scholarly journals A Toll-Spätzle Pathway in the Immune Response of Bombyx mori

Insects ◽  
2020 ◽  
Vol 11 (9) ◽  
pp. 586
Author(s):  
Bin Yu ◽  
Qi Sang ◽  
Guoqing Pan ◽  
Chunfeng Li ◽  
Zeyang Zhou
Keyword(s):  
Innate Immunity ◽  
Bombyx Mori ◽  
Direct Evidence ◽  
Defense Mechanism ◽  
Luciferase Reporter ◽  
Peptide Gene ◽  
Insect Innate Immunity ◽  
And Staphylococcus Aureus ◽  
Two Hybrid ◽  
Dual Luciferase Reporter Assay

The Toll-Spätzle pathway is a crucial defense mechanism in insect innate immunity, it plays an important role in fighting against pathogens through the regulation of antimicrobial peptide gene expression. Although Toll and Spätzle (Spz) genes have been identified in Bombyx mori, little is known regarding the specific Spz and Toll genes members involved in innate immunity. There is also limited direct evidence of the interaction between Spz and Toll. In this study, the dual-luciferase reporter assay results showed that BmToll11 and BmToll9–1 could activate both drosomycin and diptericin promoters in S2 cells. Furthermore, BmToll11, BmToll9–1, and five BmSpzs genes were found to be significantly upregulated in B. mori infected by Escherichia coli and Staphylococcus aureus. Additionally, the yeast two-hybrid assay results confirmed that BmSpz2, but not other BmSpzs, could interact with both BmToll11 and BmToll9–1. These findings suggest that the activated BmSpz2 can bind with BmToll11 and BmToll9–1 to induce the expression of AMPs after the silkworm is infected by pathogens.

Analyzing the promoters of two CYP9A genes in the silkworm Bombyx mori by dual-luciferase reporter assay

2012 ◽  
Vol 40 (2) ◽  
pp. 1701-1710 ◽  
Author(s):  
Si-Si Zhao ◽  
Guo-Dong Zhao ◽  
Tian-Yuan Di ◽  
Hua Ding ◽  
Xiao-Ling Wan ◽  
...  
Keyword(s):  
Bombyx Mori ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Silkworm Bombyx Mori ◽  
Dual Luciferase Reporter Assay

scholarly journals Rice Transcription Factor OsWRKY55 Is Involved in the Drought Response and Regulation of Plant Growth

2021 ◽  
Vol 22 (9) ◽  
pp. 4337
Author(s):  
Kai Huang ◽  
Tao Wu ◽  
Ziming Ma ◽  
Zhao Li ◽  
Haoyuan Chen ◽  
...  
Keyword(s):  
Drought Stress ◽  
Plant Growth ◽  
Plant Height ◽  
Molecular Mechanisms ◽  
Crop Improvement ◽  
Critical Role ◽  
Luciferase Reporter ◽  
Mitogen Activated Protein ◽  
Two Hybrid ◽  
Dual Luciferase Reporter Assay

WRKY transcription factors (TFs) have been reported to respond to biotic and abiotic stresses and regulate plant growth and development. However, the molecular mechanisms of WRKY TFs involved in drought stress and regulating plant height in rice remain largely unknown. In this study, we found that transgenic rice lines overexpressing OsWRKY55 (OsWRKY55-OE) exhibited reduced drought resistance. The OsWRKY55-OE lines showed faster water loss and greater accumulation of hydrogen peroxide (H2O2) and superoxide radical (O2−·) compared to wild-type (WT) plants under drought conditions. OsWRKY55 was expressed in various tissues and was induced by drought and abscisic acid (ABA) treatments. Through yeast two-hybrid assays, we found that OsWRKY55 interacted with four mitogen-activated protein kinases (MAPKs) that could be induced by drought, including OsMPK7, OsMPK9, OsMPK20-1, and OsMPK20-4. The activation effects of the four OsMPKs on OsWRKY55 transcriptional activity were demonstrated by a GAL4-dependent chimeric transactivation assay in rice protoplasts. Furthermore, OsWRKY55 was able to reduce plant height under normal conditions by decreasing the cell size. In addition, based on a dual luciferase reporter assay, OsWRKY55 was shown to bind to the promoter of OsAP2-39 through a yeast one-hybrid assay and positively regulate OsAP2-39 expression. These results suggest that OsWRKY55 plays a critical role in responses to drought stress and the regulation of plant height in rice, further providing valuable information for crop improvement.

