A cis-Acting Mutation in the PxABCG1 Promoter Is Associated with Cry1Ac Resistance in Plutella xylostella (L.)

The molecular mechanisms of insect resistance to Cry toxins generated from the bacterium Bacillus thuringiensis (Bt) urgently need to be elucidated to enable the improvement and sustainability of Bt-based products. Although downregulation of the expression of midgut receptor genes is a pivotal mechanism of insect resistance to Bt Cry toxins, the underlying transcriptional regulation of these genes remains elusive. Herein, we unraveled the regulatory mechanism of the downregulation of the ABC transporter gene PxABCG1 (also called Pxwhite), a functional midgut receptor of the Bt Cry1Ac toxin in Plutella xylostella. The PxABCG1 promoters of Cry1Ac-susceptible and Cry1Ac-resistant strains were cloned and analyzed, and they showed clear differences in activity. Subsequently, a dual-luciferase reporter assay, a yeast one-hybrid (Y1H) assay, and RNA interference (RNAi) experiments demonstrated that a cis-mutation in a binding site of the Hox transcription factor Antennapedia (Antp) decreased the promoter activity of the resistant strain and eliminated the binding and regulation of Antp, thereby enhancing the resistance of P. xylostella to the Cry1Ac toxin. These results advance our knowledge of the roles of cis- and trans-regulatory variations in the regulation of midgut Cry receptor genes and the evolution of Bt resistance, contributing to a more complete understanding of the Bt resistance mechanism.

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MAPK-activated transcription factor PxJun suppresses PxABCB1 expression and confers resistance to Bacillus thuringiensis Cry1Ac toxin in Plutella xylostella (L.)

Applied and Environmental Microbiology ◽

10.1128/aem.00466-21 ◽

2021 ◽

Author(s):

Jianying Qin ◽

Le Guo ◽

Fan Ye ◽

Shi Kang ◽

Dan Sun ◽

...

Keyword(s):

Transcription Factor ◽

Transcriptional Regulation ◽

Bacillus Thuringiensis ◽

Insect Resistance ◽

Plutella Xylostella ◽

Molecular Basis ◽

Luciferase Reporter ◽

Regulation Mechanism ◽

Cry Toxins ◽

Regulation Mechanisms

Deciphering the molecular mechanisms underlying insect resistance to Cry toxins produced by Bacillus thuringiensis (Bt) is pivotal for the sustainable utilization of Bt biopesticides and transgenic Bt crops. Previously, we identified that MAPK-mediated reduced expression of the PxABCB1 gene is associated with Bt Cry1Ac resistance in the diamondback moth, Plutella xylostella (L.). However, the underlying transcriptional regulation mechanism remains enigmatic. Herein, the PxABCB1 promoter in Cry1Ac-susceptible and Cry1Ac-resistant P. xylostella strains was cloned and analyzed and found to contain a putative Jun binding site (JBS). A dual-luciferase reporter assay and yeast one-hybrid assay (Y1H) demonstrated that the transcription factor PxJun repressed PxABCB1 expression by interacting with this JBS. The expression levels of PxJun were increased in the midguts of all resistant strains compared to the susceptible strain. Silencing of PxJun expression significantly elevated PxABCB1 expression and Cry1Ac susceptibility in the resistant NIL-R strain, and silencing of PxMAP4K4 expression decreased PxJun expression and also increased PxABCB1 expression. These results indicate that MAPK-activated PxJun suppresses PxABCB1 expression to confer Cry1Ac resistance in P. xylostella, deepening our understanding of the transcriptional regulation of midgut Cry receptor genes and the molecular basis of insect resistance to Bt Cry toxins. Importance The transcriptional regulation mechanisms underlying reduced expression of Bt toxin receptor genes in Bt-resistant insects remain elusive. This study unveils that a transcription factor PxJun activated by the MAPK signaling pathway represses PxABCB1 expression and confers Cry1Ac resistance in P. xylostella. Our results provide new insights into the transcriptional regulation mechanisms of midgut Cry receptor genes and deepen our understanding of the molecular basis of insect resistance to Bt Cry toxins. To our knowledge, this study identified the first transcription factor that can be involved in the transcriptional regulation mechanisms of midgut Cry receptor genes in Bt-resistant insects.

