scholarly journals VPB1 Encoding BELL-like Homeodomain Protein Is Involved in Rice Panicle Architecture

2021 ◽  
Vol 22 (15) ◽  
pp. 7909
Author(s):  
Mu Li ◽  
Debao Fu ◽  
Tingting Xu ◽  
Changyin Wu
Keyword(s):  
Target Genes ◽  
Differentially Expressed Gene ◽  
Gene Isolation ◽  
Primary Branch ◽  
Luciferase Reporter ◽  
Homeodomain Protein ◽  
Inflorescence Meristem ◽  
Reporter System ◽  
Inflorescence Architecture ◽  
Mobility Shift

Inflorescence architecture in rice (Oryza sativa) is mainly determined by spikelets and the branch arrangement. Primary branches initiate from inflorescence meristem in a spiral phyllotaxic manner, and further develop into the panicle branches. The branching patterns contribute largely to rice production. In this study, we characterized a rice verticillate primary branch 1(vpb1) mutant, which exhibited a clustered primary branches phenotype. Gene isolation revealed that VPB1 was a allele of RI, that it encoded a BELL-like homeodomain (BLH) protein. VPB1 gene preferentially expressed in the inflorescence and branch meristems. The arrangement of primary branch meristems was disturbed in the vpb1 mutant. Transcriptome analysis further revealed that VPB1 affected the expression of some genes involved in inflorescence meristem identity and hormone signaling pathways. In addition, the differentially expressed gene (DEG) promoter analysis showed that OsBOPs involved in boundary organ initiation were potential target genes of VPB1 protein. Electrophoretic mobility shift assay (EMSA) and dual-luciferase reporter system further verified that VPB1 protein bound to the promoter of OsBOP1 gene. Overall, our findings demonstrate that VPB1 controls inflorescence architecture by regulating the expression of genes involved in meristem maintenance and hormone pathways and by interacting with OsBOP genes.

scholarly journals The Role of BCL6 in Glioblastoma

2021 ◽  
Author(s):  
◽  
Nicole Jones
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Target Genes ◽  
Therapy Resistance ◽  
Response To Therapy ◽  
Transcriptional Analysis ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Response To Chemotherapy ◽  
Targeted Inhibition

<p>Glioblastoma (GBM) is the most common and most deadly brain tumour to occur in adults. Initially patients respond to radiation and chemotherapy, which primarily work by causing large amounts of DNA damage, leading to cell death. However, this process does not happen effectively in GBM and understanding how these cells resist cell death in response to therapy is key to improving the efficacy of treatment. BCL6 is a transcription factor that stops cell death in response to DNA damage, primarily through repressing transcription of DNA damage response genes. Recent work in our lab has shown BCL6 to be present in untreated GBM tumours and up-regulated in GBM cells treated with chemotherapy or radiation, and inhibition of BCL6 leads to a profound loss in proliferative activity. These results indicate that BCL6 may be used as a mechanism of therapy resistance in GBM cells. The objective of this study was to establish a role for BCL6 in GBM cells using luciferase reporter assays, electrophoretic mobility shift assays (EMSA), quantitative chromatin immunoprecipitation (qChIP) and targeted inhibition of BCL6 with subsequent transcriptional analysis by RNA sequencing. We observed that BCL6 appeared to be a transcriptional activator in GBM, as shown by increased luciferase activity in GBM cells treated with radiation. EMSA experiments revealed that overexpressed BCL6 formed complexes with co-repressors, but endogenous BCL6 did not. qChIP experiments revealed that BCL6 was not bound to tradtional BCL6 target genes. Analysis of transcriptional profiles has identified a unique subset of genes which are downregulated when BCL6 is inhibited and upregulated in response to chemotherapy, and these genes were related to cell survival. These changes indicate that these genes may be regulated by BCL6 in chemotherapy treated cells. Together, these results illustrate that BCL6 appears to have an active and unique function in GBM cells, and reinforces this transcription factors position as an attractive therapeutic target in GBM.</p>

Abstract 1384: Osteogenic Transcription Factor Runx2 Regulates Expression of RANKL during VSMC Calcification

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Chang Hyun Byon ◽  
Jay McDonald ◽  
Yabing Chen
Keyword(s):  
Oxidative Stress ◽  
Transcription Factor ◽  
Molecular Mechanism ◽  
Luciferase Reporter ◽  
Rankl Expression ◽  
Reporter System ◽  
Mobility Shift ◽  
Atherosclerotic Lesions ◽  
Flanking Region

