scholarly journals Enhanced at Puberty-1 (Eap1) Expression Critically Regulates the Onset of Puberty Independent of Hypothalamic Kiss1 Expression

2017 ◽  
Vol 43 (4) ◽  
pp. 1402-1412 ◽  
Author(s):  
Chenxi Li ◽  
Pin  Li
Keyword(s):  
Developmental Stages ◽  
Negative Control ◽  
Metastasis Suppressor ◽  
Vaginal Opening ◽  
Early Puberty ◽  
Intronless Gene ◽  
Protein Levels ◽  
Control Shrna ◽  
Old Rats

Background/Aims: Enhance at puberty-1 (Eap1) is an intronless gene that regulates the onset of puberty through a network of hypothalamic genes. However, precise mechanistic events essential for Eap1-regulation of puberty have not been fully elucidated. Eap1 is thought to promote the initiation of puberty through regulation of the hypothalamic metastasis-suppressor KiSS1. We aim to investigate this hypothesis by genetically perturbing Eap1 through RNA interference in vivo. Methods: We first engineered and optimized four sets of shRNAs that target rat Eap1 mRNA as well as one negative control shRNA. After generating lentiviral (LV) particles, we examined the suppression of Eap1 in NRK-54E cell line to select the most efficient one. Sequencelly, LV-Eap1-shRNA or controls including LV-eGFP and saline were intraventricular microinjected into 21-day-old rats. Rats were raised in appropriate conditions and we examined the time of vaginal opening, ovary physiology as well as hypothalamic puberty-regulatory genes at three developmental stages: juvenile (postnatal day PND25), early puberty (PND35), adult (PND42). Results: Hypothalamic suppression of Eap1 delayed the onset of rat vaginal opening. Hematoxylin and eosin (H&E) staining revealed a significant reduction of corpus luteum (CL) at PND35, but at PND42 CL levels were normal relative to control. In conjunction with differences in phenotype and ovary morphology, GnRH expression and transcripts were also reduced at PND25 and PND35, while their levels were similar to control at PND42. KiSS1 mRNA and protein levels were not significantly different at all three developmental stages. Conclusion: Eap1 expression critically regulates puberty as well as GnRH expression. However, Eap1-regulation of puberty may not necessitate KiSS1/GPR54 signaling.

Short Hairpin RNA-Mediated Inhibition of Bcl-2 Gene Increases Susceptibility to Methotrexate and Suppresses Lymphoma Growth In Vitro and In Vivo.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4513-4513
Author(s):  
Baoying Fang ◽  
Dongmei He ◽  
Yuan Zhang
Keyword(s):  
Tumor Growth ◽  
Nude Mice ◽  
Rna Polymerase Iii ◽  
Negative Control ◽  
Morphological Observation ◽  
Hairpin Rna ◽  
Raji Cells ◽  
Control Shrna ◽  
Induced Apoptosis

Abstract A high level of expression of Bcl-2 is associated with resistance to chemotherapeutic agents and radiation in a number of tumor types, so that a drug to reduce the levels of this protein would be expected to promote apoptosis and would therefore be considered a promising chemotherapeutic agent. At present, gene repression can also be achieved in mammalian cells by using vectors to express small hairpin RNA (shRNA) with a U6 or H1 promoter under the direction of RNA polymerase III. In our lab, we have constructed a U6 promoter based vector expressing shRNA structure targeting against Bcl-2, which could effectively down-regulate Bcl-2 protein. We previously demonstrated that this Bcl-2 shRNA decreased cell proliferation and enhanced radiation-induced apoptosis in Raji cells. In this study, we investigated the synergistic effect of Bcl-2 shRNA combined with methotrexate (MTX) in Molt4, Raji cells and nude mice model bearing human lymphoma.Bcl-2 shRNA was transfected into Molt4 cells and Raji cells with Lipofectamine. At 24,48,72,96 hours after transient transfection, the expression levels of Bcl-2 mRNA and protein were detected by RT-PCR and Western blot. The inhibition of cell growth was assessed by MTT assay, counting cell number. Apoptosis was determined by morphological observation and flow cytomertry. To examine the effect of Bcl-2 shRNA on proliferation and chemosensitivity against MTX in vivo, human Raji cell line was inoculated into the skin of BALB/c nude mice to establish lymphoma model. After Molt4 and Raji cells were transfected with Bcl-2 shRNA, protein and mRNA levels of Bcl-2 obviously were decreased. MTT assays indicated that the growth of cells transfected with Bcl-2 shRNA were significantly lower than those cells with negative control shRNA and untransfected cells, respectively (P<0.05). Bcl-2 shRNA combined with MTX significantly inhibited the growth of cells (P<0.05). There was no difference in cell survival between control shRNA / MTX combination and cells treated with MTX alone. Using Giemsa staining, cells treated with Bcl-2 shRNA combined with MTX displayed changes of apoptosis. Apoptotic rates of both Molt4 and Raji cells treated with Bcl-2 shRNA combined with MTX significantly increased (P<0.05), compared with either control shRNA / MTX combination or MTX-treatment cells alone. In the assay of s.c. tumors in nude mice, tumor growth was inhibited in Bcl-2 shRNA group compared with that in negative control shRNA, and immunohistochemistry showed that Bcl-2 protein level of tumor was also decreased. The inhibition rate of tumor growth was significantly higher in tumors treated with Bcl-2 shRNA combined with MTX than in either control shRNA / MTX combination or MTX-treatment group (P<0.05). These results suggest that Bcl-2 shRNA increases MTX-induced apoptosis in Molt4 and Raji cells. Moreover, the combination of Bcl-2 shRNA and MTX produces greater antitumor effect.

