Hyperglycemia induces apoptosis via CB1 activation through the decrease of FAAH 1 in retinal pigment epithelial cells

Fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of the main endocannabinoid, anandamide, and related fatty acid amides, has emerged as a regulator of endocannabinoid signaling. Retinal pigment epithelial (RPE) cells are believed to be important cells in the pathogenesis of diabetic retinopathy. However, the pathophysiology of FAAH in diabetic retinopathy has not been determined. Thus, we examined the effect of high glucose (HG) on the expression of FAAH and CB(1)R in the ARPE-19 human RPE cells. We found that HG downregulated the expression of FAAH 1 mRNA and protein in ARPE-19 cells. In contrast, it upregulated the expression of CB(1)R mRNA and protein. HG-induced internalization of CB(1)R in HEK 293 cells and ARPE-19 cells was blocked by overexpression of FAAH 1 and treatment with the CB(1)R blocker, AM 251. HG-induced generation of reactive oxygen species and lipid peroxide formation were blocked by the overexpression of FAAH 1. FAAH 1 overexpression also blocked HG-induced expression of CB(1)R in the cytosolic fraction. We also investigated whether the overexpression of FAAH 1 protected against HG-induced apoptosis. High glucose increased the Bax/Bcl-2 ratio and levels of cleaved PARP, cleaved caspase-9 and caspase-3, and reduced cell viability. HG-induced apoptotic effects were reduced by the overexpression of FAAH 1, treatment with the CB(1)R-specific antagonist AM 251 and CB(1)R siRNA transfection. In conclusion, HG-induced apoptosis in ARPE-19 cells by inducing CB(1)R expression through the downregulation of FAAH 1 expression. Our results provide evidence that CB(1)R blockade through the recovery of FAAH 1 expression may be a potential anti-diabetic therapy for the treatment of diabetic retinopathy.     

LncRNA BDNF‐AS inhibits proliferation,migration, invasion and EMT in oesophageal cancer cells by targeting miR‐214

This study was aimed at exploring the effect of lncRNA BDNF‐AS on cell proliferation, migration, invasion and epithelial‐to‐mesenchymal transition (EMT) of oesophageal cancer (EC) cells. The expression of BDNF‐AS and miR‐214 in tissue samples and cells was measured by qRT‐PCR. The targeted relationship between BDNF‐AS and miR‐214 was analysed by dual‐luciferase reporter assay. After cell transfection, the cell proliferation activity was assessed by MTS method, while the migrating and invading abilities were evaluated by transwell assay. LncRNA BDNF‐AS was remarkably down‐regulated, while miR‐214 was up‐regulated in EC tissues and cells in comparison with normal tissues and cells. Overexpression of BDNF‐AS significantly inhibited the abilities of cell proliferation, migration and invasion as well as the EMT processes of EC cells. The bioinformatics analysis and luciferase assay indicated that BDNF‐AS could be directly bound by miR‐214. Furthermore, overexpression of miR‐214 and BDNF‐AS exerted suppressive influence on EC cell multiplication, migration, invasion and EMT processes. LncRNA BDNF‐AS restrained cell proliferation, migration, invasion and EMT processes in EC cells by targeting miR‐214.     

Effects of siRNA Targeting c‐Myc and VEGF on Human Colorectal Cancer Volo Cells

c‐Myc and vascular endothelial growth factor (VEGF) genes are frequently deregulated and overexpressed in this malignancy, and strategies designed to inhibit c‐Myc and VEGF expression in cancer cells may have considerable therapeutic value. In the present study, we design and use short interfering RNA (siRNA) to inhibit c‐Myc and VEGF expression in colorectal cancer Volo cells and validate their effects on cell proliferation, cell cycle, apoptosis, and cell metastasis. Upon transient transfection with plasmid‐encoding siRNA, it was found that expression of c‐Myc and VEGF was significantly downregulated in siRNA‐transfected cells and the downregulation of c‐Myc and VEGF inhibited cell growth and induced apoptosis and metastasis of Volo cells. c‐Myc and VEGF downregulation also increased cell population in the G0–G1 phase. In conclusion, the specific siRNA efficiently silenced the expression of c‐Myc and VEGF, further suppressed the cell proliferation, triggered cell apoptosis, and inhibited cell invasiveness of colorectal cancer Volo cells. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:499‐505, 2012;Viewthis article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21455     

