Identification and characterization of CircRNAs involved in the regulation of wheat root length

BACKGROUND: Recent studies indicate that circular RNAs (circRNAs) may play important roles in the regulation of plant growth and development. Plant roots are the main organs of nutrient and water uptake. However, whether circRNAs involved in the regulation of plant root growth remains to be elucidated. METHODS: LH9, XN979 and YN29 are three Chinese wheat varieties with contrasting root lengths. Here, the root circRNA expression profiles of LH9, XN979 and YN29 were examined by using high-throughput sequencing technology. RESULTS: Thirty-three and twenty-two differentially expressed circRNAs (DECs) were identified in the YN29-LH9 comparison and YN29-XN979 comparison, respectively. Among them, ten DECs coexisted in both comparisons. As the roots of both LH9 and XN979 were significantly larger and deeper than YN29, the ten DECs coexisting in the two comparisons were highly likely to be involved in the regulation of wheat root length. Moreover, three of the ten DECs have potential miRNA binding sites. Real-time PCR analysis showed that the expression levels of the potential binding miRNAs exhibited significant differences between the long root plants and the short root plants. CONCLUSIONS: The expression levels of some circRNAs exhibited significant differences in wheat varieties with contrasting root phenotypes. Ten DECs involved in the regulation of wheat root length were successfully identified in which three of them have potential miRNAs binding sites. The expression levels of putative circRNA-binding miRNAs were correlated with their corresponding circRNAs. Our results provide new clues for studying the potential roles of circRNAs in the regulation of wheat root length.

PELI3 mediates pro-tumor actions of down-regulated miR-365a-5p in non-small cell lung cancer

BACKGROUND: To analyze the relative expression of PELI3 and its mechanistic involvement in the non-small cell lung cancer (NSCLC). Methods: PELI3 expression in NSCLC tissue samples was determined by the immunohistochemistry. The transcripts abundance of PELI3 was measured with real-time PCR. The protein intensity was analyzed by western blot. The overall survival in respect to PELI3 or miR-365a-5p expression was plotted by the Kaplan-Meier's analysis. Cell growth was determined by colony formation assay. Cell viability was measured by MTT assay. The migration and invasion were evaluated by wound healing and transwell assay respectively. The regulatory effect of miR-365a-5p on PELI3 was interrogated with luciferase reporter assay. The direct binding between miR-365a-5p and PELI3 was analyzed by pulldown assay. RESULTS: PELI3 was aberrantly up-regulated in NSCLC both in vivo and in vitro. High level of PELI3 associated with poor prognosis. PELI3-deficiency significantly inhibited cell viability, colony formation, migration and invasion. We further identified that miR-365a-5p negatively regulated PELI3 in this disease. Ectopic expression of miR-365a-5p in both A549 and H1299 phenocopied PELI3-deficiency. Meanwhile, PELI3-silencing significantly abolished the pro-tumoral effect elicited by miR-365a-5p inhibition. CONCLUSIONS: Our results highlighted the importance of dysregulated miR-365a-5p-PELI3 signaling axis in NSCLC.

The role of ischemic preconditioning in gene expression related to inflammation in a rat model of intestinal ischemia-reperfusion injury

Abstract Purpose: To investigate the gene expression related to inflammation on mice subjected to intestinal ischemia and reperfusion (I/R) and treated with ischemic preconditioning (IPC). Methods: Thirty rats (EPM-Wistar), distributed in five groups of six animals each, were underwent anesthesia and laparotomy. The ischemia time was standardized in 60 minutes and the reperfusion time 120 minutes. IPC was standardized in 5 minutes of ischemia followed by 10 minutes of reperfusion accomplished before I/R. The control group was submitted only to anesthesia and laparotomy. The other groups were submitted to ischemia, I/R, ischemia + IPC and I/R + IPC. It was collected a small intestine sample to analyses by Quantitative Polymerase Chain Reaction in real Time (RT-qPCR) and histological analyses. It was studied 27 genes. Results: The groups that received IPC presented downregulation of genes, observed in of genes in IPC+ischemia group and IPC+I/R group. Data analysis by clusters showed upregulation in I/R group, however in IPC groups occurred downregulation of genes related to inflammation. Conclusion: The ischemia/reperfusion promoted upregulation of genes related to inflammation, while ischemic preconditioning promoted downregulation of these genes.

