miR-154 inhibits EMT by targeting HMGA2 in prostate cancer cells

Epithelial–mesenchymal transition (EMT) is a crucial process that plays an important role in the invasion and metastasis of human cancers. High-mobility group AT-hook 2 (HMGA2) has been found to be involved in the EMT program, with its aberrant expression having been observed in a variety of malignant tumors. However, the mechanisms regulating HMGA2 expression remain incompletely understood. The objective of this study was to investigate whether mir-154 plays a critical role in EMT by regulating HMGA2. The expression levels of HMGA2 were examined in four samples of prostate cancer (PCa) tissue and adjacent non-tumorous tissue by Western blot analysis. The effects of forced expression of miR-154 or HMGA2 knockdown on PCa cells were evaluated by cell migration and invasion assays and Western blot analysis. HMGA2 was upregulated in the PCa tissue samples compared with the adjacent normal ones. Forced expression of miR-154 or HMGA2 knockdown significantly reduced the migratory and invasive capabilities of PCa cells in vitro and inhibited EMT gene expression, increased the levels of E-cadherin, an epithelial marker, and decreased the levels of vimentin, a mesenchymal marker. HMGA2 is a direct target gene of miR-154 by dual-luciferase reporter assay. Our findings suggest that miR-154 plays a role in regulating EMT by targeting HMGA2. Understanding the targets and regulating pathways of miR-154 may provide new insights into the underlying pathogenesis of PCa.     

Hydrogen sulfide inhibits high glucose-induced matrix protein synthesis by activating AMP-activated protein kinase in renal epithelial cells

Hydrogen sulfide, a signaling gas, affects several cell functions. We hypothesized that hydrogen sulfide modulates high glucose (30 mm) stimulation of matrix protein synthesis in glomerular epithelial cells. High glucose stimulation of global protein synthesis, cellular hypertrophy, and matrix laminin and type IV collagen content was inhibited by sodium hydrosulfide (NaHS), an H(2)S donor. High glucose activation of mammalian target of rapamycin (mTOR) complex 1 (mTORC1), shown by phosphorylation of p70S6 kinase and 4E-BP1, was inhibited by NaHS. High glucose stimulated mTORC1 to promote key events in the initiation and elongation phases of mRNA translation: binding of eIF4A to eIF4G, reduction in PDCD4 expression and inhibition of its binding to eIF4A, eEF2 kinase phosphorylation, and dephosphorylation of eEF2; these events were inhibited by NaHS. The role of AMP-activated protein kinase (AMPK), an inhibitor of protein synthesis, was examined. NaHS dose-dependently stimulated AMPK phosphorylation and restored AMPK phosphorylation reduced by high glucose. Compound C, an AMPK inhibitor, abolished NaHS modulation of high glucose effect on events in mRNA translation as well as global and matrix protein synthesis. NaHS induction of AMPK phosphorylation was inhibited by siRNA for calmodulin kinase kinase β, but not LKB1, upstream kinases for AMPK; STO-609, a calmodulin kinase kinase β inhibitor, had the same effect. Renal cortical content of cystathionine β-synthase and cystathionine γ-lyase, hydrogen sulfide-generating enzymes, was significantly reduced in mice with type 1 diabetes or type 2 diabetes, coinciding with renal hypertrophy and matrix accumulation. Hydrogen sulfide is a newly identified modulator of protein synthesis in the kidney, and reduction in its generation may contribute to kidney injury in diabetes.     

Long noncoding RNA PVT1 facilitates high glucose-induced cardiomyocyte death through the miR-23a-3p/CASP10 axis

