Downregulation of Rap1GAP contributes to Ras transformation

Although abundant in well-differentiated rat thyroid cells, Rap1GAP expression was extinguished in a subset of human thyroid tumor-derived cell lines. Intriguingly, Rap1GAP was downregulated selectively in tumor cell lines that had acquired a mesenchymal morphology. Restoring Rap1GAP expression to these cells inhibited cell migration and invasion, effects that were correlated with the inhibition of Rap1 and Rac1 activity. The reexpression of Rap1GAP also inhibited DNA synthesis and anchorage-independent proliferation. Conversely, eliminating Rap1GAP expression in rat thyroid cells induced a transient increase in cell number. Strikingly, Rap1GAP expression was abolished by Ras transformation. The downregulation of Rap1GAP by Ras required the activation of the Raf/MEK/extracellular signal-regulated kinase cascade and was correlated with the induction of mesenchymal morphology and migratory behavior. Remarkably, the acute expression of oncogenic Ras was sufficient to downregulate Rap1GAP expression in rat thyroid cells, identifying Rap1GAP as a novel target of oncogenic Ras. Collectively, these data implicate Rap1GAP as a putative tumor/invasion suppressor in the thyroid. In support of that notion, Rap1GAP was highly expressed in normal human thyroid cells and downregulated in primary thyroid tumors.     

Activation of renal angiotensin type 1 receptor contributes to the pathogenesis of progressive renal injury in a rat model of chronic cardiorenal syndrome

Although chronic cardiac dysfunction is known to progressively exacerbate renal injury, a condition known as type 2 cardiorenal syndrome (CRS), the mechanism responsible is largely unknown. The present study was undertaken to clarify the mechanism of renal injury in rats with both unilateral nephrectomy (NX) and surgically induced myocardial infarction (MI), corresponding to a model of type 2 CRS. Compared with a control group, rats with both MI and NX (MI+NX) exhibited progressive proteinuria during the experimental period (34 wk after MI surgery), whereas proteinuria was not observed in rats with MI alone and was moderate in rats with NX alone. The proteinuria in rats with MI+NX was associated with renal lesions such as glomerulosclerosis and infiltration of mononuclear cells and upregulation of the renal proinflammatory and -fibrotic cytokine and angiotensin II type 1a receptor (AT1aR) genes. In contrast, plasma renin activity was lowered in rats with MI+NX. Immunohistochemistry revealed that the increased AT1R protein was present mainly in renal interstitial mononuclear cells. Olmesartan medoxomil, an AT1R blocker, markedly reduced the proteinuria and infiltration of mononuclear cells, whereas spironolactone, a mineralocorticoid receptor blocker, did not. The present findings demonstrate the pathogenetic role of renal interstitial AT1R signaling in a model of type 2 CRS, providing evidence that AT1R blockade can be a useful therapeutic option for this syndrome.     

Downregulation of KCC2 following LTP contributes to EPSP-spike potentiation in rat hippocampus

GABAergic synaptic inhibition plays a critical role in regulating long-term potentiation (LTP) of glutamatergic synaptic transmission and circuit output. The K(+)-Cl(-) cotransporter 2 (KCC2) is an important factor in determining inhibitory GABAergic synaptic strength besides the contribution of GABA(A) receptor. Although much knowledge has been gained regarding activity-dependent downregulation of KCC2 in many pathological conditions, the potential change and contribution of KCC2 in LTP expression is still unknown. In this study, we found that downregulation of KCC2 was accompanied with the occurrence of LTP but not that of long-term depression in hippocampal CA1 region. Meanwhile, KCC2 level in CA3/DG and adjacent cortex was stable in the process of LTP expression in Schaffer collateral synapses. Blockade of NMDA receptor with APV not only prevented LTP induction also abolished the reduction of KCC2. Furthermore, the inhibition of KCC2 function with furosemide directly induced EPSP-spike (E-S) potentiation, an important component of LTP in hippocampus. The present data suggest a novel mechanism that LTP formation is accompanied by the downregulation of KCC2, which is underlying GABAergic strength and most likely contributes to the E-S potentiation following LTP.     