The expression of receptor activator of nuclear factor κ B (RANKL) is up-regulated in calcified atherosclerotic lesions, whereas it is frequently undetectable in normal vessels. The underlying molecular mechanism of increased expression of RANKL in calcified vessels is not known. We have previously demonstrated that oxidative stress induces calcification of vascular smooth muscle cells (VSMC) in vitro . Therefore, we determined whether oxidative stress regulates RANKL expression in VSMC and the underlying molecular mechanism. Consistent with previous observations in vivo , we found that the expression of RANKL in VSMC isolated from mouse. However, hydrogen peroxide (H 2 O 2 ), which induces VSMC calcification, induced a 33-fold increase in the transcripts of RANKL as determined by real-time PCR. Increased expression of RANKL protein was further confirmed by ELISA. Using flow cytometry, we demonstrated that membrane-bound RANKL was increased by oxidative stress. To characterize the molecular mechanism underlying H 2 O 2 -induced RANKL expression, we employed the luciferase reporter system with a series of deletion mutants of the RANKL 5′-flanking region. The H 2 O 2 responsive region is located between −200 to −400 in the 5′-flanking region of RANKL gene. Analyses of the sequence of this region identified multiple binding sites for the key osteogenic transcription factor, Runx2, which we previously reported to be an essential regulator of VSMC calcification. Electrophoretic mobility shift analyses demonstrated increased binding of Runx2 on the RANKL promoter sequence in nuclear extracts from VSMC exposed to H 2 O 2 . To further determine the role of Runx2 in regulating RANKL expression, we generated stable Runx2 knockdown VSMC with the use of lentivirus-carrying shRNA for Runx2 gene. H 2 O 2 -induced RANKL expression was abrogated in VSMC with Runx2 knockdown. In addition, adenovirus-mediated overexpression of Runx2 in VSMC induced the expression of RANKL. In summary, we have demonstrated that H 2 O 2 induces the expression of RANKL in VSMC, which is regulated by the osteogenic transcription factor Runx2. These observations provide novel molecular insights into the regulation of RANKL and its role on the pathogenesis of calcified atherosclerotic lesions.

Abstract 11113: A Novel ATF4 Dependent ER-Stress Induced Transcriptional Unit Within the CHAC1 Promoter

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Rebecca R Crawford ◽  
Eugenia T Prescott ◽  
Imran N Mungrue
Keyword(s):  
Metabolic Disease ◽  
Er Stress ◽  
Target Genes ◽  
Biological Effects ◽  
Transcriptional Unit ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Human Cell Cultures ◽  
Genetic Perturbations ◽  
Novel Target

A systems genetics screen of transcriptome wide RNA co-expression in a population of human cell cultures can broadly define novel functional gene-gene relationships. Using this method, we predicted a role for the human gene, CHAC1, within the ER-stress pathway functionally linked to ATF4. Targeted cell culture and genetic perturbations refined a role for CHAC1 as an ER-stress gene regulated by ATF4. ATF4 siRNAs blocked induction of CHAC1 mRNA by ER-stress, and ATF4 over-expression induced CHAC1 mRNA expression. ATF4 is an important gene involved in ER-stress and apoptotic signaling, and we demonstrated the pro-apoptotic potential for CHAC1. ATF4 plays important roles in osteoblast development, metabolic disease and atherosclerosis. Herein a direct transcriptional link between ATF4 and the CHAC1 promoter by ATF4 is defined in the setting of ER-stress. We cloned the CHAC1 promoter upstream of luciferase reporter system to define functional elements by deletion analysis. A highly conserved bipartite DNA element dubbed the ATF/CRE and ACM were the major regulators of basal CHAC1 expression, induction by ER-stress agonists or ATF4 co-expression. We compared the CHAC1 promoter -luciferase reporter to well-characterized ATF4 target genes CHOP and ASNS, using parallel luciferase assays. Notably, the CHAC1 promoter-reporter was induced to the highest extent following ATF4 co-expression compared to CHOP and ASNS promoters. Using a novel Immunoblot-EMSA assay we determined binding of ATF4 and ATF3, but not CHOP to CHAC1 ATF/CRE and ACM oligos. We also used the CHip method to query binding of ATF3 and ATF4 at the CHAC1 promoter under control and ER-stress conditions. We noted significant binding of ATF3 to the CHAC1 promoter under control and ER-stress treatment. Interestingly, we noted a dramatic increase in ATF4 occupancy at the CHAC1 promoter accompanying ER-stress. The ATF4 responsive ATF/CRE and ACM of the CHAC1 promoter represent a novel target for ATF4, and may reveal important contributions to disease processes involving ATF4. These data also implicate CHAC1 as an important target gene of ATF4, which may mediate important biological effects in the setting of ER-stress, and might have implications for cardiovascular and metabolic disease.