Dilated cardiomyopathy (DCM) is the leading cause of morbidity and mortality in diabetic patients. Long noncoding RNA plasmacytoma variant translocation 1 (PVT1) has been shown to be related to the pathogenesis of DCM. However, the mechanism by which PVT1 regulates DCM pathogenesis is unclear. High glucose level was employed to construct a DCM cell model in vitro. Cell viability was determined via cell counting kit-8 assay. The level of lactate dehydrogenase (LDH) was measured with the corresponding kit. Expression levels of PVT1, miR-23a-3p, and caspase-10 (CASP10) messenger RNA were evaluated with a quantitative real-time polymerase chain reaction. Cell apoptosis was assessed by flow cytometry assay. Protein levels of B-cell lymphoma 2-associated X (Bax), cleaved-caspase-3 (cleaved-casp-3), and CASP10 were examined via western blot analysis. The relationship between PVT1 or CASP10 and miR-23a-3p was verified with dual-luciferase reporter assay. We observed that PVT1 and CASP10 were upregulated while miR-23a-3p was downregulated in high glucose-induced cardiomyocytes. High glucose levels repressed cardiomyocyte activity and induced cardiomyocyte apoptosis, but this influence was antagonized by PVT1 knockdown or miR-23a-3p overexpression. Furthermore, PVT1 acted as a sponge for miR-23a-3p, and miR-23a-3p inhibition counterbalanced the influence of PVT1 silencing on viability and apoptosis of cardiomyocytes under high glucose level treatment. PVT1 could increase CASP10 expression via sponging miR-23a-3p. In conclusion, PVT1 acted as a deleterious lncRNA in DCM. PVT1 facilitated cardiomyocyte death by regulating the miR-23a-3p/CASP10, which offered a new mechanism to comprehend the pathogenesis of DCM.     

Empagliflozin reduces high glucose-induced oxidative stress and miR-21-dependent TRAF3IP2 induction and RECK suppression,and inhibits human renal proximal tubular epithelial cell migration and epithelial-to-mesenchymal transition

Proximal tubular epithelial cells (PTEC) in the S1 segment of the kidney abundantly express sodium-glucose co-transporters (SGLT) that play a critical role in whole body glucose homeostasis. We recently reported suppression of RECK (Reversion Inducing Cysteine Rich Protein with Kazal Motifs), a membrane anchored endogenous MMP inhibitor and anti-fibrotic mediator, in the kidneys of db/db mice, a model of diabetic kidney disease (DKD), as well as in high glucose (HG) treated human kidney proximal tubule cells (HK−2). We further demonstrated that empagliflozin (EMPA), an SGLT2 inhibitor, reversed these effects. Little is known regarding the mechanisms underlying RECK suppression under hyperglycemic conditions, and its rescue by EMPA. Consistent with our previous studies, HG (25 mM) suppressed RECK expression in HK-2 cells. Further mechanistic investigations revealed that HG induced superoxide and hydrogen peroxide generation, oxidative stress-dependent TRAF3IP2 upregulation, NF-κB and p38 MAPK activation, inflammatory cytokine expression (IL-1β, IL-6, TNF-α, and MCP-1), miR-21 induction, MMP2 activation, and RECK suppression. Moreover, RECK gain-of-function inhibited HG-induced MMP2 activation and HK-2 cell migration. Similar to HG, advanced glycation end products (AGE) induced TRAF3IP2 and suppressed RECK, effects that were inhibited by EMPA. Importantly, EMPA treatment ameliorated all of these deleterious effects, and inhibited epithelial-to-mesenchymal transition (EMT) and HK-2 cell migration. Collectively, these findings indicate that hyperglycemia and associated AGE suppress RECK expression via oxidative stress/TRAF3IP2/NF-κB and p38 MAPK/miR-21 induction. Furthermore, these results suggest that interventions aimed at restoring RECK or inhibiting SGLT2 have the potential to treat kidney inflammatory response/fibrosis and nephropathy under chronic hyperglycemic conditions, such as DKD.     

KB-R7943 inhibits high glucose-induced endothelial ICAM-1 expression and monocyte-endothelial adhesion

Hyperglycemia is the major cause of diabetic angiopathy. The aim of our study was to evaluate the impact of KB-R7943, an inhibitor of Na⁺/Ca²⁺ exchanger (NCX) on cell growth and function of human “diabetic” endothelial cells (EC). Intercellular adhesion molecule-1 (ICAM-1) expression and NCX activity were determined after EC were exposed to high glucose in the absence and presence of KB-R7943. Coincubation of EC with high glucose for 24 h resulted in a significant increase of monocyte-endothelial cell adhesion and the expression of ICAM-1. These effects were abolished by KB-R7943 and KB-R7943 significantly decreased the activation of NCX induced by high glucose. These findings suggested that KB-R7943 may play a role in inhibiting expression of adhesion molecules by inhibiting the reverse activation of NCX.     

