scholarly journals Loss of Bmyc results in increased apoptosis associated with upregulation of Myc expression in juvenile murine testis

Reproduction ◽  
2012 ◽  
Vol 144 (4) ◽  
pp. 495-503 ◽  
Author(s):  
Heikki T Turunen ◽  
Petra Sipilä ◽  
Leena Strauss ◽  
Ida Björkgren ◽  
Ilpo Huhtaniemi ◽  
...  
Keyword(s):  
Target Genes ◽  
Sperm Concentration ◽  
Seminiferous Tubules ◽  
Spermatogenic Cells ◽  
Histological Changes ◽  
Expression Levels ◽  
Adult Mice ◽  
Gene Expression Levels

Bmyc is a member of the Myc family of transcriptional regulators in the mouse and the rat. It is predominantly expressed in hormonally controlled tissues, with highest level of expression in the epididymis. The BMYC protein has been shown to function as a transcription factor in vitro and to inhibit MYC. To study the significance of BMYC in vivo, a Bmyc knockout (KO) mouse model was generated by homologous recombination. The KO mice were viable and fertile and did not display gross morphological or histological changes compared to the WT mice. However, the testes and the epididymides of the KO mice were smaller than those of the WT mice. Correspondingly, a tendency for a lower sperm concentration in the cauda epididymides of the KO mice was detected. The testosterone produced/testis was significantly reduced, and accordingly, the LH levels were increased in the KO mice. Also, the expression levels of Myc and several of its target genes were elevated in the testes of prepubertal KO mice, whereas no differences in gene expression levels were detected in adult mice. Associated with the increased Myc expression, more apoptotic spermatogenic cells were detected in the seminiferous tubules of the KO mice. In conclusion, our data suggest that Bmyc is a regulator of Myc in vivo and that overexpression of Myc in the developing testis leads to increased apoptosis of spermatogenic cells.

scholarly journals Apoptotic spermatogenic cells can be energy sources for Sertoli cells

Reproduction ◽  
2009 ◽  
Vol 137 (3) ◽  
pp. 469-479 ◽  
Author(s):  
Weipeng Xiong ◽  
Haikun Wang ◽  
Hui Wu ◽  
Yongmei Chen ◽  
Daishu Han
Keyword(s):  
Sertoli Cells ◽  
Energy Sources ◽  
Seminiferous Tubules ◽  
Spermatogenic Cells ◽  
Atp Production ◽  
Lipid Formation ◽  
Residual Bodies ◽  
Induced Apoptosis

Apoptotic spermatogenic cells and residual bodies are phagocytosed and degraded by Sertoli cells during mammalian spermatogenesis. The meaning of this event remains to be clarified. In this report, we demonstrate that apoptotic spermatogenic cells and residual bodies can be used to produce ATP by Sertoli cells after phagocytosis of them. Sertoli cells produced the highest level of ATP compared with other testicular cells. Phagocytosis assayin vitroshowed that engulfment of apoptotic spermatogenic cells increases ATP production by Sertoli cells. The increased ATP production was detected in seminiferous tubules at the stages where phagocytosis occurs. Induced apoptosis of spermatogenic cellsin vivoincreased ATP production in seminiferous tubules. The augmentation of ATP production bothin vitroandin vivoassociated with the lipid formation in Sertoli cells after phagocytosis of apoptotic spermatogenic cells. The lipid β-oxidation was a predominant pathway to produce ATP in Sertoli cells. We conclude that after phagocytosis by Sertoli cells, apoptotic spermatogenic cells are degraded to form lipids that are then used to produce ATP. The results suggest that apoptotic spermatogenic cells can be energy sources for Sertoli cells that may define a novel meaning of spermatogenic cell death.

scholarly journals The LncRNA CASC11 Promotes Colorectal Cancer Cell Proliferation and Migration by Adsorbing miR-646 and miR-381-3p to Upregulate Their Target RAB11FIP2