Sevoflurane Inhibits Growth and Metastasis of Cervical Cancer Through Up-Regulating miR-203 by Targeting Tumor Protein Translationally Controlled 1

2020 ◽  
Vol 10 (6) ◽  
pp. 874-883
Author(s):  
Li Zhang ◽  
Shiyou Wei ◽  
Zhenkai Xu ◽  
Wen Sun ◽  
Lihua Hang
Keyword(s):  
Cervical Cancer ◽  
Cell Viability ◽  
Cancer Cells ◽  
Target Genes ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter System ◽  
Cervical Cancer Cells ◽  
Induced Apoptosis ◽  
Cancer Tissues

Background: Cervical cancer is a type of malignancy with high incidence and high mortality in women all over the world. Recent findings revealed the role of sevoflurane in the inhibition of development of various cancer types. This study aimed to explore whether sevoflurane could suppress cells proliferation and metastasis through adjusting miR-203 expression in cervical cancer. Methods: The effects of sevoflurane on HeLa cell viability was assessed using CCK-8 assay. miR-203 level in Hela cells was determined by qRT-PCR. In addition, cells apoptosis, migration and invasion were evaluated using flow cytometry and transwell analysis respectively after sevoflurane treatment or miR-203 expression changes. Bioinformatics software (TargetScan) was used to predict the potential target genes for miR-203 and the prediction was validated using dual-luciferase reporter system. Results: Sevoflurane effectively inhibited cell viability, metastasis and stimulated apoptosis in cervical cancer. miR-203 demonstrated a low expression in cervical cancer tissues and cells and sevoflurane significantly up-regulated miR-203 expression in cervical cancer cells. Upregulation of miR-203 significantly suppressed cell growth and metastasis and induced apoptosis, while down-regulation of miR-203 presented the opposite effects in cervical cancer cells. In addition, the inhibitory effects of sevoflurane were eliminated by down-regulating miR-203 in cervical cancer cells. In addition, TPT1 was confirmed as a target gene for miR-203. Conclusion: Sevoflurane inhibited cervical cancer cells viability and metastasis through up-regulation of miR-203 expression by targeting TPT1.

A dual‐luciferase reporter system for characterization of small RNA target genes in both mammalian and insect cells

2021 ◽  
Author(s):  
Zhongyuan Deng ◽  
Yuting Zhang ◽  
Leyao Li ◽  
Xingcheng Xie ◽  
Jinyong Huang ◽  
...  
Keyword(s):  
Small Rna ◽  
Target Genes ◽  
Insect Cells ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Luciferase Reporter System

scholarly journals The Role of BCL6 in Glioblastoma

2021 ◽  
Author(s):  
◽  
Nicole Jones
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Target Genes ◽  
Therapy Resistance ◽  
Response To Therapy ◽  
Transcriptional Analysis ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Response To Chemotherapy ◽  
Targeted Inhibition

<p>Glioblastoma (GBM) is the most common and most deadly brain tumour to occur in adults. Initially patients respond to radiation and chemotherapy, which primarily work by causing large amounts of DNA damage, leading to cell death. However, this process does not happen effectively in GBM and understanding how these cells resist cell death in response to therapy is key to improving the efficacy of treatment. BCL6 is a transcription factor that stops cell death in response to DNA damage, primarily through repressing transcription of DNA damage response genes. Recent work in our lab has shown BCL6 to be present in untreated GBM tumours and up-regulated in GBM cells treated with chemotherapy or radiation, and inhibition of BCL6 leads to a profound loss in proliferative activity. These results indicate that BCL6 may be used as a mechanism of therapy resistance in GBM cells. The objective of this study was to establish a role for BCL6 in GBM cells using luciferase reporter assays, electrophoretic mobility shift assays (EMSA), quantitative chromatin immunoprecipitation (qChIP) and targeted inhibition of BCL6 with subsequent transcriptional analysis by RNA sequencing. We observed that BCL6 appeared to be a transcriptional activator in GBM, as shown by increased luciferase activity in GBM cells treated with radiation. EMSA experiments revealed that overexpressed BCL6 formed complexes with co-repressors, but endogenous BCL6 did not. qChIP experiments revealed that BCL6 was not bound to tradtional BCL6 target genes. Analysis of transcriptional profiles has identified a unique subset of genes which are downregulated when BCL6 is inhibited and upregulated in response to chemotherapy, and these genes were related to cell survival. These changes indicate that these genes may be regulated by BCL6 in chemotherapy treated cells. Together, these results illustrate that BCL6 appears to have an active and unique function in GBM cells, and reinforces this transcription factors position as an attractive therapeutic target in GBM.</p>

scholarly journals TaWRKY13-A Serves as a Mediator of Jasmonic Acid-Related Leaf Senescence by Modulating Jasmonic Acid Biosynthesis