scholarly journals Upregulation of miR-150-5p alleviates LPS-induced inflammatory response and apoptosis of RAW264.7 macrophages by targeting Notch1

2020 ◽  
Vol 15 (1) ◽  
pp. 544-552
Author(s):  
Xiaoyan Deng ◽  
Zhixing Lin ◽  
Chao Zuo ◽  
Yanjie Fu
Keyword(s):  
Inflammatory Response ◽  
Underlying Mechanism ◽  
Notch Receptor ◽  
Raw264.7 Cells ◽  
Luciferase Reporter ◽  
Raw264.7 Macrophages ◽  
Factor Α ◽  
Anti Inflammation ◽  
Necrosis Factor ◽  
Dual Luciferase Reporter Assay

AbstractCirculating miR-150-5p has been identified as a prognostic marker in patients with critical illness and sepsis. Herein, we aimed to further explore the role and underlying mechanism of miR-150-5p in sepsis. Quantitative real-time-PCR assay was performed to detect the expression of miR-150-5p upon stimulation with lipopolysaccharide (LPS) in RAW264.7 cells. The levels of tumor necrosis factor-α, interleukin (IL)-6 and IL-1β were measured by ELISA assay. Cell apoptosis was determined using flow cytometry. Western blot was used to assess notch receptor 1 (Notch1) expression in LPS-induced RAW264.7 cells. Dual-luciferase reporter assay was employed to validate the target of miR-150-5p. Our data showed that miR-150-5p was downregulated and Notch1 was upregulated in LPS-stimulated RAW264.7 cells. miR-150-5p overexpression or Notch1 silencing alleviated LPS-induced inflammatory response and apoptosis in RAW264.7 cells. Moreover, Notch1 was a direct target of miR-150-5p. Notch1 abated miR-150-5p-mediated anti-inflammation and anti-apoptosis in LPS-induced RAW264.7 cells. miR-150-5p alleviated LPS-induced inflammatory response and apoptosis at least partly by targeting Notch1 in RAW264.7 cells, highlighting miR-150-5p as a target in the development of anti-inflammation and anti-apoptosis drugs for sepsis treatment.

scholarly journals Identification and Functional Analysis of a Lysozyme Gene from Coridius Chinensis (Hemiptera: Dinidoridae)

Biology ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 330
Author(s):  
Hai Huang ◽  
Juan Du ◽  
Shang-Wei Li ◽  
Tao Gong
Keyword(s):  
Innate Immunity ◽  
Fat Body ◽  
Expression Patterns ◽  
Evolutionary Relationship ◽  
Immune Defense ◽  
Amino Acid Residues ◽  
Immune Effector ◽  
Medicinal Value ◽  
Muramidase Activity ◽  
Insect Innate Immunity

Coridius chinensis is a valuable medicinal insect resource in China. Previous studies have indicated that the antibacterial and anticancer effects of the C. chinensis extract mainly come from the active polypeptides. Lysozyme is an effective immune effector in insect innate immunity and usually has excellent bactericidal effects. There are two kinds of lysozymes in insects, c-type and i-type, which play an important role in innate immunity and intestinal digestion. Studying lysozyme in C. chinensis will be helpful to further explore the evolutionary relationship and functional differences among lysozymes of various species and to determine whether they have biological activity and medicinal value. In this study, a lysozyme CcLys2 was identified from C. chinensis. CcLys2 contains 223 amino acid residues, and possesses a typical domain of the c-type lysozyme and a putative catalytic site formed by two conserved residues Glu32 and Asp50. Phylogenetic analysis showed that CcLys2 belongs to the H-branch of the c-type lysozyme. The analysis of spatiotemporal expression patterns indicated that CcLys2 was mainly expressed in the fat body of C. chinensis adults and was highly expressed in the second- and fifth-instar nymphs. In addition, CcLys2 was significantly up-regulated after injecting and feeding bacteria. In the bacterial inhibition assay, it was found that CcLys2 had antibacterial activity against Gram-positive bacteria at a low pH. These results indicate that CcLys2 has muramidase activity, involves in the innate immunity of C. chinensis, and is also closely related to the bacterial immune defense or digestive function of the intestine.

scholarly journals Hsa-miR-494-3p attenuates gene HtrA3 transcription to increase inflammatory response in hypoxia/reoxygenation HK2 Cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Qian Gong ◽  
Zhi-ming Shen ◽  
Zhe Sheng ◽  
Shi Jiang ◽  
Sheng-lin Ge
Keyword(s):  
Cardiac Surgery ◽  
Inflammatory Response ◽  
Target Gene ◽  
Kidney Injury ◽  
Human Kidney ◽  
Luciferase Reporter ◽  
The Relationship ◽  
Hk2 Cells ◽  
Dual Luciferase Reporter Assay ◽  
Hypoxia Reoxygenation