Protective effects of cynaroside on oxidative stress in retinal pigment epithelial cells

Cynaroside is a flavonoid compound proved to possess antioxidant activity, but its protective effect on age‐related macular degeneration still remains unclear. In this study, the protective effects of cynaroside on oxidative stress and apoptosis in retinal pigment epithelial (RPE) cells induced by hydrogen peroxide (H₂O₂) were investigated. Results showed that cynaroside effectively attenuated the decrease of cell activity induced by H₂O₂. The total reactive oxygen species can be remitted by decreasing malondialdehyde level, as well as increasing glutathione level, and superoxide dismutase and catalase activities. In addition, Western blot analysis indicated that cynaroside protected ARPE‐19 cells from apoptosis through downregulation of caspase‐3 protein activation which was controlled by the upstream proteins Bcl‐2 and Bax. It was finally proved that cynaroside could enhance the antioxidant and antiapoptotic ability in ARPE‐19 cells by promoting the expression of p‐Akt.     

Up‐regulation of semaphorin 4A expression in human retinal pigment epithelial cells by PACAP released from cocultured neural cells

Development and homeostasis of multicellular organisms require interactions between neighbouring cells. We recently established an in vitro model of cell–cell interaction based on a collagen vitrigel membrane. We have now examined the role of neural cells in retinal homeostasis by coculture of human retinal pigment epithelial (RPE) cells and neural cells on opposite sides of such a membrane. The neural cells (differentiated PC12 cells) induced up‐regulation of semaphorin 4A (Sema4A), a member of the semaphorin family of neural guidance proteins, in RPE (ARPE19) cells. This effect of the neural cells was mimicked by the neuropeptide pituitary adenylate cyclase–activating polypeptide (PACAP) and was abolished by the PACAP antagonist PACAP(6–38). Coculture with neural cells or stimulation with PACAP also induced the phosphorylation of extracellular‐signal‐regulated kinase in ARPE19 cells, and this effect of the neural cells was inhibited by PACAP(6–38). Finally, among various cytokines examined, only the amount of interleukin‐6 released by cocultures of ARPE19 and neural cells differed from that released by ARPE19 cells cultured alone. Interleukin‐6 was not detected in culture supernatants of neural cells, and the reduction in the amount of interleukin‐6 released by the cocultures compared with that released by ARPE19 cells alone was prevented by PACAP(6–38). Our findings suggest that PACAP released from retinal neural cells (photoreceptors or optic nerve cells) may regulate Sema4A expression in RPE cells and thereby contribute to the maintenance of retinal structure and function. Development and homeostasis of multicellular organisms require interactions between neighbouring cells. With the use of a coculture system based on a collagen vitrigel membrane, we have now shown that neural cells induce up‐regulation of the neural guidance protein Sema4A in RPE cells. This effect of neural cells appears to be mediated by the neuropeptide PACAP. PACAP released from retinal neural cells (photoreceptors or optic nerve cells) may thus regulate Sema4A expression in RPE cells and thereby contribute to the maintenance of retinal structure and function. Copyright © 2014 John Wiley & Sons, Ltd.     