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A Novel Reference for Bt-Resistance Mechanism in Plutella xylostella Based on Analysis of the Midgut Transcriptomes

Insects ◽

10.3390/insects12121091 ◽

2021 ◽

Vol 12 (12) ◽

pp. 1091

Author(s):

Lei Xiong ◽

Zhaoxia Liu ◽

Lingling Shen ◽

Chao Xie ◽

Min Ye ◽

...

Keyword(s):

Plutella Xylostella ◽

Diamondback Moth ◽

Resistance Mechanism ◽

Anatomical Entity ◽

Bt Toxin ◽

Lepidopteran Insect ◽

Bt Resistance ◽

Bt Toxins ◽

Resistant Strains ◽

Shock Proteins

The diamondback moth, Plutella xylostella, is a lepidopteran insect that mainly harms cruciferous vegetables, with strong resistance to a variety of agrochemicals, including Bacillus thuringiensis (Bt) toxins. This study intended to screen genes associated with Bt resistance in P. xylostella by comparing the midgut transcriptome of Cry1Ac-susceptible and -resistant strains together with two toxin-treated strains 24 h before sampling. A total of 12 samples were analyzed by BGISEQ-500, and each sample obtained an average of 6.35 Gb data. Additionally, 3284 differentially expressed genes (DEGs) were identified in susceptible and resistant strains. Among them, five DEGs for cadherin, 14 for aminopeptidase, zero for alkaline phosphatase, 14 for ATP binding cassette transport, and five heat shock proteins were potentially involved in resistance to Cry1Ac in P. xylostella. Furthermore, DEGs associated with “binding”, “catalytic activity”, “cellular process”, “metabolic process”, and “cellular anatomical entity” were more likely to be responsible for resistance to Bt toxin. Thus, together with other omics data, our results will offer prospective genes for the development of Bt resistance, thereby providing a brand new reference for revealing the resistance mechanism to Bt of P. xylostella.

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Profiling of MicroRNAs in Midguts of Plutella xylostella Provides Novel Insights Into the Bacillus thuringiensis Resistance

Frontiers in Genetics ◽

10.3389/fgene.2021.739849 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jie Yang ◽

Xuejiao Xu ◽

Sujie Lin ◽

Shiyao Chen ◽

Guifang Lin ◽

...

Keyword(s):

Bacillus Thuringiensis ◽

Plutella Xylostella ◽

Target Genes ◽

Expression Patterns ◽

Mapk Signaling ◽

Differentially Expressed ◽

Luciferase Reporter ◽

Sequencing Analysis ◽

Bt Resistance ◽

Differentially Expressed Mirnas

The diamondback moth (DBM), Plutella xylostella, one of the most destructive lepidopteran pests worldwide, has developed field resistance to Bacillus thuringiensis (Bt) Cry toxins. Although miRNAs have been reported to be involved in insect resistance to multiple insecticides, our understanding of their roles in mediating Bt resistance is limited. In this study, we constructed small RNA libraries from midguts of the Cry1Ac-resistant (Cry1S1000) strain and the Cry1Ac-susceptible strain (G88) using a high-throughput sequencing analysis. A total of 437 (76 known and 361 novel miRNAs) were identified, among which 178 miRNAs were classified into 91 miRNA families. Transcripts per million analysis revealed 12 differentially expressed miRNAs between the Cry1S1000 and G88 strains. Specifically, nine miRNAs were down-regulated and three up-regulated in the Cry1S1000 strain compared to the G88 strain. Next, we predicted the potential target genes of these differentially expressed miRNAs and carried out GO and KEGG pathway analyses. We found that the cellular process, metabolism process, membrane and the catalytic activity were the most enriched GO terms and the Hippo, MAPK signaling pathway might be involved in Bt resistance of DBM. In addition, the expression patterns of these miRNAs and their target genes were determined by RT-qPCR, showing that partial miRNAs negatively while others positively correlate with their corresponding target genes. Subsequently, novel-miR-240, one of the differentially expressed miRNAs with inverse correlation with its target genes, was confirmed to interact with Px017590 and Px007885 using dual luciferase reporter assays. Our study highlights the characteristics of differentially expressed miRNAs in midguts of the Cry1S1000 and G88 strains, paving the way for further investigation of miRNA roles in mediating Bt resistance.

