scholarly journals Identification and Functional Verification Reveals that miR-195 Inhibiting THRSP to Affect Fat Deposition in Xinyang Buffalo

2021 ◽  
Vol 12 ◽  
Author(s):  
Shuzhe Wang ◽  
Cuili Pan ◽  
Xiaojie Ma ◽  
Chaoyun Yang ◽  
Lin Tang ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Meat Quality ◽  
Lipid Accumulation ◽  
Developmental Stages ◽  
Mature Mirnas ◽  
Fat Deposition ◽  
Luciferase Reporter ◽  
Functional Verification ◽  
Luciferase Reporter Gene ◽  
Expression Of Genes

The buffalo population is extensive in China, but its meat quality is relatively inferior. Therefore, improving meat quality should be one of the breeding goals. microRNAs (miRNAs) play an essential regulatory role in the post-transcriptional expression of genes. Some studies have reported their function regulating genes related to fat deposition and adipocyte differentiation in cattle, but there is limited reports in buffalo. We performed small RNA transcriptome sequencing of Xinyang buffalo adipose tissue between calves and adults in this study. As a result, 282 mature miRNAs were significantly differentially expressed, and co-expression analysis showed that 454 miRNAs were significantly associated with developmental stages. Target gene identification, GO (gene ontology) annotation, and KEGG analysis of miRNAs showed that miR-195, miR-192, and miR-24-3p could target key genes for lipogenesis and thus regulate adipose deposition and differentiation. Among them, miR-195 was significantly upregulated in adipose tissue and induced adipocytes of adult buffaloes, and its overexpression significantly inhibited lipid accumulation in primary adipocytes. Dual-luciferase reporter gene analysis showed that miR-195 reduced the expression of thyroid hormone response protein (THRSP) by targeting its 3′ untranslated terminal region, suggesting that miR-195 may inhibit lipid accumulation in adipocytes by regulating THRSP. The results confirmed the reliability of predictive screening of miRNAs and provided theoretical support for buffalo fattening.

scholarly journals Comprehensive evaluation of the metabolic effects of porcine CRTC3 overexpression on subcutaneous adipocytes with metabolomic and transcriptomic analyses

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jiaqi Liu ◽  
Jie Li ◽  
Wentao Chen ◽  
Xintao Xie ◽  
Xingang Chu ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Lipid Accumulation ◽  
Comprehensive Evaluation ◽  
Adenosine Monophosphate ◽  
Fat Deposition ◽  
Metabolic Effects ◽  
Rna Seq ◽  
Expression Of Genes

Abstract Background Meat quality is largely driven by fat deposition, which is regulated by several genes and signaling pathways. The cyclic adenosine monophosphate (cAMP) -regulated transcriptional coactivator 3 (CRTC3) is a coactivator of cAMP response element binding protein (CREB) that mediates the function of protein kinase A (PKA) signaling pathway and is involved in various biological processes including lipid and energy metabolism. However, the effects of CRTC3 on the metabolome and transcriptome of porcine subcutaneous adipocytes have not been studied yet. Here, we tested whether porcine CRTC3 expression would be related to fat deposition in Heigai pigs (a local fatty breed in China) and Duroc×Landrace×Yorkshire (DLY, a lean breed) pigs in vivo. The effects of adenovirus-induced CRTC3 overexpression on the metabolomic and transcriptomic profiles of subcutaneous adipocytes were also determined in vitro by performing mass spectrometry-based metabolomics combined with RNA sequencing (RNA-seq). Results Porcine CRTC3 expression is associated with fat deposition in vivo. In addition, CRTC3 overexpression increased lipid accumulation and the expression of mature adipocyte-related genes in cultured porcine subcutaneous adipocytes. According to the metabolomic analysis, CRTC3 overexpression induced significant changes in adipocyte lipid, amino acid and nucleotide metabolites in vitro. The RNA-seq analysis suggested that CRTC3 overexpression alters the expression of genes and pathways involved in adipogenesis, fatty acid metabolism and glycerophospholipid metabolism in vitro. Conclusions We identified significant alterations in the metabolite composition and the expression of genes and pathways involved in lipid metabolism in CRTC3-overexpressing adipocytes. Our results suggest that CRTC3 might play an important regulatory role in lipid metabolism and thus affects lipid accumulation in porcine subcutaneous adipocytes.

scholarly journals Nuciferine Inhibited the Differentiation and Lipid Accumulation of 3T3-L1 Preadipocytes by Regulating the Expression of Lipogenic Genes and Adipokines