AbstractThe occurrence of cardiac surgery-associated acute kidney injury (CSA-AKI) increases hospital stay and mortality. MicroRNAs has a crucial role in AKI. This objective of the current study is to explore the function of hsa-miR-494-3p in inflammatory response in human kidney tubular epithelial (HK2) cells with hypoxia/reoxygenation. According to KDIGO standard, patients after cardiac surgery with cardiopulmonary bypass were divided into two groups: AKI (n = 10) and non-AKI patients (n = 8). HK2 were raised in the normal and hypoxia/reoxygenation circumstances and mainly treated by overexpression ofmiR-494-3p and HtrA3. The relationship between miR-494-3p and HtrA3 was determined by dual-luciferase reporter assay. Our result showed that Hsa-miR-494-3p was elevated in the serum of patients with CSA-AKI, and also induced in hypoxic reoxygenated HK2 cells. Hsa-miR-494-3p also increased a hypoxia-reoxygenation induced inflammatory response in HK2 cells. Moreover, as a target gene of miR-494-3p, overexpression of HtrA3 downregulated the hypoxia-reoxygenation induced inflammatory response in HK2 cells. Overexpression of hsa-miR-494-3p-induced inflammatory response was inhibited by overexpression of HtrA3. Collectively, we identified that hsa-miR-494-3p, a miRNA induced in both circulation of AKI patients and hypoxia-reoxygenation-treated HK2 cells, enhanced renal inflammation by targeting HtrA3, which may suggest a possible role as a new therapeutic target for CSA-AKI.

scholarly journals LINC00942 Promotes Tumor Proliferation and Metastasis in Lung Adenocarcinoma via FZD1 Upregulation

2021 ◽  
Vol 20 ◽  
pp. 153303382097752
Author(s):  
Ronghua Wang ◽  
Xiuyun Wang ◽  
Jingtao Zhang ◽  
Yanpei Liu
Keyword(s):  
Lung Adenocarcinoma ◽  
Wnt Signaling ◽  
Molecular Mechanisms ◽  
Bioinformatic Analysis ◽  
Luciferase Reporter ◽  
Regulatory Relationship ◽  
Potential Biomarker ◽  
Proliferation And Metastasis ◽  
Tumor Proliferation And Metastasis ◽  
Dual Luciferase Reporter Assay

Background: Long non-coding RNAs (lncRNAs) have been reported to play important roles in the progression of human cancers. Herein, bioinformatic analysis identified that LINC00942 was a highly overexpressed lncRNA in lung adenocarcinoma (LUAD). The present study aimed to explore the roles and possible molecular mechanisms of LINC00942 in LUAD. Methods: First, on the basis of TCGA database, the expression and prognosis of LINC00942 were analyzed in LUAD tissues. Then, si-LINC00942 was transfected into A549 and H1299 cells to knockdown the expression of LINC00942. Cell viability was detected by MTT assay. Flow cytometry was used to analyze cell apoptosis. The expressions of PCNA, Bax, Bcl-2, and wnt/β-catenin pathway proteins were detected by western blotting. Dual-luciferase reporter assay was used to evaluate the regulatory relationship between LINC00942 and miR-5006-5p, or miR-5006-5p and FZD1. Results: We discovered that LINC00942 was up-regulated in LUAD tissues compared with adjacent tissues. Besides, we found the increased LINC00942 expression was associated with poor survival. In addition, silencing of LINC00942 suppressed the proliferation, migration, invasion and facilitated the apoptosis of A549 and H1299 cells. Moreover, silencing of LINC00942 repressed the expression of PCNA, Bcl-2, and enhanced Bax expression in A549 and H1299 cells. Mechanically, LINC00942 exerted its effects via enhancing Wnt signaling. LINC00942 functioned as competing endogenous RNA (ceRNA) by binding to miR-5006-5p, upregulating the expression of FZD1, which was a direct target of miR-5006-5p. Conclusion: Our findings indicated that LINC00942/miR-5006-5p/FZD1 axis played important roles in LUAD growth through enhancing Wnt signaling. LINC00942/miR-5006-5p/FZD1 axis might serve as a potential biomarker and therapeutic target for LUAD treatment.

scholarly journals Melatonin promotes bone marrow mesenchymal stem cell osteogenic differentiation and prevents osteoporosis development through modulating circ_0003865 that sponges miR-3653-3p