Eriodictyol inhibits high glucose-induced oxidative stress and inflammation in retinal ganglial cells

Diabetic retinopathy (DR) is one of the most common microvascular complications of diabetes mellitus and is considered as a leading cause of blindness. Oxidative stress and inflammation are significant drivers for the development of DR. Eriodictyol, a flavonoid compound, was proved to possess anti-inflammatory, antioxidative, and antidiabetic activities. However, the role of eriodictyol in DR has not been unveiled. In the current study, we explored the protective effects of eriodictyol on high glucose (HG)-induced rat retinal ganglial cells (RGCs). The results suggested that eriodictyol improved cell viability of HG-induced rat RGC-5 cells in a dose-dependent manner. Eriodictyol reduced the reactive oxygen species production and increased the activities of superoxide dismutase, glutathione peroxidase and catalase in rat RGC-5 cells in response to HG stimulation. The production of proinflammatory cytokines including tumor necrosis factor alpha and interleukin-8 was diminished after eriodictyol treatment. Eriodictyol also suppressed cell apoptosis induced HG in rat RGC-5 cells. Furthermore, eriodictyol enhanced the nuclear translocation of nuclear factor erythroid-2 (E2)-related factor 2 (Nrf2) and elevated the expression of antioxidant enzyme heme-oxygenase-1 (HO-1). These findings suggested that eriodictyol protects the RGC-5 cells from HG-induced oxidative stress, inflammation, and cell apoptosis through regulating the activation of Nrf2/HO-1 pathway.     

FOXO3a inhibits the EMT and metastasis of breast cancer by regulating TWIST-1 mediated miR-10b/CADM2 axis

BackgroundBreast cancer is the most common malignancy and has been considered as a leading cause of cancer death in women. Exploring the mechanism of breast cancer metastasis is extremely important for seeking novel therapeutic strategies and improving prognosis.MethodsClinical specimens and pathological characteristics were collected for evaluating the expression of forkhead box class O 3a (FOXO3a) and twist-related protein 1 (TWIST-1) in breast cancer tissues. CCK-8 assay was used to analyze cell proliferation. Cell invasion and migration were assessed by transwell assays. The expression of FOXO3a, TWIST-1, miR-10b, CADM2, FAK, phosphor-AKT and the epithelial-mesenchymal transition (EMT)-related protein (N-cadherin, E-cadherin and vimentin) were analyzed by RT-qPCR, immunohistochemical staining, immunofluorescence assay or western blot, respectively. Xenograft mouse models were used to analyze the role of the FOXO3a in breast cancer.ResultsFOXO3a was down-regulated and TWIST-1 was up-regulated in breast cancer tissues. Overexpression of FOXO3a or knockdown of TWIST-1 suppressed the proliferation, invasion, migration and EMT of breast cancer cells. Overexpression of TWIST-1 could reverse the effect of FOXO3a on the proliferation, invasion, migration and EMT of breast cancer. Moreover, FOXO3a suppressed the growth and metastasis of breast cancer by targeting TWIST1 in vivo.ConclusionFOXO3a inhibited the EMT and metastasis of breast cancer via TWIST-1/miR-10b/CADM2 axis.     

Selenium inhibits high glucose-induced cyclooxygenase-2 and P-selectin expression in vascular endothelial cells

Selenium as a component of glutathione peroxidase may be beneficial in insulin resistance, hence potentially may modify the risk of diabetes and cardiovascular disease. The aim of our study was to evaluate whether selenium can also alter high glucose (HG), advanced glycation end products (AGE), high insulin (HI) and H₂O₂-induced expression of cyclooxygenase (COX)-2 and P-selectin. Human umbilical vein endothelial cells (HUVECs) were pretreated with selenium and stimulated by HG, AGE, HI and H₂O₂. Selenium significantly inhibited HG, AGE, HI and H₂O₂-induced expression of COX-2 and P-selectin. Moreover, selenium also inhibited HG, AGE, HI and H₂O₂-induced activation of p38 mitogen-activated protein kinase (p38 MAPK), which indicated that the preventive effects of selenium on COX-2 and P-selectin may be associated with p38. Our results indicated that selenium supplementation can reduce HG, AGE, HI and H₂O₂-induced expression of COX-2 and P-selectin by inhibition of the p38 pathway.     

FGF-23 inhibits renal tubular phosphate transport and is a PHEX substrate.