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The Anti-Atherogenic Properties of Sesamin are Mediated via Improved Macrophage Cholesterol Efflux Through PPARγ1-LXRα and MAPK Signaling

International Journal for Vitamin and Nutrition Research ◽

10.1024/0300-9831/a000195 ◽

2014 ◽

Vol 84 (1-2) ◽

pp. 79-91 ◽

Cited By ~ 7

Author(s):

Amin F. Majdalawieh ◽

Hyo-Sung Ro

Keyword(s):

Cholesterol Efflux ◽

Transcriptional Activity ◽

Molecular Mechanisms ◽

Foam Cell ◽

Mapk Signaling ◽

Luciferase Reporter ◽

Foam Cell Formation ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Macrophage Cholesterol Efflux

Background: Foam cell formation resulting from disrupted macrophage cholesterol efflux, which is triggered by PPARγ1 and LXRα, is a hallmark of atherosclerosis. Sesamin and sesame oil exert anti-atherogenic effects in vivo. However, the exact molecular mechanisms underlying such effects are not fully understood. Aim: This study examines the potential effects of sesamin (0, 25, 50, 75, 100 μM) on PPARγ1 and LXRα expression and transcriptional activity as well as macrophage cholesterol efflux. Methods: PPARγ1 and LXRα expression and transcriptional activity are assessed by luciferase reporter assays. Macrophage cholesterol efflux is evaluated by ApoAI-specific cholesterol efflux assays. Results: The 50 μM, 75 μM, and 100 μM concentrations of sesamin up-regulated the expression of PPARγ1 (p< 0.001, p < 0.001, p < 0.001, respectively) and LXRα (p = 0.002, p < 0.001, p < 0.001, respectively) in a concentration-dependent manner. Moreover, 75 μM and 100 μM concentrations of sesamin led to 5.2-fold (p < 0.001) and 6.0-fold (p<0.001) increases in PPAR transcriptional activity and 3.9-fold (p< 0.001) and 4.2-fold (p < 0.001) increases in LXR transcriptional activity, respectively, in a concentration- and time-dependent manner via MAPK signaling. Consistently, 50 μM, 75 μM, and 100 μM concentrations of sesamin improved macrophage cholesterol efflux by 2.7-fold (p < 0.001), 4.2-fold (p < 0.001), and 4.2-fold (p < 0.001), respectively, via MAPK signaling. Conclusion: Our findings shed light on the molecular mechanism(s) underlying sesamins anti-atherogenic effects, which seem to be due, at least in part, to its ability to up-regulate PPARγ1 and LXRα expression and transcriptional activity, improving macrophage cholesterol efflux. We anticipate that sesamin may be used as a therapeutic agent for treating atherosclerosis.

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TEAD4 modulated LncRNA MNX1-AS1 contributes to gastric cancer progression partly through suppressing BTG2 and activating BCL2

Molecular Cancer ◽

10.1186/s12943-019-1104-1 ◽

2020 ◽

Vol 19 (1) ◽

Cited By ~ 12

Author(s):

You Shuai ◽

Zhonghua Ma ◽

Weitao Liu ◽

Tao Yu ◽

Changsheng Yan ◽

...