2021 ◽  
Vol 12 ◽  
Author(s):  
Hualiang Qiao ◽  
Yongwei Liu ◽  
Lingling Cheng ◽  
Xuelin Gu ◽  
Pengcheng Yin ◽  
...  
Keyword(s):  
Jasmonic Acid ◽  
Leaf Senescence ◽  
Brachypodium Distachyon ◽  
Wrky Transcription Factor ◽  
Luciferase Reporter ◽  
Virus Induced Gene Silencing ◽  
Reporter System ◽  
Mobility Shift ◽  
Yield And Quality ◽  
Mobility Shift Assay

Leaf senescence is crucial for crop yield and quality. Transcriptional regulation is a key step for integrating various senescence-related signals into the nucleus. However, few regulators of senescence implicating transcriptional events have been functionally characterized in wheat. Based on our RNA-seq data, we identified a WRKY transcription factor, TaWRKY13-A, that predominately expresses at senescent stages. By using the virus-induced gene silencing (VIGS) method, we manifested impaired transcription of TaWRKY13-A leading to a delayed leaf senescence phenotype in wheat. Moreover, the overexpression (OE) of TaWRKY13-A accelerated the onset of leaf senescence under both natural growth condition and darkness in Brachypodium distachyon and Arabidopsis thaliana. Furthermore, by physiological and molecular investigations, we verified that TaWRKY13-A participates in the regulation of leaf senescence via jasmonic acid (JA) pathway. The expression of JA biosynthetic genes, including AtLOX6, was altered in TaWRKY13-A-overexpressing Arabidopsis. We also demonstrated that TaWRKY13-A can interact with the promoter of AtLOX6 and TaLOX6 by using the electrophoretic mobility shift assay (EMSA) and luciferase reporter system. Consistently, we detected a higher JA level in TaWRKY13-A-overexpressing lines than that in Col-0. Moreover, our data suggested that TaWRKY13-A is partially functional conserved with AtWRKY53 in age-dependent leaf senescence. Collectively, this study manifests TaWRKY13-A as a positive regulator of JA-related leaf senescence, which could be a new clue for molecular breeding in wheat.

scholarly journals Downregulated miR-204 Promotes Skeletal Muscle Regeneration

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Ya Tan ◽  
Linyuan Shen ◽  
Mailin Gan ◽  
Yuan Fan ◽  
Xiao Cheng ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Regeneration ◽  
Muscle Injury ◽  
Target Genes ◽  
C2c12 Cell ◽  
Skeletal Muscle Regeneration ◽  
Luciferase Reporter ◽  
Limited Information ◽  
Reporter System ◽  
Skeletal Muscle Injury

Skeletal muscle is the most abundant and a highly plastic tissue of the mammals, especially when it comes to regenerate after trauma, but there is limited information about the mechanism of muscle repair and its regeneration. In the present study, we found that miR-204 is downregulated after skeletal muscle injury. In vitro experiments showed that over-expression of miR-204 by transfecting with miR-204 mimics suppressed C2C12 cell proliferation, migration, and blocked subsequent differentiation, whereas inhibition of miR-204 by transfecting with miR-204 inhibitor showed the converse effects. Furthermore, through the dual luciferase reporter system, we demonstrated that miR-204 can target the 3’UTR regions of Pax7, IGF1, and Mef2c and inhibit their expression. Taken together, our results suggest that Pax7, IGF1, and Mef2c are the target genes of miR-204 in the process of myoblasts proliferation, cell migration, and differentiation, respectively, and may contribute to mouse skeletal muscle regeneration. Our results may provide new ideas and references for the skeletal muscle study and may also provide therapeutic strategies of skeletal muscle injury.

scholarly journals miR-155 and miR-146a collectively regulate meningitic Escherichia coli infection-mediated neuroinflammatory responses

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Bo Yang ◽  
Ruicheng Yang ◽  
Bojie Xu ◽  
Jiyang Fu ◽  
Xinyi Qu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Signaling Pathways ◽  
Signaling Pathway ◽  
Target Genes ◽  
Microglia Activation ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Negative Feedback Regulation ◽  
E Coli