Oncogenic osteomalacia (OOM), X-linked hypophosphatemia (XLH), and autosomal dominant hypophosphatemic rickets (ADHR) are phenotypically similar disorders characterized by hypophosphatemia, decreased renal phosphate reabsorption, normal or low serum calcitriol concentrations, normal serum concentrations of calcium and parathyroid hormone, and defective skeletal mineralization. XLH results from mutations in the PHEX gene, encoding a membrane-bound endopeptidase, whereas ADHR is associated with mutations of the gene encoding FGF-23. Recent evidence that FGF-23 is expressed in mesenchymal tumors associated with OOM suggests that FGF-23 is responsible for the phosphaturic activity previously termed "phosphatonin." Here we show that both wild-type FGF-23 and the ADHR mutant, FGF-23(R179Q), inhibit phosphate uptake in renal epithelial cells. We further show that the endopeptidase, PHEX, degrades native FGF-23 but not the mutant form. Our results suggest that FGF-23 is involved in the pathogenesis of these three hypophosphatemic disorders and directly link PHEX and FGF-23 within the same biochemical pathway.     

MiR-1297下调PTEN的表达参与子宫内膜癌变

已有研究证明,抑癌子PTEN（phosphatase and tensin homolog）缺失或表达下调可引起子宫内膜癌变;在喉鳞状细胞癌中miR-1297可下调PTEN的表达。然而,miR-1297下调PTEN的机制,以及是否miR-1297靶向抑制PTEN参与子宫内膜癌变尚未见报道。本研究证明,miR-1297靶向抑制PTEN基因的表达参与子宫内膜癌变。收集临床病理检查确诊为子宫内膜腺癌的癌组织及癌旁组织18例。实时定量PCR（RT-qPCR）及Western印迹结果显示,与癌旁子宫内膜比较,子宫内膜癌组织miR-1297高表达,而PTEN则呈低表达或缺失。内膜癌和癌旁内膜组织中,miR-1297与PTEN表达水平的散点图分析揭示,miR-1297的表达水平与PTEN的表达水平呈明显的负相关（内膜癌：miR-1297与PTEN的相关系数r=-0.6928,P=0.0438;癌旁组织：相关系数r=-0.4085,P < 0.0001)。报告酶活性测定显示,与对照比较,pGL3-PTEN-3′-UTR-1/2/3加miR-1297模拟物共转染后,荧光素酶活性在转染的Ishikawa和ECC-1细胞中分别降低了约21%（P=0.0196）和17%（P=0.0306）;相反,转染miR-1297抑制物后,两种细胞的荧光素酶活性分别升高约59%（Ishikawa：P=0.0014）和50%（ECC-1： P=0.0025）。结果提示,miR-1297可通过特异识别PTEN mRNA的3′-UTR,抑制PTEN的表达。基因转染结合Western印迹结果证明,在原代子宫内膜细胞中,过表达miR-1297模拟物导致PTEN蛋白表达下调,而其下游底物分子--磷酸化的AKT（p-AKT）表达上调;而过表达miR-1297抑制物后,PTEN表达上调,p-AKT表达下调。Connexin V/PI双染结合流式分析证明,与对照细胞（3%细胞凋亡）比较,在子宫内膜癌细胞系Ishikawa中,敲减miR-1297表达后细胞调亡明显增加（至10%）,这可能与抑制miR-1297后PTEN的表达上调有关。上述结果提示,miR-1297在子宫内膜癌高表达,而PTEN低表达。上述结果还提示,miR-1297作为一个促癌子可通过特异识别PTEN mRNA的3′-UTR,抑制PTEN的表达,促进AKT磷酸化/激活,从而促进内膜癌的发生,这可能是子宫内膜癌变的发生原因之一。     

miR-219-5p targets TBXT and inhibits breast cancer cell EMT and cell migration and invasion

miR-219-5p has been reported to act as either a tumor suppressor or a tumor promoter in different cancers by targeting different genes. In the present study, we demonstrated that miR-219-5p negatively regulated the expression of TBXT, a known epithelial–mesenchymal transition (EMT) inducer, by directly binding to TBXT 3′-untranslated region. As a result of its inhibition on TBXT expression, miR-219-5p suppressed EMT and cell migration and invasion in breast cancer cells. The re-introduction of TBXT in miR-219-5p overexpressing cells decreased the inhibitory effects of miR-219 on EMT and cell migration and invasion. Moreover, miR-219-5p decreased breast cancer stem cell (CSC) marker genes expression and reduced the mammosphere forming capability of cells. Overall, our study highlighted that TBXT is a novel target of miR-219-5p. By suppressing TBXT, miR-219-5p plays an important role in EMT and cell migration and invasion of breast cancer cells.     

