Betulinic acid-induced mitochondria-dependent cell death is counterbalanced by an autophagic salvage response

Betulinic acid (BetA) is a plant-derived pentacyclic triterpenoid that exerts potent anti-cancer effects in vitro and in vivo. It was shown to induce apoptosis via a direct effect on mitochondria. This is largely independent of proapoptotic BAK and BAX, but can be inhibited by cyclosporin A (CsA), an inhibitor of the permeability transition (PT) pore. Here we show that blocking apoptosis with general caspase inhibitors did not prevent cell death, indicating that alternative, caspase-independent cell death pathways were activated. BetA did not induce necroptosis, but we observed a strong induction of autophagy in several cancer cell lines. Autophagy was functional as shown by enhanced flux and degradation of long-lived proteins. BetA-induced autophagy could be blocked, just like apoptosis, with CsA, suggesting that autophagy is activated as a response to the mitochondrial damage inflicted by BetA. As both a survival and cell death role have been attributed to autophagy, autophagy-deficient tumor cells and mouse embryo fibroblasts were analyzed to determine the role of autophagy in BetA-induced cell death. This clearly established BetA-induced autophagy as a survival mechanism and indicates that BetA utilizes an as yet-undefined mechanism to kill cancer cells.     

Involvement of lipid rafts in the localization and dysfunction effect of the antitumor ether phospholipid edelfosine in mitochondria

Lipid rafts and mitochondria are promising targets in cancer therapy. The synthetic antitumor alkyl-lysophospholipid analog edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) has been reported to target lipid rafts. Here, we have found that edelfosine induced loss of mitochondrial membrane potential and apoptosis in human cervical carcinoma HeLa cells, both responses being abrogated by Bcl-x_L overexpression. We synthesized a number of new fluorescent edelfosine analogs, which preserved the proapoptotic activity of the parent drug, and colocalized with mitochondria in HeLa cells. Edelfosine induced swelling in isolated mitochondria, indicating an increase in mitochondrial membrane permeability. This mitochondrial swelling was independent of reactive oxygen species generation. A structurally related inactive analog was unable to promote mitochondrial swelling, highlighting the importance of edelfosine molecular structure in its effect on mitochondria. Raft disruption inhibited mitochondrial localization of the drug in cells and edelfosine-induced swelling in isolated mitochondria. Edelfosine promoted a redistribution of lipid rafts from the plasma membrane to mitochondria, suggesting a raft-mediated link between plasma membrane and mitochondria. Our data suggest that direct interaction of edelfosine with mitochondria eventually leads to mitochondrial dysfunction and apoptosis. These observations unveil a new framework in cancer chemotherapy that involves a link between lipid rafts and mitochondria in the mechanism of action of an antitumor drug, thus opening new avenues for cancer treatment.     

Role of PARP-1 in mitochondrial homeostasis

BackgroundNuclear poly(ADP-ribose) polymerase-1 (PARP-1) is a well characterised protein that accounts for the majority of PARylation reactions using NAD⁺ as a substrate, regulating diverse cellular functions. In addition to its nuclear functions, several recent studies have identified localization of PARP-1 in mitochondria and emphasized its possible role in maintaining mitochondrial homeostasis. Various reports suggest that nuclear PARP-1 has been implicated in diverse mitochondria-specific communication processes.Scope of reviewThe present review emphasizes on the potential role of PARP-1 in mitochondrial processes such as bioenergetics, mtDNA maintenance, cell death and mitophagy.Major conclusionsThe origin of mitochondrial PARP-1 is still an enigma; however researchers are trying to establish the cross-talk between nuclear and mitochondrial PARP-1 and how these PARP-1 pools modulate mitochondrial activity.General SignificanceA better understanding of the possible role of PARP-1 in mitochondrial homeostasis helps us to explore the potential therapeutic targets to protect mitochondrial dysfunctions.     