2021 ◽  
Vol 12 ◽  
Author(s):  
Hanyuan Xu ◽  
Linjie Wang ◽  
Kemin Yan ◽  
Huijuan Zhu ◽  
Hui Pan ◽  
...  
Keyword(s):  
Lipid Accumulation ◽  
Molecular Mechanisms ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Luciferase Reporter Gene ◽  
Aporphine Alkaloid ◽  
Lipogenic Genes ◽  
Proliferation And Differentiation

Purposes: Nuciferine, a main aporphine alkaloid component found in lotus leaf (Nelumbo nucifera), has been demonstrated to possess the property of reducing fat mass and alleviating dyslipidemia in vivo. The purpose of this study is to explore the effects of nuciferine on the proliferation and differentiation of 3T3-L1 cells and further investigate the possible underlying molecular mechanisms.Methods: 3T3-L1 preadipocytes were treated with 0∼20 μM nuciferine for 24∼120 h, the cell viability was assessed using CCK8. 3T3-L1 preadipocytes and human primary preadipocytes were then induced differentiation and the effects of nuciferine on the lipid metabolism in differentiating and fully differentiated adipocytes were observed by the methods of intracellular triglyceride (TG) assay, Oil Red O staining, RT-qPCR and western blot. Transient transfection and dual luciferase reporter gene methods were used to assess the effects of nuciferine on FAS promoter activities.Results: Nuciferine inhibited the proliferation of 3T3-L1 preadipocytes in a dose- and time-dependent manner. 20 μM nuciferine significantly attenuated lipid accumulation and reduced intracellular TG contents by 47.2, 59.9 and 55.4% on the third, sixth and ninth day of preadipocytes differentiation, respectively (all p < 0.05). Moreover, the mRNA levels of PPARγ, C/EBPα, C/EBPβ, FAS, ACC, HSL and ATGL were notably decreased by 39.2∼92.5% in differentiating preadipocytes when treated with 5∼20 μM nuciferine (all p < 0.05). In fully differentiated adipocytes treated with 20 μM nuciferine for 48 h, the mRNA levels of FAS, ACC and SREBP1 were remarkably downregulated by 22.6∼45.2% compared with the controls (0 μM) (all p < 0.05), whereas the expression of adipokines FGF21 and ZAG were notably promoted by nuciferine. Similarly, in fully differentiated human primary adipocytes, the mRNA levels of FAS, ACC, SREBP1 were decreased and the expression of FGF21 and ZAG were elevated after treated with nuciferine (all p < 0.05). Further mechanism studies showed that 2.5∼20 μM nuciferine significantly decreased FAS promoter activities in 3T3-L1 preadipocytes.Conclusion: Nuciferine inhibited the proliferation and differentiation of 3T3-L1 preadipocytes. The inhibitory effects of nuciferine on adipogenesis might be due to the downregulation of PPARγ, C/EBPα and C/EBPβ, which led to the reduction of intracellular lipid accumulation in 3T3-L1 cells and by downregulating the expression of critical lipogenic enzymes, especially of FAS, which was achieved by inhibiting the FAS promoter activities. Besides, nuciferine promoted the expression of adipokines in fully differentiated adipocytes.

scholarly journals Lipid metabolism in Calanus finmarchicus is sensitive to variations in predation risk and food availability

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Elise Skottene ◽  
Ann M. Tarrant ◽  
Dag Altin ◽  
Rolf Erik Olsen ◽  
Marvin Choquet ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Predation Risk ◽  
Food Availability ◽  
Lipid Accumulation ◽  
Developmental Stages ◽  
Life Stage ◽  
Calanus Finmarchicus ◽  
Expression Of Genes ◽  
Food Conditions ◽  
Lipid Stores

AbstractLate developmental stages of the marine copepods in the genus Calanus can spend extended periods in a dormant stage (diapause) that is preceded by the accumulation of large lipid stores. We assessed how lipid metabolism during development from the C4 stage to adult is altered in response to predation risk and varying food availability, to ultimately understand more of the metabolic processes during development in Calanus copepods. We used RNA sequencing to assess if perceived predation risk in combination with varied food availability affects expression of genes associated with lipid metabolism and diapause preparation in C. finmarchicus. The lipid metabolism response to predation risk differed depending on food availability, time and life stage. Predation risk caused upregulation of lipid catabolism with high food, and downregulation with low food. Under low food conditions, predation risk disrupted lipid accumulation. The copepods showed no clear signs of diapause preparation, supporting earlier observations of the importance of multiple environmental cues in inducing diapause in C. finmarchicus. This study demonstrates that lipid metabolism is a sensitive endpoint for the interacting environmental effects of predation pressure and food availability. As diapause may be controlled by lipid accumulation, our findings may contribute towards understanding processes that can ultimately influence diapause timing.