Down-regulated miR-448 relieves spinal cord ischemia/reperfusion injury by up-regulating SIRT1

MicroRNAs play a crucial role in the progression of spinal cord ischemia/reperfusion injury (SCII). The role of miR-448 and SIRT1 in SCII was investigated in this study, to provide further insights into prevention and improvement of this disorder. In this study, expressions of miR-448 and SIRT1 protein were determined by qRT-PCR and western blot, respectively. Flow cytometry was used to analyze cell apoptosis. The endogenous expression of genes was modulated by recombinant plasmids and cell transfection. Dual-luciferase reporter assay was performed to determine the interaction between miR-448 and SIRT1. The Basso, Beattie, and Bresnahan score was used to measure the hind-limb function of rat. The spinal cord ischemia reperfusion injury model of adult rats was developed by abdominal aorta clamping, and the nerve function evaluation was completed by motor deficit index score. In SCII tissues and cells treated with hypoxia, miR-448 was up-regulated while SIRT1 was down-regulated. Hypoxia treatment reduced the expression of SIRT1 through up-regulating miR-448 in nerve cells. Up-regulation of miR-448 induced by hypoxia promoted apoptosis of nerve cells through down-regulating SIRT1. Down-regulated miR-448 improved neurological function and hind-limb motor function of rats with SCII by up-regulating SIRT1. Down-regulated miR-448 inhibited apoptosis of nerve cells and improved neurological function by up-regulating SIRT1, which contributes to relieving SCII.

Downregulation of TGF-ß1 suppressed proliferation and increased chemosensitivity of ovarian cancer cells by promoting BRCA1/Smad3 signaling

BACKGROUND: Studies have demonstrated that transforming growth factor beta-1 (TGF-ß1) exhibits oncogenic activity in different types of cancer, including ovarian cancer (OC). However, its regulatory mechanism in OC and whether TGF-ß1 is involved in chemosensitivity regulation remains unclear. Thus, the aim of this study was to investigate the role of TGF-ß1 in OC. METHODS: The OC cell line SKOV3 was employed, and TGF-ß1 overexpression or knockdown vectors were constructed. The cell proliferation of SKOV3 was evaluated with the cell counting kit (CCK8) kit after treatment with different concentrations of cis-platinum. Western blot and protein immunoprecipitation were employed to detect changes in BRCA1 and Smad3 expression and their interactions. Tumor growth in nude mice was evaluated. RESULTS: The results showed that TGF-ß1 knockdown increased chemosensitivity by promoting BRCA1 expression and Smad3 phosphorylation. In vivo studies showed that TGF-ß1 knockdown significantly inhibited the growth of tumors, also by upregulating BRCA1 expression and Smad3 phosphorylation. CONCLUSION: Taken together, our results suggest that TGF-ß1 knockdown inhibits tumor growth and increases chemosensitivity by promotion of BRCA1/Smad3 signaling.

Effect of CRABP2 on the proliferation and odontoblastic differentiation of hDPSCs

Abstract: Cellular retinoic acid-binding protein 2 (CRABP2) has been detected in several organs during embryonic development. Recent studies have demonstrated that CRABP2 plays important roles in the retinoic acid, β-catenin and Notch signaling pathways, as well as in the interaction between epithelial and mesenchymal cells, which are important for human dental pulp stem cells (hDPSCs) and tooth development. In the present study, the expression of CRABP2 during mouse molar development and the role of CRABP2 in hDPSC odontoblastic differentiation were evaluated. CRABP2 was gradually decreased during the development of the first maxillary molar, which exhibited the same trend as the expression of CRABP2 during the odontoblastic induction of hDPSCs. CRABP2 knockdown inhibited the proliferative ability of hDPSCs, while it enhanced odontoblastic differentiation via promoting mineralization nodule formation and upregulating the activity of alkaline phosphatase and the expression of mineralization-related genes. The present study uncovered a novel function of CRABP2 in hDPSCs. Our data suggest that CRABP2 may act as a regulator during the proliferation and differentiation of hDPSCs.