ERO1α-dependent endoplasmic reticulum–mitochondrial calcium flux contributes to ER stress and mitochondrial permeabilization by procaspase-activating compound-1 (PAC-1)

Procaspase-activating compound-1 (PAC-1) is the first direct caspase-activating compound discovered; using an in vitro cell-free system of caspase activation. Subsequently, this compound was shown to induce apoptosis in a variety of cancer cells with promising in vivo antitumor activity in canine lymphoma model. Recently, we have reported its ability to kill drug-resistant, Bcl-2/Bcl-xL overexpressing and Bax/Bak-deficient cells despite the essential requirement of mitochondrial cytochrome c (cyt. c) release for caspase activation, indicating that the key molecular targets of PAC-1 in cancer cells are yet to be identified. Here, we have identified Ero1α-dependent endoplasmic reticulum (ER) calcium leakage to mitochondria through mitochondria-associated ER membranes (MAM) and ER luminal hyper-oxidation as the critical events of PAC-1-mediated cell death. PAC-1 treatment upregulated Ero1α in multiple cell lines, whereas silencing of Ero1α significantly inhibited calcium release from ER and cell death. Loss of ER calcium and hyper-oxidation of ER lumen by Ero1α collectively triggered ER stress. Upregulation of GRP78 and splicing of X-box-binding protein 1 (XBP1) mRNA in multiple cancer cells suggested ER stress as the general event triggered by PAC-1. XBP1 mRNA splicing and GRP78 upregulation confirmed ER stress even in Bax/Bak double knockout and PAC-1-resistant Apaf-1-knockout cells, indicating an induction of ER stress-mediated mitochondrial apoptosis by PAC-1. Furthermore, we identified BH3-only protein p53 upregulated modulator of apoptosis (PUMA) as the key molecular link that orchestrates overwhelmed ER stress to mitochondria-mediated apoptosis, involving mitochondrial reactive oxygen species, in a p53-independent manner. Silencing of PUMA in cancer cells effectively reduced cyt. c release and cell death by PAC-1.     

Adiponectin receptor agonist AdipoRon blocks skin inflamm‐ageing by regulating mitochondrial dynamics

IntroductionSkin is susceptible to senescence‐associated secretory phenotype (SASP) and inflamm‐ageing partly owing to the degeneration of mitochondria. AdipoRon (AR) has protective effects on mitochondria in metabolic diseases such as diabetes. We explored the role of AR on mitochondria damage induced by skin inflamm‐ageing and its underlying mechanism.MethodsWestern blot, immunofluorescence and TUNEL staining were used to detect inflammatory factors and apoptosis during skin ageing. Transmission electron microscopy, ATP determination kit, CellLight Mitochondria GFP (Mito‐GFP), mitochondrial stress test, MitoSOX and JC‐1 staining were used to detect mitochondrial changes. Western blot was applied to explore the underlying mechanism. Flow cytometry, scratch test, Sulforhodamine B assay and wound healing test were used to detect the effects of AR on cell apoptosis, migration and proliferation.ResultsAR attenuated inflammatory factors and apoptosis that increased in aged skin, and improved mitochondrial morphology and function. This process at least partly depended on the suppression of dynamin‐related protein 1 (Drp1)‐mediated excessive mitochondrial division. More specifically, AR up‐regulated the phosphorylation of Drp1 at Serine 637 by activating AMP‐activated protein kinase (AMPK), thereby inhibiting the mitochondrial translocation of Drp1. Moreover, AR reduced mitochondrial fragmentation and the production of superoxide, preserved the membrane potential and permeability of mitochondria and accelerated wound healing in aged skin.ConclusionAR rescues the mitochondria in aged skin by suppressing its excessive division mediated by Drp1.     