Keyword(s):

Gastric Cancer ◽

Cancer Progression ◽

Molecular Mechanisms ◽

Mechanistic Model ◽

Ectopic Expression ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Tissue Samples ◽

Reporter Assays

Abstract Background Gastric cancer (GC) is the third leading cause of cancer-related mortality globally. Long noncoding RNAs (lncRNAs) are dysregulated in obvious malignancies including GC and exploring the regulatory mechanisms underlying their expression is an attractive research area. However, these molecular mechanisms require further clarification, especially upstream mechanisms. Methods LncRNA MNX1-AS1 expression in GC tissue samples was investigated via microarray analysis and further determined in a cohort of GC tissues via quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays. Cell proliferation and flow cytometry assays were performed to confirm the roles of MNX1-AS1 in GC proliferation, cell cycle regulation, and apoptosis. The influence of MNX1-AS1 on GC cell migration and invasion was explored with Transwell assays. A xenograft tumour model was established to verify the effects of MNX1-AS1 on in vivo tumourigenesis. The TEAD4-involved upstream regulatory mechanism of MNX1-AS1 was explored through ChIP and luciferase reporter assays. The mechanistic model of MNX1-AS1 in regulating gene expression was further detected by subcellular fractionation, FISH, RIP, ChIP and luciferase reporter assays. Results It was found that MNX1-AS1 displayed obvious upregulation in GC tissue samples and cell lines, and ectopic expression of MNX1-AS1 predicted poor clinical outcomes for patients with GC. Overexpressed MNX1-AS1 expression promoted proliferation, migration and invasion of GC cells markedly, whereas decreased MNX1-AS1 expression elicited the opposite effects. Consistent with the in vitro results, MNX1-AS1 depletion effectively inhibited the growth of xenograft tumour in vivo. Mechanistically, TEAD4 directly bound the promoter region of MNX1-AS1 and stimulated the transcription of MNX1-AS1. Furthermore, MNX1-AS1 can sponge miR-6785-5p to upregulate the expression of BCL2 in GC cells. Meanwhile, MNX1-AS1 suppressed the transcription of BTG2 by recruiting polycomb repressive complex 2 to BTG2 promoter regions. Conclusions Our findings demonstrate that MNX1-AS1 may be able to serve as a prognostic indicator in GC patients and that TEAD4-activatd MNX1-AS1 can promote GC progression through EZH2/BTG2 and miR-6785-5p/BCL2 axes, implicating it as a novel and potent target for the treatment of GC.

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Circular RNA mmu_circ_0005019 inhibits fibrosis of cardiac fibroblasts and reverses electrical remodeling of cardiomyocytes

BMC Cardiovascular Disorders ◽

10.1186/s12872-021-02128-w ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Na Wu ◽

Chengying Li ◽

Bin Xu ◽

Ying Xiang ◽

Xiaoyue Jia ◽

...

Keyword(s):

Molecular Mechanisms ◽

Cardiac Fibroblasts ◽

Circular Rna ◽

Protective Role ◽

Luciferase Reporter ◽

Loss Of Function ◽

Candidate Target ◽

Paired Control ◽

Reporter Assays ◽

And Migration

Abstract Background Circular RNA (circRNA) have been reported to play important roles in cardiovascular diseases including myocardial infarction and heart failure. However, the role of circRNA in atrial fibrillation (AF) has rarely been investigated. We recently found a circRNA hsa_circ_0099734 was significantly differentially expressed in the AF patients atrial tissues compared to paired control. We aim to investigate the functional role and molecular mechanisms of mmu_circ_0005019 which is the homologous circRNA in mice of hsa_circ_0099734 in AF. Methods In order to investigate the effect of mmu_circ_0005019 on the proliferation, migration, differentiation into myofibroblasts and expression of collagen of cardiac fibroblasts, and the effect of mmu_circ_0005019 on the apoptosis and expression of Ito, INA and SK3 of cardiomyocytes, gain- and loss-of-function of cell models were established in mice cardiac fibroblasts and HL-1 atrial myocytes. Dual-luciferase reporter assays and RIP were performed to verify the binding effects between mmu_circ_0005019 and its target microRNA (miRNA). Results In cardiac fibroblasts, mmu_circ_0005019 showed inhibitory effects on cell proliferation and migration. In cardiomyocytes, overexpression of mmu_circ_0005019 promoted Kcnd1, Scn5a and Kcnn3 expression. Knockdown of mmu_circ_0005019 inhibited the expression of Kcnd1, Kcnd3, Scn5a and Kcnn3. Mechanistically, mmu_circ_0005019 exerted biological functions by acting as a miR-499-5p sponge to regulate the expression of its target gene Kcnn3. Conclusions Our findings highlight mmu_circ_0005019 played a protective role in AF development and might serve as an attractive candidate target for AF treatment.