lncRNA MALAT1 participates in metformin inhibiting the proliferation of breast cancer cell

In recent years, the repurposing of conventional and chemotherapeutic drugs is recognized as an alternative strategy for health care. The main purpose of this study is to strengthen the application of non-oncological drug metformin on breast cancer treatment in the perspective of epigenetics. In the present study, metformin was found to inhibit cell proliferation, promote apoptosis and induce cell cycle arrest in breast cancer cells at a dose-dependent manner. In addition, metformin treatment elevated acH3K9 abundance and decreased acH3K18 level. The expression of lncRNA MALAT1, HOTAIR, DICER1-AS1, LINC01121 and TUG1 was up-regulated by metformin treatment. In metformin-treated cells, MALAT1 knock-down increased the Bax/Bcl2 ratio and enhanced p21 but decreased cyclin B1 expression. The expression of Beclin1, VDAC1, LC3-II, CHOP and Bip was promoted in the cells received combinatorial treatment of metformin and MALAT1 knock-down. The reduced phosphorylation of c-Myc was further decreased in the metformin-treated cells in combination with MALAT1 knock-down than metformin treatment alone. Taken together, these results provide a promising repurposed strategy for metformin on cancer treatment by modulating epigenetic modifiers.     

Overexpression of MALAT1 contributes to cervical cancer progression by acting as a sponge of miR-429

Cervical cancer remains a malignant type of tumor and is the fourth leading cause of cancer-related death among females. MALAT1 has been identified as a tumor oncogene in various cancers. Our present study aimed to explore the biological role of MALAT1 in cervical cancer. We observed that MALAT1 was significantly upregulated in human cervical cancer cell lines compared with the ectocervical epithelial cells. MALAT1 was repressed by transfection with LV-shMALAT1, whereas increased by LV-MALAT1 in HeLa and Caski cells. Silencing of MALAT1 obviously reduced cervical cell viability, induced cell apoptosis, and repressed cell invasion capacity. Conversely, overexpression of MALAT1 exhibited an opposite phenomenon. Furthermore, miR-429 was predicted as a direct target of MALAT1, and it was dramatically decreased in cervical cancer cells. It has been shown that miR-429 plays a crucial role in cervical cancer progression. In our current study, the targeting correlation between MALAT1 and miR-429 was confirmed by luciferase reporter assays and RIP experiments. Finally, in vivo animal models were established, and we indicated that MALAT1 inhibited cervical cancer progression via targeting miR-429. These findings revealed that MALAT1 can sponge miR-429 and regulate cervical cancer pathogenesis in vivo and in vitro. In conclusion, we indicated that the MALAT1/miR-429 axis was involved in cervical cancer development.     

Fusion of bone marrow-derived cells with renal tubules contributes to renal dysfunction in diabetic nephropathy

Although diabetic nephropathy (DN) is a major cause of end-stage renal disease, the mechanism of dysfunction has not yet been clarified. We previously reported that in diabetes proinsulin-producing bone marrow-derived cells (BMDCs) fuse with hepatocytes and neurons. Fusion cells are polyploidy and produce tumor necrosis factor (TNF)-α, ultimately causing diabetic complications. In this study, we assessed whether the same mechanism is involved in DN. We performed bone marrow transplantation from male GFP-Tg mice to female C57BL/6J mice and produced diabetes by streptozotocin (STZ) or a high-fat diet. In diabetic kidneys, massive infiltration of BMDCs and tubulointerstitial injury were prominent. BMDCs and damaged tubular epithelial cells were positively stained with proinsulin and TNF-α. Cell fusion between BMDCs and renal tubules was confirmed by the presence of Y chromosome. Of tubular epithelial cells, 15.4% contain Y chromosomes in STZ-diabetic mice, 8.6% in HFD-diabetic mice, but only 1.5% in nondiabetic mice. Fusion cells primarily expressed TNF-α and caspase-3 in diabetic kidney. These in vivo findings were confirmed by in vitro coculture experiments between isolated renal tubular cells and BMDCs. It was concluded that cell fusion between BMDCs and renal tubular epithelial cells plays a crucial role in DN.     