2021 ◽  
Vol 11 ◽  
Author(s):  
Wei Zhang ◽  
Xiaomin Li ◽  
Wenjuan Zhang ◽  
Yanxia Lu ◽  
Weihao Lin ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Target Genes ◽  
Suppressive Effect ◽  
Luciferase Reporter ◽  
Expression Levels ◽  
Non Coding Rna ◽  
Potential Biomarker ◽  
And Migration

BackgroundWe previously reported that the long non-coding RNA (lncRNA) CASC11 promotes colorectal cancer (CRC) progression as an oncogene by binding to HNRNPK. However, it remains unknown whether CASC11 can act as a competitive endogenous RNA (ceRNA) in CRC. In this study, we focused on the role of CASC11 as a ceRNA in CRC by regulating miR-646 and miR-381-3p targeting of RAB11FIP2.MethodsWe identified the target microRNAs (miRNAs) of CASC11 and the target genes of miR-646 and miR-381-3p using bioinformatic methods. A dual-luciferase reporter assay was performed to validate the target relationship. Quantitative real-time PCR (qRT-PCR), western blotting (WB), and immunohistochemistry (IHC) were used to measure the RNA and protein expression levels. Rescue experiments in vitro and in vivo were performed to investigate the influence of the CASC11/miR-646 and miR-381-3p/RAB11FIP2 axis on CRC progression.ResultsWe found that CASC11 binds to miR-646 and miR-381-3p in the cytoplasm of CRC cells. Moreover, miR-646 and miR-381-3p inhibitors reversed the suppressive effect of CASC11 silencing on CRC growth and metastasis in vitro and in vivo. We further confirmed that RAB11FIP2 is a mutual target of miR-646 and miR-381-3p. The expression levels of CASC11 and RAB11FIP2 in CRC were positively correlated and reciprocally regulated. Further study showed that CASC11 played an important role in regulating PI3K/AKT pathway by miR-646 and miR-381-3p/RAB11FIP2 axis.ConclusionOur study showed that CASC11 promotes the progression of CRC as a ceRNA by sponging miR-646 and miR-381-3p. Thus, CASC11 is a potential biomarker and a therapeutic target of CRC.

scholarly journals Combating antimicrobial resistance using antimicrobial combination therapy and β–lactamase inhibitors

2018 ◽  
Vol 12 (02.1) ◽  
pp. 14S
Author(s):  
Bassam El-Hafi ◽  
Sari Shawki Rasheed ◽  
Noor A Salloum ◽  
Antoine Abou Fayad ◽  
George F Araj ◽  
...  
Keyword(s):  
Gene Expression ◽  
Antimicrobial Resistance ◽  
Combination Therapy ◽  
Bacterial Infections ◽  
Antimicrobial Agents ◽  
Treatment Approaches ◽  
Expression Levels ◽  
Gene Expression Levels

Introduction: The range of antimicrobial agents used to treat bacterial infections is becoming limited with the constant increase in antimicrobial resistance (AMR). Several genetic factors underlie AMR, including β-lactamase-encoding genes such as blaCTXM-15 that confers resistance to third-generation cephalosporins, and blaOXA-48, blaNDM-1, and blaKPC-2 that confer resistance to carbapenems. Remaining treatment approaches for such resistant infections include antimicrobial combination therapy and the use of β-lactamase inhibitors. This study assesses the molecular effects of such treatment approaches on antimicrobial resistant Enterobacteriaceae clinical isolates in vitro and in vivo. Methodology: Nine clinical Enterobacteriaceae isolates were included in the study. One harboring blaCTXM-15, one harboring blaOXA-48, one harboring blaKPC-2, two harboring blaNDM-1 and blaCTXM-15, and four harboring blaOXA-48 and blaCTXM-15. Minimal inhibitory concentrations were determined for carbapenems with β-lactamase inhibitors: avibactam, Ca-EDTA, and relebactam. Synergism between antibiotic combinations was determined by double disc diffusion when using colistin with several antibiotics. In vitro and in vivo gene expression levels were done on these combinations with and without inhibitors. Results: The use of meropenem, imipenem, and ertapenem with the selected β-lactamase inhibitors restored isolate susceptibility in 100%, 87.5%, and 25% of the cases, respectively. Antimicrobial synergism was mostly detected between colistin and meropenem, fosfomycin, or tigecycline. Survival studies revealed the survival of most mice receiving antimicrobial combination therapy with inhibitors as compared to the controls. Overall gene expression levels of resistance genes were variable depending on treatment. Conclusions: The threat of antibiotic resistant bacterial infections remains viable; however, different approaches to therapy are available.