El uso de placebo en ensayos clínicos de fase III en Brasil / The use of placebos in phase III clinical trials in Brazil

El Consejo Federal de Medicina de Brasil (CFM) -órgano normativo y fiscalizador del ejercicio ético de la medicina- prohibió, en 2008, la participación de médicos brasileños en investigaciones que utilizaran placebo para enfermedades con tratamiento eficaz y efectivo, en contraposición a la Declaración de Helsinki, que permite su uso en condiciones metodológicamente justificadas. Con el objetivo de verificar si la normativa ética del CFM modificó el uso de placebo en ensayos clínicos de fase III en Brasil, se analizaron varias características de sus registros en el ClinicalTrials.gov, en los períodos de 2003 a 2007 y de 2009 a 2013. Se concluye que: a) la normativa promulgada por el CFM en 2008 fue ineficaz y prevaleció la posición adoptada por la Declaración de Helsinki; b) el patrocinio de ensayos con placebo por parte de la industria farmacéutica multinacional fue significativo; c) predominaron las investigaciones de fármacos para enfermedades crónicas, y fueron poco significativas para las enfermedades postergadas, de importancia para Brasil.

In 2008, Brazil's Federal Council of Medicine [Conselho Federal de Medicina] (CFM) - regulatory and supervisory agency on the ethical practice of medicine - banned the participation of Brazilian doctors in studies using placebos for diseases with efficient and effective treatment. This position differs with the Helsinki Declaration, which allows the use of placebos in methodologically justified conditions. To ascertain whether the CMF's ethical regulation modified the use of placebos in phase III clinical trials in Brazil, characteristics of the records in ClinicalTrials.gov were researched in the periods from 2003 to 2007 and from 2009 to 2013. The conclusions reached were: a) the regulations issued by the CFM in 2008 were ineffective and the position adopted by the Helsinki Declaration prevails; b) there was significant sponsorship by the multinational pharmaceutical industry of trials with placebos; c) the research was predominantly on new drugs for chronic diseases, with little study done of the neglected diseases which are of great importance to Brazil.

A DNA enzyme targeting Egr-1 inhibits rat vascular smooth muscle cell proliferation by down-regulation of cyclin D1 and TGF-â1

We have demonstrated that a synthetic DNA enzyme targeting early growth response factor-1 (Egr-1) can inhibit neointimal hyperplasia following vascular injury. However, the detailed mechanism of this inhibition is not known. Thus, the objective of the present study was to further investigate potential inhibitory mechanisms. Catalytic DNA (ED5) and scrambled control DNA enzyme (ED5SCR) were synthesized and transfected into primary cultures of rat vascular smooth muscle cells (VSMCs). VSMC proliferation and DNA synthesis were analyzed by the MTT method and BrdU staining, respectively. Egr-1, TGF-â1, p53, p21, Bax, and cyclin D1 expression was detected by RT-PCR and Western blot. Apoptosis and cell cycle assays were performed by FACS. Green fluorescence could be seen localized in the cytoplasm of 70.6 ± 1.52 and 72 ± 2.73 percent VSMCs 24 h after transfection of FITC-labeled ED5 and ED5SCR, respectively. We found that transfection with ED5 significantly inhibited cultured VSMC proliferation in vitro after 24, 48, and 72 h of serum stimulation, and also effectively decreased the uptake of BrdU by VSMC. ED5 specifically reduced serum-induced Egr-1 expression in VSMCs, further down-regulated the expression of cyclin D1 and TGF-â1, and arrested the cells at G0/G1, inhibiting entry into the S phase. FACS analysis indicated that there was no significant difference in the rate of apoptosis between ED5- and ED5SCR-transfected cells. Thus, ED5 can specifically inhibit Egr-1 expression, and probably inhibits VSMC proliferation by down-regulating the expressions of cyclin D1 and TGF-â1. However, ED5 has no effect on VSMC apoptosis.

Effect of the human follicle-stimulating hormone-binding inhibitor and its N-terminal fragment on follicle-stimulating hormone-induced progesterone secretion by granulosa cells in vitro.