TIMP1 contributes to ovarian anomalies in both an MMP-dependent and -independent manner in a rat model

Ovulatory dysfunction occurs in women with endometriosis, yet the mechanisms are unknown. We have shown that endometriotic lesions synthesize and secrete tissue inhibitor of metalloproteinase (TIMP) 1 into the peritoneal cavity in humans and a rat model of endometriosis, where excess TIMP1 localizes in the ovarian theca in endometriosis and modulating peritoneal TIMP1 alters ovarian dynamics. Here, we evaluated whether mechanisms whereby excessive peritoneal fluid TIMP1 negatively impacts ovarian function are matrix metalloproteinase (MMP)-dependent and/or MMP-independent actions. Rats were treated with a mutated TIMP1 without MMP inhibitory function (Ala-TIMP1), wild-type TIMP1 (rTIMP1), or PBS. Rats treated with Ala-TIMP1 or rTIMP1 had fewer antral follicles, fewer new corpora lutea, and the presence of luteinized unruptured follicle syndrome compared with PBS rats. Ala-TIMP1 and rTIMP1 differentially caused downstream changes in gene expression and protein localization related to ovulation, as measured by whole-genome microarray with quantitative real-time PCR validation and immunohistochemistry. More vascular endothelial growth factor and FN were expressed and localized in ovaries of Ala-TIMP1-treated rats compared to rTIMP1- and PBS-treated rats inferring MMP-independent functions. Less caspase 3 localized in ovaries of rTIMP1 compared with the other two groups, and was thus dependent on MMP action. Furthermore, after coimmunoprecipitation, more CD63 was bound to TIMP1 in ovaries of rats treated with Ala-TIMP1 than in rTIMP1-treated rats, providing evidence for another MMP-independent mechanism of ovulatory dysfunction. We predict that MMP-dependent and MMP-independent events are involved in improper fortification of the follicular wall through multiple mechanisms, such as apoptosis inhibition, extracellular matrix components and angiogenesis. Collectively, excessive peritoneal TIMP1 causes changes in ovarian dynamics, both dependently and independently of MMP inhibition.     

Downregulation of lncRNA APCDD1L-AS1 due to DNA hypermethylation and loss of VHL protein expression promotes the progression of clear cell renal cell carcinoma

Background: The current studies only indicated that long non-coding RNA (lncRNA) APCDD1L-AS1, as a novel lncRNA, may play a role in oral squamous cell carcinoma and lung cancer. However, its potential role in clear cell renal cell carcinoma (ccRCC) and its possible mechanism of action remain vague.Methods: TCGA-KIRC and GEO data and qRT-PCR and pyrosequencing results of clinical specimens were used to identify the expression level and DNA methylation status of APCDD1L-AS1. The effects of APCDD1L-AS1 overexpression on ccRCC growth and metastasis were determined by function experiments. Western blot and Tandem mass tags (TMT) were utilized to explore the relationship between APCDD1L-AS1 and VHL expression and its downstream underlying mechanisms.Results: The expression of APCDD1L-AS1 was downregulated in ccRCC. Decreased APCDD1L-AS1 expression was related to higher tumor stage and histological grade and shorter RFS (Relapse-free survival). Besides, APCDD1L-AS1 overexpression restrained the growth and metastasis of ccRCC cells in vitro and in vivo. Moreover, reduced APCDD1L-AS1 expression could be caused by DNA hypermethylation and loss of von Hippel Lindau (VHL) protein expression. Furthermore, the dysregulation of histones expression caused by APCDD1L-AS1 overexpression may be one of the important mechanisms to suppress the progression of ccRCC.Conclusion: APCDD1L-AS1 was able to inhibit the progression of ccRCC, and its decreased expression could be caused by DNA hypermethylation and loss of VHL protein expression. Therefore, APCDD1L-AS1 may serve as a new therapeutic target in the treatment of ccRCC.     