scholarly journals Identification and Characterization of circRNA in Longissimus Dorsi of Different Breeds of Cattle

2020 ◽  
Vol 11 ◽  
Author(s):  
Ruili Liu ◽  
Xianxun Liu ◽  
Xuejin Bai ◽  
Chaozhu Xiao ◽  
Yajuan Dong
Keyword(s):  
Meat Quality ◽  
Muscle Development ◽  
Muscle Growth ◽  
Production Performance ◽  
Luciferase Reporter ◽  
Meat Production ◽  
Quality Improvements ◽  
Luciferase Reporter Gene ◽  
Real Time Quantitative Pcr ◽  
Cattle Meat

Shandong black cattle is a new breed of cattle that is developed by applying modern biotechnology, such as somatic cloning, and conventional breeding methods to Luxi cattle. It is very important to study the function and regulatory mechanism of circRNAs in muscle differentiation among different breeds to improve meat quality and meat production performance and to provide new ideas for beef cattle meat quality improvements and new breed development. Therefore, the goal of this study was to sequence and identify circRNAs in muscle tissues of different breeds of cattle. We used RNA-seq to identify circRNAs in the muscles of two breeds of cattle (Shandong black and Luxi). We identified 14,640 circRNAs and found 655 differentially expressed circRNAs. We also analyzed the classification and characteristics of circRNAs in muscle tissue. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were used on the parental genes of circRNAs. They were mainly involved in a variety of biological processes, such as muscle fiber development, smooth muscle cell proliferation, bone system morphogenesis, tight junctions and the MAPK, AMPK, and mTOR signaling pathways. In addition, we used miRanda to predict the interactions between 14 circRNAs and 11 miRNAs. Based on the above assays, we identified circRNAs (circ0001048, circ0001103, circ0001159, circ0003719, circ0003424, circ0003721, circ0003720, circ0001519, circ0001530, circ0005011, circ0014518, circ0000181, circ0000190, circ0010558) that may play important roles in the regulation of muscle growth and development. Using real-time quantitative PCR, 14 circRNAs were randomly selected to verify the real circRNAs. Luciferase reporter gene system was used to verify the binding site of miR-1 in circ0014518. Our results provide more information about circRNAs regulating muscle development in different breeds of cattle and lay a solid foundation for future experiments.

scholarly journals Identification of Key Small Non‐Coding MicroRNAs Controlling Pacemaker Mechanisms in the Human Sinus Node

2020 ◽  
Vol 9 (20) ◽  
Author(s):  
Maria Petkova ◽  
Andrew J. Atkinson ◽  
Joseph Yanni ◽  
Luke Stuart ◽  
Abimbola J. Aminu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ex Vivo ◽  
Sinus Node ◽  
Luciferase Reporter ◽  
Pacemaker Activity ◽  
Luciferase Reporter Gene ◽  
Atrial Muscle ◽  
Expression Of Genes ◽  
Gene Assay ◽  
Inhibit Gene Expression

Background The sinus node (SN) is the primary pacemaker of the heart. SN myocytes possess distinctive action potential morphology with spontaneous diastolic depolarization because of a unique expression of ion channels and Ca 2+ ‐handling proteins. MicroRNAs (miRs) inhibit gene expression. The role of miRs in controlling the expression of genes responsible for human SN pacemaking and conduction has not been explored. The aim of this study was to determine miR expression profile of the human SN as compared with that of non‐pacemaker atrial muscle. Methods and Results SN and atrial muscle biopsies were obtained from donor or post‐mortem hearts (n=10), histology/immunolabeling were used to characterize the tissues, TaqMan Human MicroRNA Arrays were used to measure 754 miRs, Ingenuity Pathway Analysis was used to identify miRs controlling SN pacemaker gene expression. Eighteen miRs were significantly more and 48 significantly less abundant in the SN than atrial muscle. The most interesting miR was miR‐486‐3p predicted to inhibit expression of pacemaking channels: HCN1 (hyperpolarization‐activated cyclic nucleotide‐gated 1), HCN4, voltage‐gated calcium channel (Ca v )1.3, and Ca v 3.1. A luciferase reporter gene assay confirmed that miR‐486‐3p can control HCN4 expression via its 3′ untranslated region. In ex vivo SN preparations, transfection with miR‐486‐3p reduced the beating rate by ≈35±5% ( P <0.05) and HCN4 expression ( P <0.05). Conclusions The human SN possesses a unique pattern of expression of miRs predicted to target functionally important genes. miR‐486‐3p has an important role in SN pacemaker activity by targeting HCN4, making it a potential target for therapeutic treatment of SN disease such as sinus tachycardia.