Intrafollicular factors play an important role in folliculogenesis. The follicle-stimulating hormone (FSH)-binding inhibitor (FSHBI), purified by our laboratory from human ovarian follicular fluid, has been shown to suppress ovulation and induce follicular atresia/apoptosis in mice as well as impair fertility in marmosets, the New World monkeys. The octapeptide, a peptide corresponding to the N-terminal region of human FSHBI (hFSHBI), has been synthesized and also shows FSHBI activity in vitro. In the present study, we have attempted to identify the mechanism of action of the peptide in granulosa cell cultures. Rat granulosa cell cultures were treated with varying concentrations of the octapeptide or partially purified hFSHBI (gel chromatography fraction hGF 2) in the presence or absence of human FSH (hFSH) and the amount of progesterone (P;4) secreted in the culture supernatants after 3 h/48 h was estimated. Both hGF2 and the octapeptide failed to alter basal levels as well as 8-bromo cAMP-induced P;4 production, while FSH-induced P 4 secretion was inhibited in a dose-dependent manner. These studies reveal that the octapeptide, a fragment of FSHBI, and the native protein have similar activity in vitro and both compounds alter FSH action at the receptor level upstream of cAMP formation.

Down-regulation of the cardiac sarcoplasmic reticulum ryanodine channel in severely food-restricted rats

We have shown that myocardial dysfunction induced by food restriction is related to calcium handling. Although cardiac function is depressed in food-restricted animals, there is limited information about the molecular mechanisms that lead to this abnormality. The present study evaluated the effects of food restriction on calcium cycling, focusing on sarcoplasmic Ca2+-ATPase (SERCA2), phospholamban (PLB), and ryanodine channel (RYR2) mRNA expressions in rat myocardium. Male Wistar-Kyoto rats, 60 days old, were submitted to ad libitum feeding (control rats) or 50 percent diet restriction for 90 days. The levels of left ventricle SERCA2, PLB, and RYR2 were measured using semi-quantitative RT-PCR. Body and ventricular weights were reduced in 50 percent food-restricted animals. RYR2 mRNA was significantly decreased in the left ventricle of the food-restricted group (control = 5.92 ± 0.48 vs food-restricted group = 4.84 ± 0.33, P < 0.01). The levels of SERCA2 and PLB mRNA were similar between groups (control = 8.38 ± 0.44 vs food-restricted group = 7.96 ± 0.45, and control = 1.52 ± 0.06 vs food-restricted group = 1.53 ± 0.10, respectively). Down-regulation of RYR2 mRNA expressions suggests that chronic food restriction promotes abnormalities in sarcoplasmic reticulum Ca2+ release.

Adrenalectomy Abolishes Fasting-induced Down-regulation of NADPH-diaphorase in the Rat Paraventricular Nucleus

This study was conducted to define the molecular mechanism of fasting-induced down-regulation of neuronal nitric oxide synthase (nNOS) expression in the hypothalamic paraventricular nucleus (PVN). Rats were adrenalectomized (ADX), and then either underwent food deprivation or received varying doses of dexamethasone for 48 h. The brain tissues were processed for NADPH-diaphorase (NADPH-d) staining, a histochemical marker of nNOS enzyme activity. Both the ADX and the sham operated rats showed a significant weight loss after 48 h of food deprivation. Food deprivation decreased the number of NADPH-d containing cells in the PVN of sham rats, however, not in the ADX rats. Dexamethasone dose- dependently decreased NADPH-d cells in the PVN of ADX rats. The effect of ADX or dexamethasone was limited to the parvocellular subdivision of PVN. These results suggest that the adrenal glucocorticoids may down-regulate nNOS expression in the PVN during food deprivation.

The Levels of MDM2 Protein Are Decreased by a Proteasome-Mediated Proteolysis Prior to Caspase-3-Dependent pRb and PARP Cleavages

MDM2 is a substrate of caspase-3 in p53-mediated apoptosis. In addition, MDM2 mediates its own ubiquitination in a RING finger-dependent manner. Thus, we investigated whether MDM2 is degraded through a ubiquitin-dependent proteasome pathway in the absence of p53. When HL-60 cells, p53 null, were treated with etoposide, MDM2 was markedly decreased prior to caspase-3-dependent retinoblastoma tumor suppressor protein (pRb) and poly (ADP- ribose) polymerase (PARP) cleavages. Moreover, down-regulation of MDM2 level was not coupled with its mRNA down-regulation. However, the level of MDM2 was partially restored by proteasome inhibitors such as LLnL and lactacystin, even in the presence of etoposide. Our results suggest that, in the p53 null status, MDM2 protein level is decreased by proteasome-mediated proteolysis prior to caspase-3-dependent PARP and pRb cleavages.