Abstract 253: NOTCH1 Protects Against Atherosclerosis by Repressing Endothelial Activation and Recruitment of Inflammatory Cells

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Anais Briot ◽  
Mete Civelek ◽  
Atsuko Seki ◽  
Sarah Dry ◽  
Michael Fishbein ◽  
...  
Keyword(s):  
Target Genes ◽  
Inflammatory Cells ◽  
Endothelial Activation ◽  
Predisposing Factor ◽  
Rapid Reduction ◽  
Large Arteries ◽  
Protein Levels ◽  
Adult Mice

Maintenance of endothelial homeostasis is critical in the prevention of vascular disorders including atherosclerosis. While we have a good understanding of the mechanisms that promote endothelial activation, less understood are the mechanisms that counterbalance this state. We found that inducible deletion of Notch1 in the endothelium of adult mice led to the accumulation of CD45+ cells in the subendothelial space of large arteries. NOTCH1 is constitutively expressed in adult arteries of mouse and human in vivo but it is excluded from veins. Together these observations led us to speculate that NOTCH1 might actively suppress the acquisition of a pro-inflammatory state in adult endothelium of large arteries. In fact, knockdown of NOTCH1 in human aortic endothelial cells (HAECs) in vitro triggered the expression of inflammatory cytokines in the absence of any other stimuli. Furthermore exposure of HAECs to oxidized phospholipids (OxPAPC) resulted in a rapid reduction of the expression levels of NOTCH1 and several NOTCH1-target genes. Using gene expression microarrays, we found that approximately 25% of the genes differentially expressed after exposure to OxPAPC (by 20%) were similarly regulated (ie either proportionally down or upregulated) when NOTCH1 was knockdown in HAECs, suggesting that these two events might be linked. Among these genes, CXCL1, a molecule previously shown to facilitate monocyte recruitment and binding to the endothelium, was increased in both conditions. Upregulation of CXCL1 upon exposure to OxPAPC was also consistently observed in a cohort of cells isolated from 147 individual donors and suppression of NOTCH1 also led to increase of the chemokine at the mRNA and protein levels. These data indicated that constant NOTCH1 expression was required for a homeostatic anti-inflammatory phenotype in the endothelium. We conclude that NOTCH1 may provide inherent protection to the endothelium during the initial stages of atherosclerosis by repressing the recruitment of inflammatory cells. Thus, decline of endogenous endothelial NOTCH1 levels is likely to constitute a predisposing factor for atherosclerosis.

scholarly journals The efficiency of use lentiviral vectors for the transformation of rooster spermatogenic cells in vivo

2019 ◽  
Vol 14 (2) ◽  
pp. 162-169
Author(s):  
Natalya Alexandrovna Volkova ◽  
Anastasia Nikolaevna Vetokh ◽  
Lyudmila Aleksandrovna Volkova ◽  
Anatolievna Zinovyeva Nataliya
Keyword(s):  
Genetic Transformation ◽  
Reporter Gene ◽  
Germ Cells ◽  
Lentiviral Vectors ◽  
Seminiferous Tubules ◽  
Target Cells ◽  
Spermatogenic Cells ◽  
Viral Preparation