Influence of chlorophyll and tannins in plant extracts on cell-based luciferase reporter gene assays

Planta Medica ◽  
2009 ◽  
Vol 75 (09) ◽  
Author(s):  
S Vogl ◽  
P Picker ◽  
N Fakhrudin ◽  
A Atanasov ◽  
E Heiß ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Plant Extracts ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Reporter Gene Assays

Investigation of Targeting Relationship between Micro-Rna-22 and Vegfr3 in Lung Squamous Cell Carcinoma

2020 ◽  
Vol 23 ◽  
Author(s):  
Zheng Dong ◽  
Qing-Hua Xu ◽  
Yuan-Bin Zhu ◽  
Yong-Feng Wang ◽  
Jie Xiong ◽  
...  
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Lung Squamous Cell Carcinoma ◽  
Luciferase Reporter ◽  
Control Group ◽  
Vascular Endothelial ◽  
Luciferase Reporter Gene ◽  
Endothelial Growth Factor

Aims : The present study explored the clinical significance of microRNA-22 (miR-22) expression in lung squamous cell carcinoma and to explore the targeting relationship with vascular endothelial growth factor receptor 3 (VEGFR3). Methods: A total of 49 patients with lung squamous cell carcinoma who underwent surgical treatment was selected. The expression of miR-22 was detected by fluorescence quantitative real-time PCR (qPCR), the expression of VEGFR3 was detected by Western blotting assays, and D240 labeled microlymphatic vessels density (MLVD) was detected immunohistochemistry (IHC). Lung squamous cell carcinoma cell line SK-MES-1 was selected and the targeting relationship between miR-22 and VEGFR3 was analyzed by double luciferase reporter gene assay. Western blotting assays were used to detect the expression of vascular endothelial growth factor-D (VEGF-D) and D240 in the blank control group, empty vector transfection group, miR-22 transfection group, miR-22 and VEGFR3 co-transfection group. Results: The expression range of miR-22 in lung squamous cell carcinoma was 0.8-3.5. The expression of miR-22 in lung squamous cell carcinoma was significantly different by tumor maximum diameter, lymph node metastasis, vascular invasion and TNM stage. The expression of miR-22 was linked to survival time. There was a negative correlation between miR-22 and VEGFR3, miR-22 and MLVD. Double luciferase reporter gene assays showed that miR-22 reduced the luciferase activity of pGL3-VEGFR3-WT transfected cells. Compared with the control group, the expression of VEGF-D and D2-40 in the miR-22 transfection group was significantly decreased. However, VEGF-D and D240 in the miR-22 and VEGFR3 cotransfection group reversed the changes. Conclusion: We assumed that the abnormal expression of miR-22 in lung squamous cell carcinoma may be involved in the development and progression of lung squamous cell carcinoma. MiR-22 negatively regulated the target gene VEGFR3 to mediate lymphangiogenesis. The expression of miR-22 may also be linked to the prognosis of the disease.

Benzisothiazolone Derivatives Exhibit Cytotoxicity in Hodgkin’s Lymphoma Cells through NF-κB Inhibition and are Synergistic with Doxorubicin and Etoposide

2020 ◽  
Vol 20 (6) ◽  
pp. 715-723
Author(s):  
Natarajan Nandakumar ◽  
Pushparathinam Gopinath ◽  
Jacob Gopas ◽  
Kannoth M. Muraleedharan
Keyword(s):  
Synergistic Effect ◽  
Hodgkin’S Lymphoma ◽  
A549 Cells ◽  
Hodgkin's Lymphoma ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Lymphoma Cells ◽  
Nuclear Extracts ◽  
Gene Assay