Differential expression of microRNAs in TM3 Leydig cells of mice treated with brain‐derived neurotrophic factor

Brain‐derived neurotrophic factor (BDNF) is a neurotrophin that can promote the development and proliferation of neurons. BDNF has been found to be involved in male reproduction. Leydig cells in testicular interstitial tissues can secrete testosterone in a luteinizing hormone‐dependent manner. We showed that BDNF and its receptor TrkB were expressed in mice TM3 Leydig cells in the present study. Furthermore, BDNF can promote proliferation of mouse TM3 Leydig cells in vitro. Results of microRNA (miRNA) deep sequencing showed that BDNF can alter the expression profile of miRNAs in TM3 Leydig cells. Eighty‐three miRNAs were significantly different in the BDNF‐treated and control groups (fold change of >2.0 or <0.5, P < 0.05) wherein 40 were upregulated and 43 were downregulated. The expression levels of miR‐125a‐5p, miR‐22‐5p, miR‐342‐59, miR‐451a, miR‐148a‐5p, miR‐29b‐3p, miR‐199b‐5p, and miR‐145a‐5p were further confirmed by quantitative real‐time polymerase chain reaction. Bioinformatic analysis revealed that miRNAs regulated a large number of genes with different functions. Pathway analysis indicated that miRNAs participate in the pathways involved in signal transduction, cancer, metabolism, endocrine system, immune system, and nerve system. This study indicated that miRNAs might be involved in the BDNF‐regulated cellular functions of Leydig cells.     

Extracellular vesicle‐derived circ_SLC19A1 promotes prostate cancer cell growth and invasion through the miR‐497/septin 2 pathway

The occurrence and development of prostate cancer (PCa) is complex, and the related mechanism is not fully understood. Current studies have found that extracellular vesicles (EVs) and circular RNAs (circRNAs) have important functions in various tumours and other diseases. In this study, the detection of circRNAs in PCa showed that circ_SLC19A1 was increased in PCa cells and their secreted EVs. EVs with high expression of circ_SLC19A1 could be taken up by PCa cells, which promoted cell proliferation and invasion. The sequence of circ_SLC19A1 contained multiple binding sites for miR‐497, and circ_SLC19A1 could bind directly to miR‐497 in cells. The expression of miR‐497 was downregulated in PCa cells, while the expression of its target gene septin 2 (SEPT2) was upregulated significantly. Transfection of circ_SLC19A1 small interfering RNA (siRNA) or miR‐497 mimics could significantly inhibit the expression of SEPT2 and the phosphorylation of extracellular signal‐regulated kinase 1 and 2 (ERK1/2). After co‐transfection of circ_SLC19A1 siRNA and miR‐497 inhibitors or SEPT2 overexpression vector, the expression of SEPT2 and ERK1/2 phosphorylation levels showed no significant changes. Similar results were obtained with co‐transfection of miR‐497 mimics and the SEPT2 overexpression vector. Therefore, cancer cells can regulate the expression of SEPT2 through miR‐497 by secreting EVs with high expression of circ_SLC19A1, thus affecting the activation of the downstream ERK1/2 pathway and ultimately regulating PCa cell growth and invasion. Therefore, EV‐derived circ_SLC19A1 plays an important regulatory role in PCa and may be an important target for PCa prevention and treatment.     

CDADC1在人视网膜色素上皮细胞中功能研究

目的：研究细胞增殖相关基因CDADC1在人视网膜色素上皮细胞ARPE19中的表达及对ARPE19细胞增殖的影响。方法：采用基因重组技术构建荧光表达载体pEGFP－C1－CDADC1和真核表达载体pcDNA3．1-myc-CDADC1,用脂质体转染法转染ARPE19细胞,观察GFP—CDADC1融合蛋白在ARPE19细胞的表达定位,流式细胞仪测定CDADC1转染后对ARPE19细胞生长周期、凋亡的影响。结果：FP—CDADC1融合蛋白亚细胞定位显示,CDADC1低表达于ARPE19细胞胞浆,高表达于细胞核;pcD—NA3．1-myc-CDADC1瞬时转染ARPE19细胞显示24小时细胞无明显改变,48小时后重组质粒转染组S期细胞占细胞总数的19．37％,pcDNA3．1-myc空载质粒转染组S期细胞占10．87％,而空白对照组S期细胞占3．33％,重组质粒转染组与两对照组之间的差异有统计学意义（P〈0．01）。结论：CDADC1在增生性玻璃体视网膜病变（PVR）发生和发展过程中可促进DNA的合成,引起细胞增生。     