Male gonad cells are considered as promising target cells for the introduction of recombinant DNA within obtaining genetically modified individuals with given characteristics. The use of testicular spermatogonial stem cells is of the greatest interest. In the process of differentiation, this type of cell gives rise to a significant population of mature male germ cells. In the case of their genetic transformation, differentiated cells can be used to inseminate females in order to produce transgenic progeny. The aim of the research was to study the efficiency of using lentiviral vectors for the local transformation of roosters’ testicular spermatogenic cells. We used a lentiviral vector containing the ZsGreen reporter gene under the control of the CMV promoter. In vitro transformation of rooster spermatogenic cells was carried out by infection with a viral preparation, in vivo through multiple injections of the viral preparation into the testicular parenchyma of roosters ( n = 5). The efficiency of transformation was assessed by expression of the reporter ZsGreen gene in transfected spermatogenic cells. The success of using lentiviral vectors for the genetic transformation of rooster spermatogenic cells was shown in experiments in vitro and in vivo . The transformation efficiency of this cells types in an in vitro culture varied from 45 to 57% and averaged 48 ± 4%. The expression of the ZsGreen reporter gene in the cells of the spermatogenic epithelium of the testes was established in almost all experimental roosters in the in vivo experiments. The number of seminiferous tubules with transformed spermatogenic cells varied in the studied experimental roosters from 10 to 22%. The effectiveness of genetic transformation of the testes spermatogenic cells was 1.8 ± 0.2%. The obtained results indicate to the success of using lentiviral vectors for the genetic transformation of spermatogenic cells of rooster testes in vivo in order to create individuals with genetically transformed germ cells for the further production of transgenic offspring with given characteristics.

scholarly journals TRIM8 Inhibits Breast Cancer Proliferation by Regulating Estrogen Signaling

2020 ◽  
Author(s):  
Zelin Tian ◽  
Jianing Tang ◽  
Xing Liao ◽  
Qian Yang ◽  
Yumin Wu ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Target Genes ◽  
Estrogen Signaling ◽  
Endocrine Treatment ◽  
Interaction Domain ◽  
Expression Levels ◽  
Protein Ubiquitination ◽  
Er Positive

Abstract Background: Breast cancer (BC) ranks first in female malignancies wordwide, 70% of which are estrogen receptor alpha (ERα) positive. Endocrine treatment, such as tamoxifen, is primary adjuvant therapy for patients with ER-positive BC, while some of them can develop acquired resistance during long-time treatment. Thus, further research on estrogen signaling is of significance to improve the prognosis of BC patients. Methods: In this study, we report that the E3 ubiquitin ligase TRIM8 acts as a novel regulator of ERα signaling. TRIM8 and ERα target genes expression levels were measured by RT-PCR, while protein expression levels were measured by western blot. CCK8 assay, clone formation, and EDU assay were used to measure cells proliferation. Wound healing assay was used to measure cells migration. Protein stability assay and protein ubiquitination analysis were used to detect ERα protein degradation. Co-immunoprecipitation assay was used to detect the interaction domain between TRIM8 and ERα. Results: TRIM8 is downregulated in BC and is associated with poor prognosis. The protein level of TRIM8 is negatively correlated with ERα. RNA-seq result revealed that estrogen signaling maybe a potential target of TRIM8. Knockdown of TRIM8 can significantly enhance BC cell proliferation and migration in vitro and in vivo. And this effect can be reversed by ERα depletion. Further mechanistic studies have shown that TRIM8 interacts with AF1 domain of ERα via its RING domain in the cytoplasm, and affects poly-ubiquitination of ERα protein. Conclusion: Our study reveals an interesting post-translational mechanism between ERα and TRIM8 in ER-positive BC, TRIM8 may be a potential therapeutic target for BC treatment.

scholarly journals miR-106b regulates the proliferation and differentiation of neural stem/progenitor cells through Tp53inp1-Tp53-Cdkn1a axis

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Xiaohuan Xia ◽  
Hongfang Lu ◽  
Chunhong Li ◽  
Yunlong Huang ◽  
Yi Wang ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Target Genes ◽  
Target Prediction ◽  
Luciferase Assay ◽  
Expression Levels ◽  
Individual Mirnas ◽  
Proliferation And Differentiation ◽  
Neural Stem Progenitor Cells