Novel triterpenoid AECHL-1 induces apoptosis in breast cancer cells by perturbing the mitochondria–endoplasmic reticulum interactions and targeting diverse apoptotic pathways

BackgroundThe modus operandi for an anti-cancer drug must allow for an efficient discrimination system between tumorigenic and non-tumorigenic cells. Targeting ER stress and mitochondrial function in cancer cells appears to be a suitable option, as these processes are dysregulated in tumor cells. AECHL-1, a novel triterpenoid, exhibits potent anticancer activity against an array of cancer cell lines however, its mechanism of action remains elusive.MethodsMolecular targets of AECHL-1 were investigated using breast adenocarcinoma cells MCF-7, MDA-MB-231 and mammary epithelial cell line MCF 10A in vitro and xenograft tumors in SCID mice in vivo. Western blotting, flow cytometry, and immunohistochemical studies were employed to delineate the molecular pathways.ResultsAECHL-1 caused a transient elevation of ER stress proteins along with a prolonged phosphorylation of eIF2α in breast cancer cells. This was accompanied by a simultaneous release of calcium from ER stores and subsequent mitochondrial accumulation. These effects could be reversed by using ER stress inhibitors. AECHL-1 brings about mitochondria mediated, caspase independent cell death via AIF in MCF-7 cells; MDA-MB-231 succumbed to caspase dependent extrinsic pathway. Xenograft studies closely echoed our in vitro results. AECHL-1 did not alter cellular and molecular parameters in MCF 10A.ConclusionThese findings reveal that, AECHL-1 targets the Achilles Heel of cancer cell, namely dysfunctional ER and mitochondria while being non toxic to normal parenchyma and can thus be further explored as a potential chemotherapeutic intervention.General significanceAggravation of ER stress by AECHL-1 uncovers a novel pathway for selective elimination of cancer cells.     

Disulfiram oxy-derivatives induce entosis or paraptosis-like death in breast cancer MCF-7 cells depending on the duration of treatment

BackgroundDithiocarbamates and derivatives (including disulfiram, DSF) are currently investigated as antineoplastic agents. We have revealed earlier the ability of hydroxocobalamin (vitamin В_12b) combined with diethyldithiocarbamate (DDC) to catalyze the formation of highly cytotoxic oxidized derivatives of DSF (DSFoxy, sulfones and sulfoxides).MethodsElectron and fluorescent confocal microscopy, molecular biology and conventional biochemical techniques were used to study the morphological and functional responses of MCF-7 human breast cancer cells to treatment with DDC and B_12b alone or in combination.ResultsDDC induces unfolded protein response in MCF-7 cells. The combined use of DDC and B_12b causes MCF-7 cell death. Electron microscopy revealed the separation of ER and nuclear membranes, leading to the formation of both cytoplasmic and perinuclear vacuoles, with many fibers inside. The process of vacuolization coincided with the appearance of ER stress markers, a marked damage to mitochondria, a significant inhibition of 20S proteasome, and actin depolimerization at later stages. Specific inhibitors of apoptosis, necroptosis, autophagy, and ferroptosis did not prevent cell death. A short- time (6-h) exposure to DSFoxy caused a significant increase in the number of entotic cells.ConclusionsThese observations indicate that MCF-7 cells treated with a mixture of DDC and B_12b die by the mechanism of paraptosis. A short- time exposure to DSFoxy caused, along with paraptosis, a significant activation of the entosis and its final stage, lysosomal cell death.General significanceThe results obtained open up opportunities for the development of new approaches to induce non-apoptotic death of cancer cells by dithiocarbamates.     

MicroRNA-351 Regulates Two-Types of Cell Death,Necrosis and Apoptosis,Induced by 5-fluoro-2'-deoxyuridine

Cell-death can be necrosis and apoptosis. We are investigating the mechanisms regulating the cell death that occurs on treatment of mouse cancer cell-line FM3A with antitumor 5-fluoro-2''-deoxyuridine (FUdR): necrosis occurs for the original clone F28-7, and apoptosis for its variant F28-7-A. Here we report that a microRNA (miR-351) regulates the cell death pattern. The miR-351 is expressed strongly in F28-7-A but only weakly in F28-7. Induction of a higher expression of miR-351 in F28-7 by transfecting an miRNA mimic into F28-7 resulted in a change of the death mode; necrosis to apoptosis. Furthermore, transfection of an miR-351 inhibitor into F28-7-A resulted in the morphology change, apoptosis to necrosis, in this death-by-FUdR. Possible mechanism involving lamin B1 in this miR-351’s regulatory action is discussed.     