Alantolactone Induces Apoptosis and Cell Cycle Arrest on Lung Squamous Cancer SK‐MES‐1 Cells

Alantolactone, a sesquiterpene lactone compound, has variety of pharmacological properties, including anti‐inflammatory and antineoplastic effects. In our study, alantolactone inhibited cancer cell proliferation. To explore the mechanisms underlying its antitumor action, we further examined apoptotic cells and cell cycle distribution using flow cytometry analysis. Alantolactone triggered apoptosis and induced cell cycle G1/G0 phase arrest. Furthermore, the expressions of caspases‐8, ‐9, ‐3, PARP, and Bax were significantly upregulated, while antiapoptotic factor Bcl‐2 expression was inhibited. In addition, the expressions of cyclin‐dependent kinase 4 (CDK4), CDK6, cyclin D3, and cyclin D1 were downregulated by alantolactone. Therefore, our findings indicated that alantolactone has an antiproliferative role on lung squamous cancer cells, and it may be a promising chemotherapeutic agent for squamous lung cancer SK‐MES‐1 cells.     

Suppression of c-Myc induces apoptosis via an AMPK/mTOR-dependent pathway by 4-<Emphasis Type="Italic">O</Emphasis>-methyl-ascochlorin in leukemia cells

4-O-Methyl-ascochlorin (MAC) is a methylated derivative of the prenyl-phenol antibiotic ascochlorin, which was isolated from an incomplete fungus, Ascochyta viciae. Although the effects of MAC on apoptosis have been reported, the underlying mechanisms remain unknown. Here, we show that MAC promoted apoptotic cell death and downregulated c-Myc expression in K562 human leukemia cells. The effect of MAC on apoptosis was similar to that of 10058-F4 (a c-Myc inhibitor) or c-Myc siRNA, suggesting that the downregulation of c-Myc expression plays a role in the apoptotic effect of MAC. Further investigation showed that MAC downregulated c-Myc by inhibiting protein synthesis. MAC promoted the phosphorylation of AMP-activated protein kinase (AMPK) and inhibited the phosphorylation of mammalian target of rapamycin (mTOR) and its target proteins, including p70S6 K and 4E-BP-1. Treatment of cells with AICAR (an AMPK activator), rapamycin (an mTOR inhibitor), or mTOR siRNA downregulated c-Myc expression and induced apoptosis to a similar extent to that of MAC. These results suggest that the effect of MAC on apoptosis induction in human leukemia cells is mediated by the suppression of c-Myc protein synthesis via an AMPK/mTOR-dependent mechanism.     

Effects of miR-21 downregulation and silibinin treatment in breast cancer cell lines

Silibinin is a natural polyphenol with high antioxidant and anticancer properties, which causes cell cycle arrest and apoptosis in most cancer cell types including breast cancer, but the in-line mechanisms, are still unknown. Silibinin significantly downregulated oncomiR miR-21 expression in breast cancer cells. Here the effect of anti-miR-21 on cell viability, apoptotic induction, cell cycle distribution, and the expression levels of downstream targets of miR-21 were investigated in MCF-7 and T47D cells. MiR-21 mimic transfection was also applied in silibinin treated samples to evaluate functional role of miR-21downregulation on silibinin effects. It was found that after anti-miR-21 transfection, no significant changes were detected in cell viability, apoptosis (except early apoptosis), and cell cycle in MCF-7 and T47D cells. Compared to silibinin, miR-21 mimic transfection in combination with silibinin caused a slight modulation in some of the examined silibinin effects including apoptosis, Bcl2 mRNA and PTEN mRNA and protein levels. Silibinin slightly changed luciferase activity from reporters containing the miR-21 recognition elements from PTEN-3′UTR and Bcl2-3′UTR in both cell lines. Together these data demonstrated negligible cancer-progression impact of miR-21 and limited roles of miR-21 downregulation in examined silibinin effects, and strengthened the anti-cancer pathways of silibinin other than miR-21downregulation in MCF-7 and T47D cells.     