LncRNA MALAT1 targeting miR-124-3p regulates DAPK1 expression contributes to cell apoptosis in Parkinson's Disease

Death associated protein kinase 1 (DAPK1) was initially discovered in the progress of gamma-interferon induced programmed cell death, it is a key factor in the central nervous system, including Parkinson's disease (PD). However, the underlying mechanisms of DAPK1 in PD remain unclear and this research work aims to explore the potential mechanisms of DAPK1 in PD. In the study, we exposed SH-SY5Y cells to MPP⁺ and treated mice with MPTP to investigate the roles of DAPK1 in PD and the underlying mechanisms. The results indicated that the expression of DAPK1 is significantly upregulated and negatively correlated with miR-124-3p levels in SH-SY5Y cells treated by MPP⁺, and miR-124-3p mimics could effectively inhibit DAPK1 expressions and alleviate MPP⁺-induced cell apoptosis. In addition, knockdown MALAT1 reduces the levels of DAPK1 and the ratio of SH-SY5Y cell apoptosis, which is reversed via miR-124-3p inhibitor in vitro. Similarly, knockdown MALAT1 could improve behavioral changes and reduce apoptosis by miR-124-3p upregulation and DAPK1 downregulation in MPTP induced PD mice. Taken together, our data showed that lncRNA MALAT1 positively regulates DAPK1 expression by targeting miR-124-3p, and mediates cell apoptosis and motor disorders in PD. In summary, these results suggest that MALAT1/miR-124-3p /DAPK1 signaling cascade mediates cell apoptosis in vitro and in vivo, which may provide experimental evidence of developing potential therapeutic strategies for PD.     

PDGF-D contributes to neointimal hyperplasia in rat model of vessel injury

In this study, we determined the role of PDGF-D, a new member of the PDGF family, in a rat model of balloon injured artery made with a 2F catheter in Sprague-Dawley male rats. PDGF-D expression was studied in the injured and control segments of abdominal aorta. The function of PDGF-D was evaluated in rat vascular smooth muscle cells stably transfected with PDGF-D gene. We found that in normal abdominal aorta, PDGF-D was highly expressed in adventia, moderate in endothelia, and unidentified in media. Stable transfection of PDGF-D gene into vascular smooth muscle cells increased the cell migration by 2.2-fold, and the proliferation by 2.3-fold, respectively, and MMP-2 production and activity as well. These results support the fact that PDGF-D is involved in the formation of neointimal hyperplasia induced by balloon catheter injury and may serve as a target in preventing vascular restenosis after coronary angioplasty.     

Cardiovascular and renal characteristics, and responses to acute volume expansion of a rat model of diabetic pregnancy

The objective of this study was to characterize the cardiovascular and renal alterations that occur during diabetic pregnancy, and to evaluate the effect of insulin treatment in 12-14 days pregnant diabetic rats. Four groups of female Sprague Dawley rats were studied: virgin control group (NP), pregnant control group (CP), diabetic pregnant group (DP), and diabetic pregnant group with insulin treatment (DPI). Systolic arterial pressure (SAP) was increased on day 12, whereas heart rate (HR) decreased starting with day 3 in DP group of rats. DP rats exhibited marked renal hypertrophy with greater kidney weight (wt) and kidney wt/body wt ratio. Insulin treatment normalized blood glucose (BG) concentration, SAP and HR, and prevented the increase in kidney wt/body wt ratio in DPI rats. At the time of the terminal acute experiment, acute saline volume expansion (VE, 5% body wt/30 min) significantly increased renal interstitial hydrostatic pressure (RIHP), urinary sodium excretion (U(Na)V) and urine flow rate (V) in all groups, but the increases (Delta) were significantly attenuated in both CP (1.7 +/- 0.2mmHg, 12.0 +/- 1.5 microEq.min(-1).g kidney wt(-1) and 76.2 +/- 10.9 microl.min(-1).g kidney wt(-1) for DeltaRIHP, DeltaU(Na)V and DeltaV respectively) and DP (1.3 +/- 0.1 mmHg, 6.8 +/- 1.8 microEq.min(-1).g kidney wt(-1) and 32.3 +/- 9.3 microl.min(-1).g kidney wt(-1) for DeltaRIHP, DeltaU(Na)V and DeltaV respectively) group of rats as compared to NP (4.0 +/- 0.6 mmHg, 21.6 +/- 1.4 microEq.min(-1).g kidney wt(-1)and 136.8 +/- 10.5 microl.min(-1).g kidney wt(-1) for DeltaRIHP, DeltaU(Na)V and DeltaV respectively) group of rats. Although RIHP response to VE was similar in DP and CP group of rats, the natriuretic and diuretic responses to VE were significantly lower in DP as compared to CP group of rats. Insulin treatment had no effect on RIHP response (DeltaRIHP = 1.5 +/- 0.3 mmHg), but restored most of the natriuretic (DeltaU(Na)V = 15.7 +/- 2.9 microEq.min(-1).g kidney wt(-1)) and diuretic (DeltaV = 100.2 +/- 19.3 microl.min(-1).g kidney wt(-1)) responses to VE in DPI as compared with CP group of rats. These data suggest that with VE, the restoration of the increase in U(Na)V and V with insulin treatment in diabetic pregnant rats is not mediated by changes in RIHP.     