scholarly journals Acute Antiapoptotic Effects of Hydrocortisone in the Hippocampus of Neonatal Rats

2013 ◽  
pp. 205-213 ◽  
Author(s):  
P. N. MENSHANOV ◽  
A. V. BANNOVA ◽  
V. V. BULYGINA ◽  
N. N. DYGALO
Keyword(s):  
Glucocorticoid Receptor ◽  
Dna Fragmentation ◽  
Hippocampal Formation ◽  
Hormone Treatment ◽  
Protein Levels ◽  
Apoptotic Protein ◽  
Proapoptotic Protein ◽  
Old Rats ◽  
Developing Rat

Natural glucocorticoid hydrocortisone was suggested as a potent substitution for dexamethasone in the treatment of bronchopulmonary dysplasia in neonates. The aim of this study was to investigate whether hydrocortisone is able to affect the expression of apoptotic genes and the intensity of naturally occurring cell death in the developing rat hippocampus. Hormone treatment decreased procaspase-3 and active caspase-3 levels as well as DNA fragmentation intensity in the hippocampal formation of one-week-old rats in 6 h after injection. These changes were accompanied by an upregulation of antiapoptotic protein Bcl-XL, while expression of proapoptotic protein Bax remained unchanged. The action of hydrocortisone was glucocorticoid receptor-independent, as the selective glucocorticoid receptor agonist dexamethasone did not affect either apoptotic protein levels or DNA fragmentation intensity in the hippocampal region. The data are the first evidences for in vivo antiapoptotic effects of hydrocortisone in the developing hippocampus.

scholarly journals Anthelminthic effects of extracts of indigenous browses from mid rift valley of Ethiopia

2021 ◽  
Vol 25 (2) ◽  
pp. 132-143
Author(s):  
Amsalu Sisay ◽  
Tegene Negesse ◽  
Ajebu Nurfeta
Keyword(s):  
Developmental Stages ◽  
Negative Control ◽  
In Vitro Assays ◽  
Inhibition Assay ◽  
Egg Hatching ◽  
Acacia Senegal ◽  
Egg Hatch ◽  
Infective Larvae

This study was conducted to evaluate the potential anthelminthic properties of extracts of leaves of indigenous browses (Acacia seyal, Acacia senegal, Acacia tortilis, Millettia ferruginea, and Vernonia amygadalina) based on three in vitro assays. Acetone extracts of browses at different concentrations (75 to 1200 μg/ml, for egg and larvae and 100mg/ml for an adult) were tested on three developmental stages of Haemonchus contortus (eggs, infective larvae, and adult worms) using egg hatch assay (EHA), larval migration inhibition assay (LMIA) and adult worm motility inhibition assay (AMIA). Significant effects were obtained with all five browses but differences were observed depending on the parasitic stages. The effects of five browse extracts on egg hatching were concentration-dependent, the highest (P<0.05) egg hatch inhibition rate was observed at 1200 μg/ml concentration for all browses. All extracts had a higher effect (P<0.01) than that of the negative control, phosphate buffer saline (PBS). In contrast, no concentration-response relationship was found for infective larvae and adult worms, although more potent effects were observed with the highest concentrations. The LMI rate (70%) induced by Vernonia  amygadalina extract, at a concentration of 300 μg/ml, was the highest (P<0.05) of all other browses, even at higher concentrations. The highest LMI rate (62%) induced by Acacia senegal extract at higher concentration, was lower than that of LMI rate (70%) induced by Vernonia amygadalina, at 300 μg/ml concentration. Vernonia amygadalina was found to be highly and rapidly effective against adult worms inducing the highest mortality rate (90%) as soon as 4 hrs after incubation. Overall, the in vitro results suggest that these five  browses do possess anti-parasitic properties and Vernonia amygadalina showed the most effective anti-parasitic property. These effects remain to be confirmed through in vivo study.