Abstract Background Recent studies suggested that miR-17~106 family was involved in the regulation of neural stem/progenitor cells (NPCs). However, distinct function of each family member was reported in regulating stem cells within and without the brain. Hence, to investigate the roles of individual miRNAs in miR-17~106 family and mechanisms underlying their effects on neurogenesis is important to extend our understanding in the CNS development. Methods Here, we examined the influence of miR-106a/b on the proliferation, differentiation, and survival of embryonic NPCs using specific mimics and inhibitor. The targets of miR-106a/b were identified from miRNA target prediction database and confirmed by luciferase assay. Specific siRNAs were utilized to erase the effects of miR-106a/b on the expression levels of target genes. Results A positive correlation was observed between the temporal reduction of miR-106a/b expression levels and the decline of NPC pools in vivo and in vitro. The perturbation of miR-106’s function approaches revealed that miR-106b, but not miR-106a, facilitated the maintenance of NPCs and repressed the generation of both neuronal and glial cells, without preference to a particular lineage. No effect was observed for miR-106a/b in NPCs’ survival. The influence of miR-106b on NPCs’ proliferation and differentiation is likely achieved by directly inhibiting the expression of Tp53inp1 and Cdkn1a, key components of Tp53inp1-Tp53-Cdkn1a axis. Conclusion Our study demonstrated a novel axis, miR-106b-Tp53inp1-Tp53-Cdkn1a, in regulating the proliferation and differentiation of NPCs.

scholarly journals The Synergistic Effect of Azoles and Fluoxetine against Resistant Candida albicans Strains Is Attributed to Attenuating Fungal Virulence

2016 ◽  
Vol 60 (10) ◽  
pp. 6179-6188 ◽  
Author(s):  
Wenrui Gu ◽  
Dongmei Guo ◽  
Liuping Zhang ◽  
Dongmei Xu ◽  
Shujuan Sun
Keyword(s):  
Gene Expression ◽  
Candida Albicans ◽  
Synergistic Effects ◽  
Mechanism Study ◽  
Content Type ◽  
Expression Levels ◽  
Time Kill ◽  
Gene Expression Levels

ABSTRACTThis study evaluated the synergistic effects of the selective serotonin reuptake inhibitor, fluoxetine, in combination with azoles againstCandida albicansbothin vitroandin vivoand explored the underlying mechanism. MICs, sessile MICs, and time-kill curves were determined for resistantC. albicans.Galleria mellonellawas used as a nonvertebrate model for determining the efficacy of the drug combinations againstC. albicansin vivo. For the mechanism study, gene expression levels of theSAPgene family were determined by reverse transcription (RT)-PCR, and extracellular phospholipase activities were detectedin vitroby the egg yolk agar method. The combinations resulted in synergistic activity againstC. albicansstrains, but the same effect was not found for the non-albicans Candidastrains. For the biofilms formed over 4, 8, and 12 h, synergism was seen for the combination of fluconazole and fluoxetine. In addition, the time-kill curves confirmed the synergism dynamically. The results of theG. mellonellastudies agreed with thein vitroanalysis. In the mechanism study, we observed that fluconazole plus fluoxetine caused downregulation of the gene expression levels ofSAP1toSAP4and weakened the extracellular phospholipase activities of resistantC. albicans. The combinations of azoles and fluoxetine showed synergistic effects against resistantC. albicansmay diminish the virulence properties ofC. albicans.

scholarly journals Circular RNA circSDHC serves as a sponge for miR-127-3p to promote the proliferation and metastasis of renal cell carcinoma via the CDKN3/E2F1 axis

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Junjie Cen ◽  
Yanping Liang ◽  
Yong Huang ◽  
Yihui Pan ◽  
Guannan Shu ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
Cell Carcinoma ◽  
Renal Cell ◽  
Target Genes ◽  
Expression Patterns ◽  
Luciferase Reporter ◽  
Circular Rnas ◽  
Proliferation And Metastasis