An imbalance in autophagy contributes to retinal damage in a rat model of oxygen-induced retinopathy

In retinopathy of prematurity (ROP), the abnormal retinal neovascularization is often accompanied by retinal neuronal dysfunction. Here, a rat model of oxygen-induced retinopathy (OIR), which mimics the ROP disease, was used to investigate changes in the expression of key mediators of autophagy and markers of cell death in the rat retina. In addition, rats were treated from birth to postnatal day 14 and 18 with 3-methyladenine (3-MA), an inhibitor of autophagy. Immunoblot and immunofluorescence analysis demonstrated that autophagic mechanisms are dysregulated in the retina of OIR rats and indicated a possible correlation between autophagy and necroptosis, but not apoptosis. We found that 3-MA acts predominantly by reducing autophagic and necroptotic markers in the OIR retinas, having no effects on apoptotic markers. However, 3-MA does not ameliorate retinal function, which results compromised in this model. Taken together, these results revealed the crucial role of autophagy in retinal cells of OIR rats. Thus, inhibiting autophagy may be viewed as a putative strategy to counteract ROP.     

Downregulation of histone deacetylase 1 by microRNA-520h contributes to the chemotherapeutic effect of doxorubicin

Doxorubicin induces DNA damage to exert its anti-cancer function. Histone deacetylase 1 (HDAC1) can protect the genome from DNA damage. We found that doxorubicin specifically downregulates HDAC1 protein expression and identified HDAC1 as a target of miR-520h, which was upregulated by doxorubicin. Doxorubicin-induced cell death was impaired by exogenous HDAC1 or by miR-520h inhibitor. Moreover, HDAC1 reduced the level of γH2AX by preventing the interaction of doxorubicin with DNA. In summary, doxorubicin downregulates HDAC1 protein expression, by inducing the expression of HDAC1-targeting miR-520h, to exacerbate DNA–doxorubicin interaction. The upregulation of HDAC1 protein may contribute to drug resistance of human cancer cells and targeting HDAC1 is a promising strategy to increase the clinical efficacy of DNA damage-inducing chemotherapeutic drugs.     

A decrease in DKK1, a WNT inhibitor, contributes to placental lipid accumulation in an obesity-prone rat model

Placenta, as the sole transport mechanism between mother and fetus, links the maternal physical state and the immediate as well as lifelong outcomes of the offspring. The present study examined the consequences of maternal obesity on placental lipid accumulation and metabolism. Pregnant obesity-prone (OP) and obesity-resistant (OR) rat strains were fed a control diet throughout gestation. Placentas were collected on Gestational Day 21 for mRNA and oxidative stress analysis, and frozen placental sections were analyzed for fat accumulation as well as beta-catenin and Dickkopf homolog 1 (Xenopus laevis) (DKK1) localization. JEG3 trophoblast cells were cultured in vitro to determine the relationship between DKK1 and lipid accumulation. Maternal plasma and placental nonesterified fatty acids and triglycerides (TG) were elevated in OP dams. Placental Dkk1 mRNA content was 4-fold lower in OP placentas, and a significant increase was noted in beta-catenin accumulation as well as in mRNA content of fat transport and TG synthesis genes, including Ppard (peroxisome proliferator-activated receptor delta), Slc27a1 (fatty acid transport protein 1; also known as Fatp1), Cd36 (cluster of differentiation 36; also known as fatty acid translocation [Fat]), Lipin1, and Lipin3. Significant lipid accumulation was found within the decidual zones in OP, but not OR, placentas, and thickness of the decidual and junctional zones was significantly smaller in OP than in OR placentas. Overexpression of DKK1 in JEG3 cells decreased lipid accumulation and mRNA content of PPARD, SLC27A1, CD36, LIPIN1, and LIPIN3. Our results demonstrate that DKK1 is regulating certain aspects of placental lipid metabolism through the WNT signaling pathway.     