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xudong Wang ◽  
Taiqiu Chen ◽  
Zhihuai Deng ◽  
Wenjie Gao ◽  
Tongzhou Liang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Osteogenic Differentiation ◽  
Luciferase Reporter ◽  
Specific Gene ◽  
Biological Processes ◽  
Alizarin Red ◽  
Qrt Pcr ◽  
Dual Luciferase Reporter Assay

Abstract Background Little is known about the implications of circRNAs in the effects of melatonin (MEL) on bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation and osteoporosis (OP) progression. The aim of our study was to investigate circRNAs in MEL-regulated BMSC differentiation and OP progression. Methods BMSC osteogenic differentiation was measured by qRT-PCR, western blot (WB), Alizarin Red, and alkaline phosphatase (ALP) staining. Differential circRNA and mRNA profiles of BMSCs treated by MEL were characterized by deep sequencing, followed by validation using RT-PCR, Sanger sequencing, and qRT-PCR. Silencing and overexpression of circ_0003865 were conducted for functional investigations. The sponged microRNAs and targeted mRNAs were predicted by bioinformatics and validated by qRT-PCR, RNA pull-down, and dual-luciferase reporter assay. The function of miR-3653-3p and circ_0003865/miR-3653-3p/growth arrest-specific gene 1 (GAS1) cascade was validated for the osteogenic differentiation of BMSCs by CCK-8, qRT-PCR, WB, Alizarin Red, and ALP staining. The effects of circ_0003865 on OP development were tested in murine OP model. Results MEL promoted osteogenic differentiation of BMSCs. RNA sequencing revealed significant alterations in circRNA and mRNA profiles associated with multiple biological processes and signaling pathways. Circ_0003865 expression in BMSCs was significantly decreased by MEL treatment. Silencing of circ_0003865 had no effect on proliferation while promoted osteogenic differentiation of BMSCs. Overexpression of circ_0003865 abrogated the promotion of BMSC osteogenic differentiation induced by MEL, but proliferation of BMSCs induced by MEL had no change whether circ_0003865 was overexpression or not. Furthermore, circ_0003865 sponged miR-3653-3p to promote GAS1 expression in BMSCs. BMSC osteogenic differentiation was enhanced by miR-3653-3p overexpression while BMSC proliferation was not affected. By contrast, miR-3653-3p silencing mitigated the promoted BMSC osteogenic differentiation caused by circ_0003865 silencing, but had no effect on proliferation. Finally, circ_0003865 silencing repressed OP development in mouse model. Conclusion MEL promotes BMSC osteogenic differentiation and inhibits OP pathogenesis by suppressing the expression of circ_0003865, which regulates GAS1 gene expression via sponging miR-3653-3p.

scholarly journals Micro-RNA-338-3p Promotes the Development of Atherosclerosis by Targeting Desmin and Promoting Proliferation

2021 ◽  
Author(s):  
Shiran Yan ◽  
Jing Chen ◽  
Teng Zhang ◽  
Jian Zhou ◽  
Ge Wang ◽  
...  
Keyword(s):  
Rat Model ◽  
Luciferase Reporter Assay ◽  
Immunofluorescent Staining ◽  
Luciferase Reporter ◽  
Cell Phenotype ◽  
Reporter Assay ◽  
Synthetic Cell ◽  
Incorporation Assay ◽  
Morphologic Changes ◽  
Dual Luciferase Reporter Assay

AbstractAtherosclerosis (AS) is a dynamic and multi-stage process that involves various cells types, such as vascular smooth muscle cells (VSMCs) and molecules such as microRNAs. In this study, we investigated how miR-338-3p works in the process of AS. To determine how miR-338-3p was expressed in AS, an AS rat model was established and primary rat VSMCs were cultured. Real-time polymerase chain reaction was performed to detect miR-338-3p expression. Markers of different VSMC phenotypes were tested by Western blot. Immunofluorescent staining was employed to observe the morphologic changes of VSMCs transfected with miR-338-3p mimics. A dual luciferase reporter assay system was used to verify that desmin was a target of miR-338-3p. To further identify the role of miR-338-3p in the development of AS, VSMC proliferation and migration were evaluated by EdU incorporation assay, MTT assay, and wound healing assay. miR-338-3p expression was upregulated in the aortic tissues of an AS rat model and in primary rat VSMCs from a later passage. The transfection of miR-338-3p mimics in VSMCs promoted the synthetic cell phenotype. Bioinformatics analysis proposed desmin as a candidate target for miR-338-3p and the dual luciferase reporter assay confirmed in vivo that desmin was a direct target of miR-338-3p. The MTT and EdU incorporation assay revealed increased cell viability when miR-338-3p mimics were transfected. The increased expression of PCNA was a consistent observation, although a positive result was not obtained with respect to VSMC mobility. In AS, miR-338-3p expression was elevated. Elevated miR-338-3p inhibited the expression of desmin, thus promoting the contractile-to-synthetic VSMC phenotypic transition. In addition to morphologic changes, miR-338-3p enhanced the proliferative but not mobile ability of VSMCs. In summary, miR-338-3p promotes the development of AS.