scholarly journals EP1 receptor is involved in prostaglandin E2-induced osteosarcoma growth

2019 ◽  
Author(s):  
Jing-cai Niu ◽  
Nan Ma ◽  
Wei Liu ◽  
Pei-ji Wang
Keyword(s):  
Western Blot ◽  
Mg63 Cell ◽  
Negative Control ◽  
Migration And Invasion ◽  
Enzyme Activity Assay ◽  
Protein Levels ◽  
Mg63 Cells ◽  
Ep1 Receptor

Recent studies showed that the activation of prostaglandin (PG) receptor EP1 promotes cell migration and invasion in different cancers. The aim of this study was to investigate the role of EP1 in the proliferation of osteosarcoma (OS) cells in vitro and in vivo. EP1 mRNA and protein levels were analyzed by real-time RT-PCR and Western blot, respectively in human OS cell lines MG63, OS732, U-2OS, HOS and SAOS-2 compared to human fetal osteoblastic hFOB 1.19 cells. MG63 cells were treated with PGE2, EP1 specific agonist 17-PT-PGE2, 17-PT-PGE2 + EP1 specific antagonist SC51089, or DMSO (control). EP1R-siRNA or a non-silencing irrelevant RNA duplex (negative control) were used for the transfection of MG63 cells, followed by PGE2 treatment. Nude mice carrying MG63 xenografts were treated with SC51089 (2 mg/kg/day). MG63 cells/xenografts were analyzed by MTT assay, TUNEL assay, PKC enzyme activity assay, and Western blot (EP1 and apoptotic proteins), and tumor growth/volume was evaluated in mice. EP1 levels were significantly higher in OS cells compared to osteoblasts. PGE2 or 17-PT-PGE2 treatment increased the proliferation and decreased the apoptosis of MG63 cells. Inhibition of EP1 by SC51089 or siRNA markedly decreased the viability of MG63 cells. Similarly, SC51089 treatment significantly inhibited MG63 cell proliferation and promoted apoptosis in vivo. The silencing of EP1 receptor by siRNA or blockade of EP1 signaling by SC51089 activated extrinsic and intrinsic apoptotic pathways both in vivo and in vitro, as evidenced by increased levels of Bax, cyt-c, cleaved caspase-3, caspase-8 and caspase-9. EP1 appears to be involved in PGE2-induced proliferative activity of MG63 cells. Antagonizing EP1 may provide a novel therapeutic approach to the treatment of OS.

scholarly journals Downregulation of TTF1 in the rat hypothalamic ARC or AVPV nucleus inhibits Kiss1 and GnRH expression, leading to puberty delay

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Shaolian Zang ◽  
Xiaoqin Yin ◽  
Pin Li
Keyword(s):  
Pubertal Development ◽  
Gonad Development ◽  
Gene Promoter ◽  
Female Rats ◽  
Luciferase Reporter ◽  
Vaginal Opening ◽  
Early Puberty ◽  
Puberty Onset