坏死性凋亡:一种新的细胞死亡方式

传统上,细胞死亡分为凋亡和坏死两类。而近年来越来越多的研究表明坏死性凋亡是一种不同于凋亡和坏死的新型细胞死亡途径,在多种疾病模型中发挥着重要作用。本文对坏死性凋亡的产生机制、坏死性凋亡同凋亡和坏死的区别及其在疾病和药物靶点发现中的作用进行综述,以利于加深对不同细胞死亡方式的认识并促进相关新药的研发。     

PGAM5: A crucial role in mitochondrial dynamics and programmed cell death

In response to mitochondrial damage, mitochondria activate mitochondrial dynamics to maintain normal functions, and an imbalance in mitochondrial dynamics triggers multiple programmed cell death processes. Recent studies have shown that phosphoglycerate mutase 5 (PGAM5) is associated with mitochondrial damage. PGAM5 activates mitochondrial biogenesis and mitophagy to promote a cellular compensatory response when mitochondria are mildly damaged, whereas severe damage to mitochondria leads to PGAM5 inducing excessive mitochondria fission, disruption to mitochondrial movement, and amplification of apoptosis, necroptosis and mitophagic death signals, which eventually evoke cell death. PGAM5 functions mainly through protein-protein interactions and specific Ser/Thr/His protein phosphatase activity. PGAM5 is also regulated by mitochondrial proteases. Detection of PGAM5 and its interacting protein partners should enable a more accurate evaluation of mitochondrial damage and a more precise method for the diagnosis and treatment of diseases.     

Necroptosis Interfaces with MOMP and the MPTP in Mediating Cell Death

During apoptosis the pro-death Bcl-2 family members Bax and Bak induce mitochondrial outer membrane permeabilization (MOMP) to mediate cell death. Recently, it was shown that Bax and Bak are also required for mitochondrial permeability transition pore (MPTP)-dependent necrosis, where, in their non-oligomeric state, they enhance permeability characteristics of the outer mitochondrial membrane. Necroptosis is another form of regulated necrosis involving the death receptors and receptor interacting protein kinases (RIP proteins, by Ripk genes). Here, we show cells or mice deficient for Bax/Bak or cyclophilin D, a protein that regulates MPTP opening, are resistant to cell death induced by necroptotic mediators. We show that Bax/Bak oligomerization is required for necroptotic cell death and that this oligomerization reinforces MPTP opening. Mechanistically, we observe mixed lineage kinase domain-like (MLKL) protein and cofilin-1 translocation to the mitochondria following necroptosis induction, while expression of the mitochondrial matrix isoform of the antiapoptotic Bcl-2 family member, myeloid cell leukemia 1 (Mcl-1), is significantly reduced. Some of these effects are lost with necroptosis inhibition in Bax/Bak1 double null, Ppif^-/-, or Ripk3^-/- fibroblasts. Hence, downstream mechanisms of cell death induced by necroptotic stimuli utilize both Bax/Bak to generate apoptotic pores in the outer mitochondrial membrane as well as MPTP opening in association with known mitochondrial death modifying proteins.     

MicroRNA-29b-3p promotes 5-fluorouracil resistance via suppressing TRAF5-mediated necroptosis in human colorectal cancer