Influence of ethanol on neonatal cerebellum of BDNF gene‐deleted animals: analyses of effects on Purkinje cells,apoptosis‐related proteins,and endogenous antioxidants

The sensitivity of the developing central nervous system (CNS) to the deleterious effects of ethanol has been well documented, with exposure leading to a wide array of CNS abnormalities. Certain CNS regions are susceptible to ethanol during well‐defined critical periods. In the neonatal rodent cerebellum, a profound loss of Purkinje cells is found when ethanol is administered early in the postnatal period [on postnatal days 4 or 5 (P4–5)], while this neuronal population is much less vulnerable to similar ethanol insult slightly later in the postnatal period (P7–9). Prior studies have shown that neurotrophic factors (NTFs) can be altered by ethanol exposure, and both in vitro and in vivo studies have provided evidence that such substances have the potential to protect against ethanol neurotoxicity. In the present study, it was hypothesized that depletion of an NTF shown to be important to cerebellar development would exacerbate ethanol‐related effects within this region, when administration was confined to a normally ethanol‐resistant ontogenetic period. For this study, brain‐derived neurotrophic factor (BDNF) gene‐deleted (“knockout”) and wild‐type mice were exposed to ethanol via vapor inhalation or to control conditions during the normally ethanol‐resistant period (P7 and P8). Two hours after termination of exposure on P8, analyses were made of body weight, crown‐rump length, and brain weight. In subsequent investigations, the number and density of Purkinje cells and the volume of cerebellar lobule I were determined, and the expression of anti‐ and pro‐apoptotic proteins and the activities of endogenous antioxidants were assessed. It was found that the BDNF knockouts were significantly smaller than the wild‐type animals, with smaller brain weights. Purkinje cell number and density was reduced in ethanol‐treated knockout, but not wild‐type animals, and the volume of lobule I was significantly decreased in the gene‐deleted animals compared to wild‐types, but was not further affected by ethanol treatment. The loss of Purkinje cells in the BDNF knockouts was accompanied by decreases in anti‐apoptotic Bcl‐xl and in phosphorylated (and hence inactivated) pro‐apoptotic Bad, and reduced activity of the antioxidant glutathione reductase, while the antioxidant catalase was increased by ethanol treatment in this genotype. In the wild‐type animals, anti‐apoptotic Bcl‐2 was decreased by ethanol treatment, but the pro‐apoptotic c‐Jun N‐terminal kinase (JNK) was markedly diminished by ethanol exposure, while the activity of the protective antioxidant superoxide dismutase (SOD) was significantly enhanced. These results suggest that neurotrophic factors have the capacity to protect against ethanol neurotoxicity, perhaps by regulation of expression of molecules critical to neuronal survival such as elements of the apoptosis cascade and protective antioxidants. © 2002 Wiley Periodicals, Inc. J Neurobiol 51: 160–176, 2002     

Overexpression of STAMP2 suppresses atherosclerosis and stabilizes plaques in diabetic mice

Our research aims to evaluate the function of the STAMP2 gene, an important trigger in insulin resistance (IR), and explore its role in macrophage apoptosis in diabetic atherosclerotic vulnerable plaques. The characteristics of diabetic mice were measured by serial metabolite and pathology tests. The level of STAMP2 was measured by RT‐PCR and Western blot. The plaque area, lipid and collagen content of brachiocephalic artery plaques were measured by histopathological analyses, and the macrophage apoptosis was measured by TUNEL. Correlation of STAMP2/Akt signaling pathway and macrophage apoptosis was validated by Ad‐STAMP2 transfection and STAMP2 siRNA inhibition. The diabetic mice showed typical features of IR, hyperglycaemia. Overexpression of STAMP2 ameliorated IR and decreased serum glucose level. In brachiocephalic lesions, lipid content, macrophage quantity and the vulnerability index were significantly decreased by overexpression of STAMP2. Moreover, the numbers of apoptotic cells and macrophages in lesions were both significantly decreased. In vitro, both mRNA and protein expressions of STAMP2 were increased under high glucose treatment. P‐Akt was highly expressed and caspase‐3 was decreased after overexpression of STAMP2. However, expression of p‐Akt protein was decreased and caspase‐3 was increased when STAMP2 was inhibited by siRNA. STAMP2 overexpression could exert a protective effect on diabetic atherosclerosis by reducing IR and diminishing macrophage apoptosis.     