Acetylcholinesterase deficiency contributes to neuromuscular junction dysfunction in type 1 diabetic neuropathy

Diabetic neuropathy is associated with functional and morphological changes of the neuromuscular junction (NMJ) associated with muscle weakness. This study examines the effect of type 1 diabetes on NMJ function. Swiss Webster mice were made diabetic with three interdaily ip injections of streptozotocin (STZ). Mice were severely hyperglycemic within 7 days after the STZ treatment began. Whereas performance of mice on a rotating rod remained normal, the twitch tension response of the isolated extensor digitorum longus to nerve stimulation was reduced significantly at 4 wk after the onset of STZ-induced hyperglycemia. This mechanical alteration was associated with increased amplitude and prolonged duration of miniature end-plate currents (mEPCs). Prolongation of mEPCs was not due to expression of the embryonic acetylcholine receptor but to reduced muscle expression of acetylcholine esterase (AChE). Greater sensitivity of mEPC decay time to the selective butyrylcholinesterase (BChE) inhibitor PEC suggests that muscle attempts to compensate for reduced AChE levels by increasing expression of BChE. These alterations of AChE are attributed to STZ-induced hyperglycemia since similar mEPC prolongation and reduced AChE expression were found for db/db mice. The reduction of muscle end-plate AChE activity early during the onset of STZ-induced hyperglycemia may contribute to endplate pathology and subsequent muscle weakness during diabetes.     

Attenuation of diabetic cardiomyopathy by relying on kirenol to suppress inflammation in a diabetic rat model

Diabetic cardiomyopathy is characterized by diabetes‐induced myocardial abnormalities, accompanied by inflammatory response and alterations in inflammation‐related signalling pathways. Kirenol, isolated from Herba Siegesbeckiae, has potent anti‐inflammatory properties. In this study, we aimed to investigate the cardioprotective effect of kirenol against DCM and underlying the potential mechanisms in a type 2 diabetes mellitus model. Kirenol treatment significantly decreased high glucose‐induced cardiofibroblasts proliferation and increased the cardiomyocytes viability, prevented the loss of mitochondrial membrane potential and further attenuated cardiomyocytes apoptosis, accompanied by a reduction in apoptosis‐related protein expression. Kirenol gavage could affect the expression of pro‐inflammatory cytokines in a dose‐dependent manner but not lower lipid profiles, and only decrease fasting plasma glucose, fasting plasma insulin and mean HbA1c levels in high‐dose kirenol‐treated group at some time‐points. Left ventricular dysfunction, hypertrophy, fibrosis and cell apoptosis, as structural and functional abnormalities, were ameliorated by kirenol administration. Moreover, in diabetic hearts, oral kirenol significantly attenuated activation of mitogen‐activated protein kinase subfamily and nuclear translocation of NF‐κB and Smad2/3 and decreased phosphorylation of IκBα and both fibrosis‐related and apoptosis‐related proteins. In an Electrophoretic mobility shift assay, the binding activities of NF‐κB, Smad3/4, SP1 and AP‐1 in the nucleus of diabetic myocardium were significantly down‐regulated by kirenol treatment. Additionally, high dose significantly enhanced myocardial Akt phosphorylation without intraperitoneal injection of insulin. Kirenol may have potent cardioprotective effects on treating for the established diabetic cardiomyopathy, which involves the inhibition of inflammation and fibrosis‐related signalling pathways and is independent of lowering hyperglycaemia, hyperinsulinemia and lipid profiles.