Laminar shear stress inhibits high glucose-induced migration and invasion in human bladder cancer cells

High glucose has been known to play a pathogenic role in the development and progression of bladder cancer in diabetics, whereas the leading cause of death in such patients is mainly attributed to hyperglycemia-enhanced metastasis. In addition to the impact of glucose, cancer cells may be affected by laminar shear stress (LSS) generated from interstitial, blood, and/or lymphatic fluid flows during metastasis. Although the effect of flow-induced mechanical force on cancer pathophysiology has been extensively investigated, very little is understood regarding the cells that are simultaneously stimulated by LSS and hyperglycemia. To address this issue, the influence of LSS on bladder cancer cell motility in a hyperglycemic environment was examined. Based on the results of cell movement and protein expression analyses, we found that both cell migration and invasion were up- and downregulated by 25 mM glucose and 12 dynes/cm² LSS, respectively. Furthermore, the motility of the cells with simultaneous hyperglycemic and LSS stimulations was significantly reduced compared with that of the cells stimulated by high glucose alone (P?<?0.05), demonstrating that the LSS rather than hyperglycemia played the dominant role in regulation of cell motility. These results implied that LSS with an intensity ≥?12 dynes/cm² may serve as a feasible tool to reduce bladder cancer motility in diabetics.     

Baicalin,baicalein and wogonin inhibits high glucose-induced vascular inflammation in vitro and in vivo

Vascular inflammatory process has been suggested to play a key role in initiation and progression of atherosclerosis, a major complication of diabetes mellitus. Thus, in this study, we attempted to determine whether three structurally related polyphenols found in the Chinese herb Huang Qui, namely baicalin, baicalein, and wogonin, can suppress vascular inflammatory processes induced by high glucose (HG) in human umbilical vein endothelial cells (HUVECs) and mice. Data showed that HG induced markedly increased vascular permeability, monocyte adhesion, expressions of cell adhesion molecules (CAMs), formation of reactive oxygen species (ROS) and activation of nuclear factor (NF)-κB. Remarkably, all of the above mentioned vascular inflammatory effects of HG were attenuated by pretreatment with baicalin, baicalein, and wogonin. Vascular inflammatory responses induced by HG are critical events underlying development of various diabetic complications, therefore, our results suggest that baicalin, baicalein, and wogonin may have significant therapeutic benefits against diabetic complications and atherosclerosis. [BMB Reports 2015; 48(9): 519-524]     

Down-regulation by elicitors of phosphatidylcholine-hydrolyzing phospholipase C and up-regulation of phospholipase A in plant cells

Phosphatidylcholine, labeled by two fluorescent fatty acids, was fed to cultured plant cells (Petrosilenum crispum, L.; VBI-0, Nicotiana benthiana, L.) and fluorescent diacylglycerol (DAG) was the major metabolite. When a glycoprotein elicitor, derived from Phytophthora sojae, was applied to the parsley cells and the small protein cryptogein from Phytophthora cryptogea was applied to the tobacco cells, these signal substances strongly and rapidly decreased the pool of fluorescent diacylglycerol and weakly increased the pool of free fluorescent fatty acid and of fluorescent lysophosphatidylcholine. The cells responded in a very similar way to the application of mastoparan, a wasp venom peptide. As phosphatidic acid was only a very minor fluorescent metabolite DAG is hypothesized to arise by the action of a phosphatidylcholine-hydrolyzing phospholipase C which was down-regulated by elicitors. Up-regulation of a phospholipase A by elicitors is also suggested by these results. This is the first evidence for phosphatidylcholine-hydrolyzing phospholipase C in plant signal transduction.     

Hydrogen sulphide inhibits cardiomyocyte hypertrophy by up-regulating miR-133a

Hydrogen sulphide (H₂S) has been shown to play a crucial role in cardiovascular physiology and disease. However, there is no information about the possible role of H₂S in cardiomyocyte hypertrophy (CH). Our results showed that pretreatment with NaHS, an H₂S donor, significantly reduced [³H]-leucine incorporation, cell surface area, mRNA expression of brain natriuretic peptide (BNP), intracellular reactive oxygen species (ROS), miR-21 and increased atrial natriuretic peptide (ANP) and miR-133a expression in hypertrophic cardiomyocytes. Anti-miR133a inhibitor transfection partly reduced the anti-hypertrophic effect of NaHS. In conclusion, H₂S is a direct inhibitor of CH; it acts by increasing miR-133a and inhibiting the increase in intracellular ROS.