Abstract Background TTF1 is a transcription factor that is expressed in the hypothalamus after birth and plays crucial roles in pubertal development. TTF1 may regulate the expression of the Kiss1 gene, which may drive puberty onset in the hypothalamic arcuate (ARC) and anterior ventral paraventricular (AVPV) nuclei. Methods A dual-luciferase reporter assay was used to detect binding between TTF1 and the Kiss1 gene promoter. To investigate the effects of TTF1, we modified TTF1 expression in cell lines and in the ARC or AVPV nucleus of 21-day-old female rats via lentivirus infection. TTF1 and other puberty onset-related genes were detected by qRT-PCR and western blot analyses. Results The in vitro data indicated that TTF1 knockdown (KD) significantly reduced Kiss1 and GnRH expression. Overexpression (OE) of TTF1 promoted Kiss1 expression. In vivo, the expression of Kiss1 and GnRH decreased significantly in the rats with hypothalamic ARC- or AVPV-specific TTF1 KD. The TTF1-KD rats showed vaginal opening delay. H&E staining revealed that the corpus luteum was obviously reduced at the early puberty and adult stages in the rats with ARC- or AVPV-specific TTF1 KD. Conclusion TTF1 bound to the promoter of the Kiss1 gene and enhanced its expression. For 21-day-old female rats, decreased TTF1 in the hypothalamic ARC or AVPV nucleus resulted in delayed vaginal opening and ovarian abnormalities. These observations suggested that TTF1 regulates puberty onset by promoting the expression of Kiss1 and plays an important role in gonad development.

scholarly journals Grid1 Regulates the Onset of Puberty in Female Rats

2020 ◽  
Author(s):  
Jing Ye ◽  
Ping Qin ◽  
Hailing Li ◽  
Tiezhu Kang ◽  
Wenyu Si ◽  
...  
Keyword(s):  
Arcuate Nucleus ◽  
Female Rats ◽  
Mrna Levels ◽  
Vaginal Opening ◽  
Ionotropic Receptor ◽  
Delta Type ◽  
Periventricular Nucleus ◽  
Old Rats ◽  
Puberty Onset

Abstract The present study aimed to investigate whether Grid1, encoding the glutamate ionotropic receptor delta type subunit 1(GluD1), influences the onset of puberty in female rats. First, we detected the expression of Grid1 mRNA and its protein in the hypothalamus from infancy to puberty. Second, we evaluated the suppression of Grid1 expression by Lentivirus-Grid1 (LV-Grid1) in primary hypothalamus cells through measuring the expression level of Grid1. Finally, LV-Grid1 was intracerebroventricular injected (ICV) into 21-day-old rats and to investigate the effect of Grid1 suppression on puberty onset in vivo. Results showed that GluD1 immunoreactivity could be detected in the arcuate nucleus (ARC), paraventricular nucleus (PVN), and periventricular nucleus (PeN). Grid1 mRNA levels were the lowest at prepuberty. Treatment of hypothalamic neurons with LV-Grid1 decreased the mRNA expression levels of Grid1 and Rfrp-3 (encoding RFamide-related peptide 3, RFRP 3), but increased that of Gnrh (encoding gonadotropin-releasing hormone, GnRH). After 7 days of ICV LV-Grid1 into rats, the Grid1 mRNA was significantly reduced (by 46%), Gnrh mRNA expression was significantly increased, but Rfrp-3 mRNA levels were decreased. The time of rat vaginal opening (VO) was earlier in the LV-Grid1 group; the concentrations of luteinizing hormone (LH), estradiol (E2), and progesterone (P4) in serum were significantly increased; and the ovaries were significantly larger. Our study revealed that Grid1 affects the onset of puberty by regulating the level of GnRH and RFRP3.

The Natural Flavonoid Naringenin Inhibits the Cell Growth of WilmsTumorinChildren by Suppressing TLR4/NF-κB Signaling

2020 ◽  
Vol 20 ◽  
Author(s):  
Hongtao Li ◽  
Peng Chen ◽  
Lei Chen ◽  
Xinning Wang
Keyword(s):  
Cell Growth ◽  
Western Blot ◽  
Wilms Tumor ◽  
Critical Role ◽  
Time Dependent ◽  
Luciferase Assay ◽  
Dependent Manner ◽  
Tlr4 Expression ◽  
Protein Levels

Background: Nuclear factor kappa B (NF-κB) is usually activated in Wilms tumor (WT) cells and plays a critical role in WT development. Objective: The study purpose was to screen a NF-κB inhibitor from natural product library and explore its effects on WT development. Methods: Luciferase assay was employed to assess the effects of natural chemical son NF-κB activity. CCK-8 assay was conducted to assess cell growth in response to naringenin. WT xenograft model was established to analyze the effect of naringenin in vivo. Quantitative real-time PCR and Western blot were performed to examine the mRNA and protein levels of relative genes, respectively. Results: Naringenin displayed significant inhibitory effect on NF-κB activation in SK-NEP-1 cells. In SK-NEP-1 and G-401 cells, naringenin inhibited p65 phosphorylation. Moreover, naringenin suppressed TNF-α-induced p65 phosphorylation in WT cells. Naringenin inhibited TLR4 expression at both mRNA and protein levels in WT cells. CCK-8 staining showed that naringenin inhibited cell growth of the two above WT cells in dose-and time-dependent manner, whereas Toll-like receptor 4 (TLR4) over expression partially reversed the above phenomena. Besides, naringenin suppressed WT tumor growth in dose-and time-dependent manner in vivo. Western blot found that naringenin inhibited TLR4 expression and p65 phosphorylation in WT xenograft tumors. Conclusion: Naringenin inhibits WT development viasuppressing TLR4/NF-κB signaling