Background: The authors investigated the NF-κB inhibitory role of three Benzisothiazolone (BIT) derivatives (1, 2 and 3) in Hodgkin’s Lymphoma cells (L428) which constitutively express activated NF-κB. All three compounds showed dose-dependent NF-κB inhibition (78.3, 70.7 and 34.6%) in the luciferase reporter gene assay and were found cytotoxic at IC50 values of 3.3μg/ml, 4.35μg/ml and 13.8μg/ml, respectively by the XTT assay. BIT 1and BIT 2 (but not BIT 3) suppressed both NF-κB subunits p50 and p65 in cytoplasmic and nuclear extracts in a concentration-dependent manner. Furthermore, BIT 1 showed a moderate synergistic effect with the standard chemotherapy drugs etoposide and doxorubicin, whereas BIT 2 and 3 showed a moderate additive effect to antagonistic effect. Cisplatin exhibited an antagonist effect on all the compounds tested under various concentrations, except in the case of 1.56μg/ml of BIT 3 with 0.156μg/ml of cisplatin. The compounds also inhibited the migration of adherent human lung adenocarcinoma cells (A549) in vitro. We conclude that especially BIT 1 and BIT 2 have in vitro anti-inflammatory and anti-cancer activities, which can be further investigated for future potential therapeutic use. Methods: Inspired by the electrophilic sulfur in Nuphar alkaloids, monomeric and dimeric benzisothiazolones were synthesized from dithiodibenzoic acid and their NF-κB inhibitory role was explored. NF-κB inhibition and cytotoxicity of the synthesized derivatives were studied using luciferase reporter gene assay and XTTassay. Immunocytochemistry studies were performed using L428 cells. Cell migration assay was conducted using the A549 cell line. L428 cells were used to conduct combination studies and the results were plotted using CompuSyn software. Results: Benzisothiazolone derivatives exhibited cytotoxicity in Hodgkin’s Lymphoma cells through NF-κB inhibition. Potent compounds showed suppression of both NF-κB subunits p50 and p65 in a concentrationdependent manner, both in cytoplasmic and nuclear extracts. Combination studies suggest that benzisothiazolone derivatives possess a synergistic effect with etoposide and doxorubicin. Furthermore, the compounds also inhibited the migration of A549 cells. Conclusion: Benzisothiazolones bearing one or two electrophilic sulfur atoms as part of the heterocyclic framework exhibited cytotoxicity in Hodgkin’s Lymphoma cells through NF-κB inhibition. In addition, these derivatives also exhibited a synergistic effect with etoposide and doxorubicin along with the ability to inhibit the migration of A549 cells. Our study suggests that BIT-based new chemical entities could lead to potential anticancer agents.

Comparison of the Effect of Adipocyte-derived Stem Cells and Curcumin Nanoliposomes with Phenytoin on Open Cutaneous Wound Healing in Rats

2020 ◽  
Vol 20 ◽  
Author(s):  
Mohammadreza Ebrahimzade ◽  
Mohammad Mirdoraghi ◽  
Ameneh Alikarami ◽  
Sahar Heidari ◽  
Tayebeh Rastegar ◽  
...  
Keyword(s):  
Stem Cells ◽  
Wound Healing ◽  
Adipose Tissue ◽  
Healing Time ◽  
Daily Work ◽  
Expression Of Genes ◽  
Skin Ulcers ◽  
Immobility Time ◽  
Liposomal Nanoparticles ◽  
Chromosome Staining

Background: Reducing the healing time of wounds can decrease the patient`s immobility time and their medical costs,leading a faster return of the patients to daily work. Objective: To compare the effect of adipose-derived stem cells and curcumin-containing liposomal nanoparticles with phenytoin on wound healing. Method: After anesthesia of the rats, open skin ulcers were made by a bistoury blade.Subsequently,stem cells were re-moved from the adipose tissue of theupper border of the epididymis. Then,the originality of stem cells was confirmed by the flow cytometry. The fusion method was used to prepare the liposome;and also nanoliposomal particles wereconfirmedby using the DLS microscope.The percentage of recovery and the cell count was measured with IMAGEJ.The expression of genes was assessed by PCR. The number of fibro blasts was counted by immuno histo chemistry techniques.The amount of collagen was determined by Tri-chromosome staining and the number of capillaries was enumerated byH & E staining. Results: The expression of TGF-β1 gene, vascular number, wound healing rate and the numberof fibroblasts increased significantly in adipose tissue-derived stem cells and curcumin nanoliposome groups(p<0.05);the wound surface was also decreased significantly(p<0.05). Conclusion: Based on the results of our research, adipose tissue-derived stem cells and curcumin nanoliposomescan heal wounds efficiently.

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