Drug resistance in colorectal cancer is a great challenge in clinic. Elucidating the deep mechanism underlying drug resistance will bring much benefit to diagnosis, therapy and prognosis in patients with colorectal cancer. In this study, miR-29b-3p was shown to be involved in resistance to 5-fluorouracil (5-FU)-induced necroptosis of colorectal cancer. Further, miR-29b-3p was shown to target a regulatory subunit of necroptosis TRAF5. Rescue of TRAF5 could reverse the effect of miR-29b-3p on 5-FU-induced necroptosis, which was consistent with the role of necrostatin-1 (a specific necroptosis inhibitor). Then it was demonstrated that miR-29b-3p was positively correlated with chemoresistance in colorectal cancer while TRAF5 negatively. In conclusion, it is deduced that miR-29b-3p/TRAF5 signaling axis plays critical role in drug resistance in chemotherapy for colorectal cancer patients by regulating necroptosis. The findings in this study provide us a new target for interfere therapy in colorectal cancer.Key words: Colorectal cancer, miR-29b-3p, TRAF5, necroptosis, 5-fluorouracil resistance     

The role of necroptosis in cancer: A double-edged sword?

Necroptosis is a programmed, caspase-independent cell death that is morphologically similar to necrosis. Unlike apoptosis, necroptosis evokes inflammatory responses by releasing damage-associated molecular patterns. Recent studies suggest that tumor undergoes necroptosis in vivo and necroptosis has pro- or anti-tumoral effects in cancer development and progression. Furthermore, triggering necroptosis in tumor cells has been explored as a potential therapeutic strategy against cancer. Here, we will review the recent research progress of necroptosis in conferring anti- or pro-tumoral effects and its potential application in cancer therapy.     

抑癌因子miR-10a抑制Tiam1表达对胃癌细胞凋亡和迁移的影响

目的:探讨miR-10a抑制Tiam1表达对胃癌细胞凋亡和侵袭的影响。方法:获取胃上皮组织细胞及胃癌组织细胞,利用q PCR及Western blot实验检测两种细胞中mi R-10a表达与Tiam1的m RNA及蛋白表达水平,同时检测胃癌细胞S746T及正常胃粘膜细胞RGM-1和NGEC中mi R-10a表达与Tiam1蛋白表达水平。通过将mi R-10a mimic和mi R-10a inhibitor转染HS746T细胞,利用流式细胞术检测HS746T的细胞周期和细胞凋亡,TranswellTM实验检测HS746T细胞的侵袭能力,qPCR及Western blot实验检测凋亡相关蛋白caspase3、caspase9和Bax以及周期相关蛋白P21表达水平;荧光素酶活性分析实验检测Tiam1是mi R-10a的作用靶点。已构建的Tiam1高表达的Tiam1-pcDNA3.1质粒和敲除Tiam1基因的PX458质粒分别转染HS746T细胞,通过流式细胞术及TranswellTM实验检测HS746T细胞的凋亡及侵袭能力。结果:与胃上皮组织细胞相比,早期胃癌临床组织细胞中mi R-10a表达降低,Tiam1的m RNA及蛋白表达升高;mi R-10a的表达与早期胃癌患者的肿瘤转移密切相关,与年龄、性别和肿瘤分期无关;与正常胃粘膜细胞RGM-1和NGEC相比,胃癌细胞HS746T中的mi R-10a表达降低,而Tiam1蛋白表达升高;mi R-10a可抑制HS746T细胞侵袭,促进细胞凋亡,使其停滞于G0/G1期;mi R-10a靶向作用于Tiam1基因的3'非翻译区(3'UTR),减少Tiam1的蛋白表达;Tiam1可抑制HS746T细胞凋亡,促进HS746T细胞侵袭。结论:mi R-10a靶向作用于Tiam1基因的3'UTR,抑制HS746T细胞的增殖及侵袭,促进HS746T细胞凋亡。     

10H‐3,6‐Diazaphenothiazines triggered the mitochondrial‐dependent and cell death receptor‐dependent apoptosis pathways and further increased the chemosensitivity of MCF‐7 breast cancer cells via inhibition of AKT1 pathways