scholarly journals All-Trans Retinoic Acid Increases DRP1 Levels and Promotes Mitochondrial Fission

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1202
Author(s):  
Bojjibabu Chidipi ◽  
Syed Islamuddin Shah ◽  
Michelle Reiser ◽  
Manasa Kanithi ◽  
Amanda Garces ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Mitochondrial Fission ◽  
Normal Function ◽  
Mitochondrial Network ◽  
Protein Levels ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Area And Perimeter

In the heart, mitochondrial homeostasis is critical for sustaining normal function and optimal responses to metabolic and environmental stressors. Mitochondrial fusion and fission are thought to be necessary for maintaining a robust population of mitochondria, and disruptions in mitochondrial fission and/or fusion can lead to cellular dysfunction. The dynamin-related protein (DRP1) is an important mediator of mitochondrial fission. In this study, we investigated the direct effects of the micronutrient retinoid all-trans retinoic acid (ATRA) on the mitochondrial structure in vivo and in vitro using Western blot, confocal, and transmission electron microscopy, as well as mitochondrial network quantification using stochastic modeling. Our results showed that ATRA increases DRP1 protein levels, increases the localization of DRP1 to mitochondria in isolated mitochondrial preparations. Our results also suggested that ATRA remodels the mitochondrial ultrastructure where the mitochondrial area and perimeter were decreased and the circularity was increased. Microscopically, mitochondrial network remodeling is driven by an increased rate of fission over fusion events in ATRA, as suggested by our numerical modeling. In conclusion, ATRA results in a pharmacologically mediated increase in the DRP1 protein. It also results in the modulation of cardiac mitochondria by promoting fission events, altering the mitochondrial network, and modifying the ultrastructure of mitochondria in the heart.

scholarly journals TRIM27 interacts with Iκbα to promote the growth of human renal cancer cells through regulating the NF-κB pathway

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Chengwu Xiao ◽  
Wei Zhang ◽  
Meimian Hua ◽  
Huan Chen ◽  
Bin Yang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Prognostic Indicator ◽  
The Cancer Genome Atlas ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Protein Levels ◽  
Kaplan Meier ◽  
Cancer Genome Atlas

Abstract Background The tripartite motif (TRIM) family proteins exhibit oncogenic roles in various cancers. The roles of TRIM27, a member of the TRIM super family, in renal cell carcinoma (RCC) remained unexplored. In the current study, we aimed to investigate the clinical impact and roles of TRIM27 in the development of RCC. Methods The mRNA levels of TRIM27 and Kaplan–Meier survival of RCC were analyzed from The Cancer Genome Atlas database. Real-time PCR and Western blotting were used to measure the mRNA and protein levels of TRIM27 both in vivo and in vitro. siRNA and TRIM27 were exogenously overexpressed in RCC cell lines to manipulate TRIM27 expression. Results We discovered that TRIM27 was elevated in RCC patients, and the expression of TRIM27 was closely correlated with poor prognosis. The loss of function and gain of function results illustrated that TRIM27 promotes cell proliferation and inhibits apoptosis in RCC cell lines. Furthermore, TRIM27 expression was positively associated with NF-κB expression in patients with RCC. Blocking the activity of NF-κB attenuated the TRIM27-mediated enhancement of proliferation and inhibition of apoptosis. TRIM27 directly interacted with Iκbα, an inhibitor of NF-κB, to promote its ubiquitination, and the inhibitory effects of TRIM27 on Iκbα led to NF-κB activation. Conclusions Our results suggest that TRIM27 exhibits an oncogenic role in RCC by regulating NF-κB signaling. TRIM27 serves as a specific prognostic indicator for RCC, and strategies targeting the suppression of TRIM27 function may shed light on future therapeutic approaches.

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