an autophagy marker, LC3B in BCa patients (normal/hyperplasia=8; grade-I=15, grade-II=84, and grade-III=27) and BCa cell lines. To modulate the activation of autophagy, we used novel herbal compound nimocinol acetate (NA) in BCa cell lines and the anticancer activity was measured by phenotypic and molecular analysis.Results:LC3B is highly expressed in tumours as compared with normal tissues. Activation of LC3B in NA-treated BCa (MCF-7 and MDA-MB-231) cells was evident as compared with other autophagy makers. Further, our results confirmed that NA-transcriptionally regulates LC3B (as confirmed by mRNA levels and reporter assay), which resulted in the formation of acidic autophagy vesicles and autolysosomes in BCa cells. Nimocinol acetate inhibited mTOR-mediated pro-survival signalling that resulted in inhibition of growth in BCa cells without affecting normal breast epithelial cells. Downregulation of LC3B expression by siRNA significantly inhibited the anticancer effects of NA in BCa cells.Conclusions:Together, our results suggest that LC3B is highly expressed in BCa tissues and increasing the threshold of LC3B activation dictates the pro-apoptotic function, which in turn, suppresses the growth of BCa cells. Nimocinol acetate could be a potential agent for treatment of BCa.

Transient knockdown of presenilin-1 provokes endoplasmic reticulum stress related formation of autophagosomes in HepG2 cells

The involvement of presenilins in the endoplasmic reticulum (ER) related autophagy was investigated by their transient knockdown in HepG2 cells. The silencing of PSEN1 but not of PSEN2 led to cell growth impairment and decreased viability. PSEN1 silencing resulted in ER stress response as evidenced by the elevated levels of glucose regulated protein 78 (Grp78), protein disulfide isomerase (PDI), and CCAAT/enhancer-binding protein homologous protein (CHOP) and by the activation of activating transcription factor 6 (ATF6). The activation of autophagy was indicated by the increased procession of microtubule-associated light chain 3 protein isoform B (LC3B) and by decreased phosphorylation of mammalian target of rapamycin (mTOR) and 70kDa ribosomal protein S6 kinase (p70S6K). Formation of ER-related cytoplasmic vacuolization colocalizing with the autophagic marker LC3B was also observed. The morphological effects and LC3B activation in presenilin-1 knockdown cells could be prevented by using the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin or by calcium chelation. The results show that presenilin-1 hampers the ER stress dependent initiation of macroautophagy.

Post-infarct treatment with microRNA145 reduces myocardial infarct size through acceleration of myocyte autophagy in rabbits

Background: We previously reported that microRNA145 (miRNA145) attenuates the ischemia-reperfusion injury. However, it is not still unclear how miRNA145 attenuates the ischemia-reperfusion injury. Objective: We aimed to investigate whether post-infarct treatment with miRNA145 reduces myocardial infarct size and its precise mechanism in a rabbit model of myocardial infarction. Methods: Male Japanese white rabbits underwent 30 min of coronary occlusion followed by 14 days of reperfusion. Rabbits received intravenous injection of saline (control group, n=7) or 0.035 mg/kg of miRNA145 encapsulated with liposome (miRNA145 group, n=7) immediately after reperfusion. At 14 days after reperfusion, rabbits were sacrificed and the hearts were removed. The area at risk and infarct areas were measured by evans blue dye and TTC staining, respectively, and the infarct size was calculated as a percentage of the risk area of the left ventricle. In another series of experiments, rat myoblast H9C2 were treated with 20 nM of miRNA145 and investigated the morphological changes by electron microscopy and signals by western blot analysis such as LC3B. Results: Post-infarct treatment with miRNA145 significantly reduced the myocardial infarct size (16.0±3.1%) as compared to the control (28.7±6.1%) group. Post-infarct treatment with miRNA145 significantly improved LV remodeling and improved ±dp/dt as compared to the control group. In the myoblast experiment, treatment with miRNA145 caused autophagic changes such as autophagosome and autolysosome, and caused activation of LC3B. Conclusions: It is suggested that post-infarct treatment with miRNA145 attenuated ischemia-reperfusion injury through acceleration of myocyte autophagy. The miRNA145 may be a promising strategy for treatment of acute myocardial infarction.