Abstract Background There is increasing evidence that circular RNAs (circRNAs) have significant regulatory roles in cancer development and progression; however, the expression patterns and biological functions of circRNAs in renal cell carcinoma (RCC) remain largely elusive. Method Bioinformatics methods were applied to screen for circRNAs differentially expressed in RCC. Analysis of online circRNAs microarray datasets and our own patient cohort indicated that circSDHC (hsa_circ_0015004) had a potential oncogenic role in RCC. Subsequently, circSDHC expression was measured in RCC tissues and cell lines by qPCR assay, and the prognostic value of circSDHC evaluated. Further, a series of functional in vitro and in vivo experiments were conducted to assess the effects of circSDHC on RCC proliferation and metastasis. RNA pull-down assay, luciferase reporter and fluorescent in situ hybridization assays were used to confirm the interactions between circSDHC, miR-127-3p and its target genes. Results Clinically, high circSDHC expression was correlated with advanced TNM stage and poor survival in patients with RCC. Further, circSDHC promoted tumor cell proliferation and invasion, both in vivo and in vitro. Analysis of the mechanism underlying the effects of circSDHC in RCC demonstrated that it binds competitively to miR-127-3p and prevents its suppression of a downstream gene, CDKN3, and the E2F1 pathway, thereby leading to RCC malignant progression. Furthermore, knockdown of circSDHC caused decreased CDKN3 expression and E2F1 pathway inhibition, which could be rescued by treatment with an miR-127-3p inhibitor. Conclusion Our data indicates, for the first time, an essential role for the circSDHC/miR-127-3p/CDKN3/E2F1 axis in RCC progression. Thus, circSDHC has potential to be a new therapeutic target in patients with RCC.

scholarly journals TMOD-15. EFFICACY OF THE MDM2 INHIBITOR KRT-232 IN GLIOBLASTOMA PATIENT-DERIVED XENOGRAFT MODELS

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii231-ii231
Author(s):  
Rachael Vaubel ◽  
Ann Mladek ◽  
Yu Zhao ◽  
Shiv K Gupta ◽  
Minjee Kim ◽  
...  
Keyword(s):  
Target Genes ◽  
Xenograft Model ◽  
Culture Conditions ◽  
Patient Derived Xenograft ◽  
Fold Induction ◽  
Extended Survival ◽  
Mdm2 Amplification ◽  
Mdm2 Inhibitor

Abstract Non-genotoxic reactivation of p53 by MDM2 inhibitors represents a promising therapeutic strategy for tumors with wild-type TP53, particularly tumors harboring MDM2 amplification. MDM2 controls p53 levels by targeting it for degradation, while disruption of the MDM2-p53 interaction causes rapid accumulation of p53 and activation of the p53 pathway. We examined the efficacy of the small molecule MDM2 inhibitor KRT-232, alone and in combination with radiation therapy (RT), in MDM2-amplified and/or p53 wildtype patient-derived xenograft (PDX) models of glioblastoma in vitro and in vivo. In vitro, glioblastoma PDX explant cultures showed sensitivity to KRT-232, both tumors with MDM2 amplification (GBM108 and G148) and non-amplified but TP53-wildtype lines (GBM10, GBM14, and GBM39), with IC50s ranging from 300-800 nM in FBS culture conditions. A TP53 p.F270C mutant PDX (GBM43) was inherently resistant, with IC50 >3000 nM. In the MDM2-amplified GBM108 line, KRT-232 led to a robust (5-6 fold) induction of p53-target genes p21, PUMA, and NOXA, with initiation of both apoptosis and senescence. Expression of p21 and PUMA was greater with KRT-232 in combination with RT (25-35 fold induction), while stable knock-down of p53 in GBM108 led to complete resistance to KRT-232. In contrast, GBM10 showed lower induction of p21 and PUMA (2-3 fold) and was more resistant to KRT-232. In an orthotopic GBM108 xenograft model, treatment with KRT-232 +/- RT for one week extended survival from 22 days (placebo) to 46 days (KRT-232 alone); combination KRT-232 + RT further extended survival (77 days) over RT alone (31 days). KRT-232 is an effective treatment in a subset of glioblastoma pre-clinical models alone and in combination with RT. Further studies are underway to understand the mechanisms conferring innate sensitivity or resistance to KRT-232.

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