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Knockdown of Circ_SLC39A8 protects against the progression of osteoarthritis by regulating miR-591/IRAK3 axis

Journal of Orthopaedic Surgery and Research ◽

10.1186/s13018-021-02323-7 ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Jizhe Yu ◽

Yushuang Qin ◽

Naxin Zhou

Keyword(s):

Cell Viability ◽

Molecular Mechanisms ◽

Interleukin 1 ◽

Cell Counting ◽

Luciferase Reporter ◽

Circular Rnas ◽

Oa Chondrocytes ◽

Polymerase Chain ◽

Apoptosis And Inflammation ◽

The Relationship

Abstract Background The dysregulation of circular RNAs (circRNAs) has been identified in various human diseases, including osteoarthritis (OA). The purpose of this study was to identify the role and mechanism of circ_SLC39A8 in regulating the progression of OA. Methods The expression levels of circ_SLC39A8, miR-591, and its potential target gene, interleukin-1-receptor-associated kinase 3 (IRAK3), were identified by quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability and apoptosis were determined by Cell Counting Kit-8 (CCK-8) assay and flow cytometry, respectively. The relationship between miR-591 and circ_SLC39A8 or IRAK3 was predicted by bioinformatics tools and verified by dual-luciferase reporter. Results Circ_SLC39A8 and IRAK3 were upregulated and miR-591 was downregulated in OA cartilage tissues. Knockdown of circ_SLC39A8 inhibited apoptosis and inflammation in OA chondrocytes, while these effects were reversed by downregulating miR-591. Promotion cell viability effects of miR-591 were partially reversed by IRAK3 overexpression. Conclusion Our findings indicated that knockdown of circ_SLC39A8 delayed the progression of OA via modulating the miR-591-IRAK3 axis, providing new insight into the molecular mechanisms of OA pathogenesis.

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Pyramiding of cry toxins and methanol producing genes to increase insect resistance in cotton

GM crops & food ◽

10.1080/21645698.2021.1944013 ◽

2021 ◽

Vol 12 (1) ◽

pp. 382-395

Author(s):

Abdul Razzaq ◽

Arfan Ali ◽

Muhammad Mubashar Zafar ◽

Aisha Nawaz ◽

Deng Xiaoying ◽

...

Keyword(s):

Insect Resistance ◽

Cry Toxins

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Circular RNA hsa_circ_0000277 sequesters miR-4766-5p to upregulate LAMA1 and promote esophageal carcinoma progression

Cell Death and Disease ◽

10.1038/s41419-021-03911-5 ◽

2021 ◽

Vol 12 (7) ◽

Author(s):

Peng Li Zhou ◽

Zhengyang Wu ◽

Wenguang Zhang ◽

Miao Xu ◽

Jianzhuang Ren ◽

...

Keyword(s):

Molecular Mechanisms ◽

Bioinformatics Analysis ◽

Epithelial Mesenchymal Transition ◽

Expression Profiles ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Circular Rnas ◽