Breast cancer is one of the leading causes of death in cancer categories, followed by lung, colorectal, and ovarian among the female gender across the world. 10H‐3,6‐diazaphenothiazine (PTZ) is a thiazine derivative compound that exhibits many pharmacological activities. Herein, we proceed to investigate the pharmacological activities of PTZ toward breast cancer MCF‐7 cells as a representative in vitro breast cancer cell model. The PTZ exhibited a proliferation inhibition (IC₅₀ = 0.895 µM) toward MCF‐7 cells. Further, cell cycle analysis illustrated that the S‐phase checkpoint was activated to achieve proliferation inhibition. In vitro cytotoxicity test on three normal cell lines (HEK293 normal kidney cells, MCF‐10A normal breast cells, and H9C2 normal heart cells) demonstrated that PTZ was more potent toward cancer cells. Increase in the levels of reactive oxygen species results in polarization of mitochondrial membrane potential (ΔΨm), together with suppression of mitochondrial thioredoxin reductase enzymatic activity suggested that PTZ induced oxidative damages toward mitochondria and contributed to improved drug efficacy toward treatment. The RT² PCR Profiler Array (human apoptosis pathways) proved that PTZ induced cell death via mitochondria‐dependent and cell death receptor‐dependent pathways, through a series of modulation of caspases, and the respective morphology of apoptosis was observed. Mechanistic studies of apoptosis suggested that PTZ inhibited AKT1 pathways resulting in enhanced drug efficacy despite it preventing invasion of cancer cells. These results showed the effectiveness of PTZ in initiation of apoptosis, programmed cell death, toward highly chemoresistant MCF‐7 cells, thus suggesting its potential as a chemotherapeutic drug.     

RIP3-Dependent Accumulation of Mitochondrial Superoxide Anions in TNF-α-Induced Necroptosis

Excessive production of reactive oxygen species (ROS) is a key phenomenon in tumor necrosis factor (TNF)-α-induced cell death. However, the role of ROS in necroptosis remains mostly elusive. In this study, we show that TNF-α induces the mitochondrial accumulation of superoxide anions, not H₂O₂, in cancer cells undergoing necroptosis. TNF-α-induced mitochondrial superoxide anions production is strictly RIP3 expression-dependent. Unexpectedly, TNF-α stimulates NADPH oxidase (NOX), not mitochondrial energy metabolism, to activate superoxide production in the RIP3-positive cancer cells. In parallel, mitochondrial superoxide-metabolizing enzymes, such as manganese-superoxide dismutase (SOD2) and peroxiredoxin III, are not involved in the superoxide accumulation. Mitochondrial-targeted superoxide scavengers and a NOX inhibitor eliminate the accumulated superoxide without affecting TNF-α-induced necroptosis. Therefore, our study provides the first evidence that mitochondrial superoxide accumulation is a consequence of necroptosis.     

Doxorubicin-induced cardiomyocyte apoptosis: Role of mitofusin 2

BackgroundDoxorubicin (DOX) is an anti-tumor agent that is widely used in clinical setting for cancer treatment. The application of the DOX, however, is limited by its cardiac toxicity which can induce heart failure through an undefined mechanism. Mitofusin 2 (Mfn2) is a mitochondrial GTPase fusion protein that is located on the outer membrane of mitochondria (OMM). The Mfn2 plays an important role in mitochondrial fusion and fission. The aim of this study is to identify the role of the Mfn2 in DOX-induced cardiomyocyte apoptosis.MethodsCultured neonatal rat cardiomyocytes were used in this study. Mfn2 expression in cardiomyocytes was determined after the cardiomyocytes were challenged with DOX. Cardiomyocyte mitochondrial fission, mitochondrial reactive oxygen species (ROS) production was assessed with mitochondrial fragmentation and MitoSOX fluorescence probe, respectively. Cardiomyocyte apoptosis was determined with caspase3 activity and TUNEL staining.ResultsChallenging of the cardiomyocytes with DOX resulted in increasing in cardiomyocyte oxidative stress and apoptosis. In addition, levels of Mfn2 in cardiomyocytes were decreased after the cells were challenged with DOX which was associated with increased mitochondrial fission (fragmentation) and mitochondrial ROS production. An increase in cardiomyocyte levels of Mfn2 attenuated the DOX-induced increase in mitochondrial fission and prevented cardiomyocyte mitochondrial ROS production. An increase in cardiomyocyte levels of Mfn2 or pretreatment of cardiomyocytes with an anti-oxidant, Mito-tempo, also prevented the DOX-induced cardiomyocyte apoptosis.ConclusionOur results indicate that DOX results in a decreased cardiomyocyte Mfn2 expression which promotes mitochondrial fission and ROS production further leads to cardiomyocyte apoptosis.     