scholarly journals MiR-196b Promotes the Invasion and Migration of Lung Adenocarcinoma Cells by Targeting AQP4

2021 ◽  
Vol 20 ◽  
pp. 153303382098586
Author(s):  
Xuhui Wu ◽  
Gongzhi Wu ◽  
Huaizhong Zhang ◽  
Xuyang Peng ◽  
Bin Huang ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Cell Invasion ◽  
Target Gene ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Invasion And Migration ◽  
And Migration ◽  
Dual Luciferase Reporter Assay

Objective: We aimed to investigate the mechanism of the regulatory axis of miR-196b/AQP4 underlying the invasion and migration of lung adenocarcinoma (LUAD) cells. Methods: LUAD miRNA and mRNA expression profiles were downloaded from TCGA database and then differential analysis was used to identify the target miRNA. Target gene for the miRNA was obtained via prediction using 3 bioinformatics databases and intersection with the differentially expressed mRNAs searched from TCGA-LUAD. Then, qRT-PCR and western blot were used to validate the expression of miR-196b and AQP4. Dual-luciferase reporter assay was performed to confirm the targeting relationship between miR-196b and AQP4. Transwell assay was used to investigate the migration and invasion of LUAD cells. Results: MiR-196b was screened out by differential and survival analyses, and the downstream target gene AQP4 was identified. In LUAD, miR-196b was highly expressed while AQP4 was poorly expressed. Besides, overexpression of miR-196b promoted cell invasion and migration, while overexpression of AQP4 had negative effects. Moreover, the results of the dual-luciferase reporter assay suggested that AQP4 was a direct target of miR-196b. In addition, we also found that overexpressing AQP4 could suppress the promotive effect of miR-196b on cancer cell invasion and migration. Conclusion: MiR-196b promotes the invasion and migration of LUAD cells by down-regulating AQP4, which helps us find new molecular targeted therapies for LUAD.

scholarly journals Duck RIG-I CARD Domain Induces the Chicken IFN-βby Activating NF-κB

2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
Author(s):  
Yang Chen ◽  
Zhengyang Huang ◽  
Bin Wang ◽  
Qinming Yu ◽  
Ran Liu ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Eukaryotic Expression ◽  
Poly I:C ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Enzyme Linked Immunosorbent Assay ◽  
Regulatory Domains ◽  
Polycytidylic Acid ◽  
Card Domain ◽  
Inducible Gene

Retinoic acid-inducible gene I- (RIG-I-) like receptors (RLRs) have recently been identified as cytoplasmic sensors for viral RNA. RIG-I, a member of RLRs family, plays an important role in innate immunity. Although previous investigations have proved that RIG-I is absent in chickens, it remains largely unknown whether the chicken can respond to RIG-I ligand. In this study, the eukaryotic expression vectors encoding duRIG-I full length (duck RIG-I, containing all domains), duRIG-I N-terminal (containing the two caspase activation and recruitment domain, CARDs), and duRIG-I C-terminal (containing helicase and regulatory domains) labeled with 6*His tags were constructed successfully and detected by western blotting. Luciferase reporter assay and enzyme-linked immunosorbent assay (ELISA) detected the duRIG-I significantly activated NF-κB and induced the expression of IFN-βwhen polyinosinic-polycytidylic acid (poly[I:C], synthetic double-stranded RNA) challenges chicken embryonic fibroblasts cells (DF1 cells), while the duRIG-I was inactive in the absence of poly[I:C]. Further analysis revealed that the CARDs (duRIG-I-N) induced IFN-βproduction regardless of the presence of poly[I:C], while the CARD-lacking duRIG-I (duRIG-I-C) was not capable of activating downstream signals. These results indicate that duRIG-I CARD domain plays an important role in the induction of IFN-βand provide a basis for further studying the function of RIG-I in avian innate immunity.

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