Abstract Background Escherichia coli is the most common Gram-negative bacterium causing meningitis, and E. coli meningitis is associated with high mortality and morbidity throughout the world. Our previous study showed that E. coli can colonize the brain and cause neuroinflammation. Increasing evidence supports the involvement of miRNAs as key regulators of neuroinflammation. However, it is not clear whether these molecules participate in the regulation of meningitic E. coli-mediated neuroinflammation. Methods The levels of miR-155 and miR-146a, as well as their precursors, in E. coli-infected astrocytes were measured using quantitative real-time PCR (qPCR). Overexpression and knockdown studies of miR-155 and miR-146a were performed to observe the effects on bacterial loads, cytokines, chemokines, and NF-κB signaling pathways. Bioinformatics methods were utilized to predict the target genes, and these target genes were validated using qPCR, Western blotting, and luciferase reporter system. In vivo knockdown of miR-155 and miR-146a was carried out to observe the effects on bacterial loads, inflammatory genes, astrocyte activation, microglia activation, and survival in a mouse model. Results The levels of miR-155, miR-146a, and their precursors were significantly increased in astrocytes during E. coli infection. miR-155 and miR-146a were induced by the NF-κB-p65 signaling pathway upon infection. Overexpressing and inhibiting miR-155 and miR-146a in astrocytes did not affect the bacterial loads. Further, the in vitro overexpression of miR-155 and miR-146a suppressed the E. coli-induced inflammatory response, whereas the inhibition of miR-155 and miR-146a enhanced it. Mechanistically, miR-155 inhibited TAB2, and miR-146a targeted IRAK1 and TRAF6; therefore, they functioned collaboratively to modulate TLR-mediated NF-κB signaling. In addition, both miR-155 and miR-146a could regulate the EGFR–NF-κB signaling pathway. Finally, the in vivo suppression of E. coli-induced miR-155 and miR-146a further promoted the production of inflammatory cytokines, aggravated astrocyte and microglia activation, and decreased mouse survival time, without affecting the bacterial loads in the blood and brain. Conclusions E. coli infection induced miR-155 and miR-146a, which collectively regulated bacteria-triggered neuroinflammatory responses through negative feedback regulation involving the TLR-mediated NF-κB and EGFR–NF-κB signaling pathways, thus protecting the central nervous system from further neuroinflammatory damage.

scholarly journals MiR-34a Enhances Chondrocyte Apoptosis, Senescence and Facilitates Development of Osteoarthritis by Targeting DLL1 and Regulating PI3K/AKT Pathway

2018 ◽  
Vol 48 (3) ◽  
pp. 1304-1316 ◽  
Author(s):  
Wei Zhang ◽  
Peichun Hsu ◽  
Biao  Zhong ◽  
Shang Guo ◽  
Chi Zhang ◽  
...  
Keyword(s):  
Cell Death ◽  
Target Genes ◽  
Cartilage Degeneration ◽  
Luciferase Reporter ◽  
Akt Pathway ◽  
Chondrocyte Apoptosis ◽  
Articular Chondrocyte ◽  
Reporter System ◽  
Chondrocyte Death

Background/Aims: Osteoarthritis (OA) is the prevalent degenerative disease caused by various factors. MicroRNAs are important regulators in the inflammation and immune response. The aim of this study was to investigate the effect of microRNA-34a (MiR-34a) on the death of chondrocytes, senescence, as well as its role in OA progression. Methods: A series of experiments involving CCK-8, flow cytometry, β-galactosidase staining and wound healing assays were conducted to determine the cellular capabilities of proliferation, cell apoptosis, senescence and the ability of cells to recover from injury, respectively. Binding sites between miR-34a and delta-like protein 1 (DLL1) were identified using a luciferase reporter system, whereas mRNA and protein expression of target genes was determined by RT-PCR and immunoblot, respectively. OA model was generated via surgery. Results: We found that miR-34a expression was increased in the cartilage of OA patients. In rat chondrocytes and chondrosarcoma cells, miR-34a transfections noticeably inhibited the expression of DLL1, triggered cell death and senescence, suppressed proliferation, and prevented scratch assay wound closure. However, transfection of a miR-34a inhibitor displayed adverse effects. Additionally, secretion and expression of factors associated with cartilage degeneration were altered via miR-34a. Moreover, miR-34a directly inhibits DLL1 mRNA. Furthermore, concentrations of DLL1, total PI3K, and p-AKT declined in chondrocytes that overexpress miR-34a. DLL1 overexpression elevated PI3K and p-AKT levels, and eliminated cell death triggered by a miR-34a mimic. In vivo, miR-34a remarkably inhibited miR-34a up-regulation, while enhanced the level of DLL1 expression. In the knee joints of surgery-induced OA rats, articular chondrocyte death and loss of cartilage were attenuated via miR-34a antagomir injection. Conclusions: These findings indicate that miR-34a contributes to chondrocyte death, causing OA progression through DLL1 and modulation of the PI3K/AKT pathway.

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