Epigenetic Regulation of RIP3 Suppresses Necroptosis and Increases Resistance to Chemotherapy in NonSmall Cell Lung Cancer

INTRODUCTION: The efficacy of chemotherapeutic agents in killing cancer cells is mainly attributed to the induction of apoptosis. However, the tremendous efforts on enhancing apoptosis-related mechanisms have only moderately improved lung cancer chemotherapy, suggesting that other cell death mechanisms such as necroptosis could be involved. In this study, we investigated the role of the necroptosis pathway in the responsiveness of nonsmall cell lung cancer (NSCLC) to chemotherapy. METHODS: In vitro cell culture and in vivo xenograft tumor therapy models and clinical sample studies are combined in studying the role of necroptosis in chemotherapy and mechanism of necroptosis suppression involving RIP3 expression regulation. RESULTS: While chemotherapeutic drugs were able to induce necroptotic cell death, this pathway was suppressed in lung cancer cells at least partly through downregulation of RIP3 expression. Ectopic RIP3 expression significantly sensitized lung cancer cells to the cytotoxicity of anticancer drugs such as cisplatin, etoposide, vincristine, and adriamycin. In addition, RIP3 suppression was associated with RIP3 promoter methylation, and demethylation partly restored RIP3 expression and increased chemotherapeutic-induced necroptotic cell death. In a xenograft tumor therapy model, ectopic RIP3 expression significantly sensitized anticancer activity of cisplatin in vivo. Furthermore, lower RIP3 expression was associated with worse chemotherapy response in NSCLC patients. CONCLUSION: Our results indicate that the necroptosis pathway is suppressed in lung cancer through RIP3 promoter methylation, and reactivating this pathway should be exploited for improving lung cancer chemotherapy.     

The Circular RNA Cdr1as Promotes Myocardial Infarction by Mediating the Regulation of miR-7a on Its Target Genes Expression

Objectives

Recent studies have demonstrated the role of Cdr1as (or CiRS-7), one of the well-identified circular RNAs (circRNAs), as a miR-7a/b sponge or inhibitor in brain tissues or islet cells. This study aimed to investigate the presence of Cdr1as/miR-7a pathway in cardiomyocytes, and explore the mechanism underlying the function of miR-7a in protecting against myocardial infarction (MI)-induced apoptosis.

Methods

Mouse MI injury model was established and evaluated by infarct size determination. Real-time PCR was performed to quantify the expression of Cdr1as and miR-7a in cardiomyocytes. Cell apoptosis was determined by caspase-3 activity analysis and flow cytometry assays with Annexin V/PI staining. Transfection of Cdr1as overexpressing plasmid and miR-7a mimic were conducted for gain-of-function studies. Luciferase reporter assay and western blot analysis were performed to verity potential miR-7a targets.

Results

Cdr1as and miR-7a were both upregulated in MI mice with increased cardiac infarct size, or cardiomyocytes under hypoxia treatment. Cdr1as overexpression in MCM cells promoted cell apoptosis, but was then reversed by miR-7a overexpression. The SP1 was identified as a new miR-7a target, in line with previously identified PARP, while miR-7a-induced decrease of cell apoptosis under hypoxia treatment was proven to be inhibited by PARP-SP1 overexpression. Moreover, Cdr1as overexpression in vivo increased cardiac infarct size with upregulated expression of PARP and SP1, while miR-7a overexpression reversed these changes.

Conclusions

Cdr1as also functioned as a powerful miR-7a sponge in myocardial cells, and showed regulation on the protective role of miR-7a in MI injury, involving the function of miR-7a targets, PARP and SP1.