Bioengineered Bruch's-like extracellular matrix promotes retinal pigment epithelial differentiation

In the eye, the retinal pigment epithelium (RPE) adheres to a complex protein matrix known as Bruch's membrane (BrM). The aim of this study was to provide enriched conditions for RPE cell culture through the production of a BrM-like matrix. Our hypothesis was that a human RPE cell line would deposit an extracellular matrix (ECM) resembling BrM. The composition and structure of ECM deposited by ARPE19 cells (ARPE19-ECM) was characterized. To produce ARPE19-ECM, ARPE19 cells were cultured in the presence dextran sulphate. ARPE19-ECM was decellularized using deoxycholate and characterized by immunostaining and western blot analysis. Primary human RPE and induced pluripotent stem cells were seeded onto ARPE19-ECM or geltrex coated surfaces and examined by microscopy or RT-PCR. Culture of ARPE19 cells with dextran sulphate promoted nuclear localization of SOX2, formation of tight junctions and deposition of ECM. ARPE19 cells deposited ECM proteins found in the inner layers of BrM, including fibronectin, vitronectin, collagens IV and V as well as laminin-alpha-5, but not those found in the middle elastic layer (elastin) or the outer layers (collagen VI). ARPE19-ECM promoted pigmentation in human RPE and pluripotent stem cell cultures. Expression of RPE65 was significantly increased on ARPE19-ECM compared with geltrex in differentiating pluripotent stem cell cultures. ARPE19 cells deposit ECM with a composition and structure similar to BrM in the retina. Molecular cues present in ARPE19-ECM promote the acquisition and maintenance of the RPE phenotype. Together, these results demonstrate a simple method for generating a BrM-like surface for enriched RPE cell cultures.     

MeCP2‐421‐mediated RPE epithelial‐mesenchymal transition and its relevance to the pathogenesis of proliferative vitreoretinopathy

Proliferative vitreoretinopathy (PVR) is a blinding eye disease. Epithelial‐mesenchymal transition (EMT) of RPE cells plays an important role in the pathogenesis of PVR. In the current study, we sought to investigate the role of the methyl‐CpG‐binding protein 2 (MeCP2), especially P‐MeCP2‐421 in the pathogenesis of PVR. The expressions of P‐MeCP2‐421, P‐MeCP2‐80, PPAR‐γ and the double labelling of P‐MeCP2‐421 with α‐SMA, cytokeratin, TGF‐β and PPAR‐γ in human PVR membranes were analysed by immunohistochemistry. The effect of knocking down MeCP2 using siRNA on the expressions of α‐SMA, phospho‐Smad2/3, collagen I, fibronectin and PPAR‐γ; the expression of α‐SMA stimulated by recombinant MeCP2 in ARPE‐19; and the effect of TGF‐β and 5‐AZA treatment on PPAR‐γ expression were analysed by Western blot. Chromatin immunoprecipitation was used to determine the binding of MeCP2 to TGF‐β. Our results showed that P‐MeCP2‐421 was highly expressed in PVR membranes and was double labelled with α‐SMA, cytokeratin and TGF‐β, knocking down MeCP2 inhibited the activation of Smad2/3 and the expression of collagen I and fibronectin induced by TGF‐β. TGF‐β inhibited the expression of PPAR‐γ, silence of MeCP2 by siRNA or using MeCP2 inhibitor (5‐AZA) increased the expression of PPAR‐γ. α‐SMA was up‐regulated by the treatment of recombinant MeCP2. Importantly, we found that MeCP2 bound to TGF‐β as demonstrated by Chip assay. The results suggest that MeCP2 especially P‐MeCP2‐421 may play a significant role in the pathogenesis of PVR and targeting MeCP2 may be a potential therapeutic approach for the treatment of PVR.