Mesenchymal Transition ◽

Dismal Prognosis ◽

Advanced Tumor

AbstractGrowing evidence has indicated that circular RNAs (circRNAs) play a pivotal role as functional RNAs in diverse cancers. However, most circRNAs involved in esophageal squamous cell carcinoma (ESCC) remain undefined, and the underlying molecular mechanisms mediated by circRNAs are largely unclear. Here, we screened human circRNA expression profiles in ESCC tissues and found significantly increased expression of hsa_circ_0000277 (termed circPDE3B) in ESCC tissues and cell lines compared to the normal controls. Moreover, higher circPDE3B expression in patients with ESCC was correlated with advanced tumor-node-metastasis (TNM) stage and dismal prognosis. Functional experiments demonstrated that circPDE3B promoted the tumorigenesis and metastasis of ESCC cells in vitro and in vivo. Mechanistically, bioinformatics analysis, a dual-luciferase reporter assay, and anti-AGO2 RNA immunoprecipitation showed that circPDE3B could act as a competing endogenous RNA (ceRNA) by harboring miR-4766-5p to eliminate the inhibitory effect on the target gene laminin α1 (LAMA1). In addition, LAMA1 was significantly upregulated in ESCC tissues and was positively associated with the aggressive oncogenic phenotype. More importantly, rescue experiments revealed that the oncogenic role of circPDE3B in ESCC is partly dependent on the miR-4766-5p/LAMA1 axis. Furthermore, bioinformatics analysis combined with validation experiments showed that epithelial-mesenchymal transition (EMT) activation was involved in the oncogenic functions of the circPDE3B–miR-4766-5p/LAMA1 axis in ESCC. Taken together, we demonstrate for the first time that the circPDE3B/miR-4766-5p/LAMA1 axis functions as an oncogenic factor in promoting ESCC cell proliferation, migration, and invasion by inducing EMT, implying its potential prognostic and therapeutic significance in ESCC.

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Circular RNA AFF4 modulates osteogenic differentiation in BM-MSCs by activating SMAD1/5 pathway through miR-135a-5p/FNDC5/Irisin axis

Cell Death and Disease ◽

10.1038/s41419-021-03877-4 ◽

2021 ◽

Vol 12 (7) ◽

Author(s):

Chao Liu ◽

An-Song Liu ◽

Da Zhong ◽

Cheng-Gong Wang ◽

Mi Yu ◽

...

Keyword(s):

Bone Marrow ◽

Bone Formation ◽

Osteogenic Differentiation ◽

Molecular Mechanisms ◽

Expression Patterns ◽

Regulatory System ◽

Circular Rna ◽

Luciferase Reporter ◽

Integrin Αv

AbstractBone marrow-derived mesenchymal stem cells (BM-MSCs), the common progenitor cells of adipocytes and osteoblasts, have been recognized as the key mediator during bone formation. Herein, our study aim to investigate molecular mechanisms underlying circular RNA (circRNA) AFF4 (circ_AFF4)-regulated BM-MSCs osteogenesis. BM-MSCs were characterized by FACS, ARS, and ALP staining. Expression patterns of circ_AFF4, miR-135a-5p, FNDC5/Irisin, SMAD1/5, and osteogenesis markers, including ALP, BMP4, RUNX2, Spp1, and Colla1 were detected by qRT-PCR, western blot, or immunofluorescence staining, respectively. Interactions between circ_AFF4 and miR-135a-5p, FNDC5, and miR-135a-5p were analyzed using web tools including TargetScan, miRanda, and miRDB, and further confirmed by luciferase reporter assay and RNA pull-down. Complex formation between Irisin and Integrin αV was verified by Co-immunoprecipitation. To further verify the functional role of circ_AFF4 in vivo during bone formation, we conducted animal experiments harboring circ_AFF4 knockdown, and born samples were evaluated by immunohistochemistry, hematoxylin and eosin, and Masson staining. Circ_AFF4 was upregulated upon osteogenic differentiation induction in BM-MSCs, and miR-135a-5p expression declined as differentiation proceeds. Circ_AFF4 knockdown significantly inhibited osteogenesis potential in BM-MSCs. Circ_AFF4 stimulated FNDC5/Irisin expression through complementary binding to its downstream target molecule miR-135a-5p. Irisin formed an intermolecular complex with Integrin αV and activated the SMAD1/5 pathway during osteogenic differentiation. Our work revealed that circ_AFF4, acting as a sponge of miR-135a-5p, triggers the promotion of FNDC5/Irisin via activating the SMAD1/5 pathway to induce osteogenic differentiation in BM-MSCs. These findings gained a deeper insight into the circRNA-miRNA regulatory system in the bone marrow microenvironment and may improve our understanding of bone formation-related diseases at physiological and pathological levels.

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