scholarly journals MiR-4448 is involved in deltamethrin resistance by targeting CYP4H31 in Culex pipiens pallens

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Xixi Li ◽  
Shengli Hu ◽  
Haitao Yin ◽  
Hongbo Zhang ◽  
Dan Zhou ◽  
...  
Keyword(s):  
Culex Pipiens ◽  
Oral Feeding ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Culex Pipiens Pallens ◽  
Deltamethrin Resistance ◽  
Regulatory Functions ◽  
Pipiens Pallens

Abstract Background Culex pipiens (Cx. pipiens) complex, which acts as a vector of viruses and is widespread and abundant worldwide, including West Nile virus, Japanese encephalitis virus, and Sindbis virus, can cause serious vector-borne diseases affecting human health. Unfortunately, mosquitoes have developed deltamethrin resistance because of its long-term overuse, representing a major challenge to mosquito control. Understanding the molecular regulatory mechanisms of resistance is vital to control mosquitoes. MicroRNAs (miRNAs) are short non-coding RNAs that have been demonstrated to be important regulators of gene expression across a wide variety of organisms, which might function in mosquito deltamethrin resistance. In the present study, we aimed to investigate the regulatory functions of miR-4448 and CYP4H31 in the formation of insecticidal resistance in mosquito Culex pipiens pallens. Methods We used quantitative real-time reverse transcription PCR to measure miR-4448 and CYP4H31 (encoding a cytochrome P450) expression levels. The regulatory functions of miR-4448 and CYP4H31 were assessed using dual-luciferase reporter assays. Then, oral feeding, RNA interference, and the American Centers for Disease Control and Prevention bottle bioassay were used to determine miR-4448’s association with deltamethrin resistance by targeting CYP4H31in vivo. Cell Counting Kit-8 (CCK-8) was also used to detect the viability of pIB/V5-His-CYP4H31-transfected C6/36 cells after deltamethrin treatment in vitro. Results MiR-4448 was downregulated in the deltamethrin-resistant strain (DR strain), whereas CYP4H31 was downregulated in deltamethrin-susceptible strain. CYP4H31 expression was downregulated by miR-4448 recognizing and binding to its 3′ untranslated region. Functional verification experiments showed that miR-4448 overexpression resulted in lower expression of CYP4H31. The mortality of miR-4448 mimic-injected DR strain mosquitoes was higher than that of the controls. CCK-8 assays showed that CYP4H31 decreased cellular resistance to deltamethrin in vitro and the mortality of the DR strain increased when CYP4H31 was knocked down in vivo. Conclusions In mosquitoes, miR-4448 participates in deltamethrin resistance by targeting CYP4H31. The results of the present study increase our understanding of deltamethrin resistance mechanisms.

scholarly journals MiR-279-3p regulates deltamethrin resistance through CYP325BB1 in Culex pipiens pallens

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Xixi Li ◽  
Shengli Hu ◽  
Hongbo Zhang ◽  
Haitao Yin ◽  
Huan Wang ◽  
...  
Keyword(s):  
Insecticide Resistance ◽  
Culex Pipiens ◽  
Oral Feeding ◽  
Luciferase Reporter ◽  
Culex Pipiens Pallens ◽  
Mosquito Mortality ◽  
Deltamethrin Resistance ◽  
Pipiens Pallens

Abstract Background The overuse of insecticides to control insect vectors has promoted extensive insecticide resistance in mosquitoes. In this study, the functions of microRNA (miR)-279-3p and its target CYP325BB1 in the regulation of deltamethrin resistance in Culex pipiens pallens was investigated. Methods Quantitative real-time reverse transcription PCR was used to detect the expression levels of miR-279-3p and CYP325BB1. Then, the dual-luciferase reporter assay system, RNA interference, CDC bottle bioassay and Cell Counting Kit-8 (CCK-8) assay were used to explore the roles of these molecules in deltamethrin resistance both in vivo and in vitro. Results The expression patterns of miR-279-3p and CYP325BB1 were compared between deltamethrin-sensitive (DS-strain) and deltamethrin-resistant (DR-strain) mosquitoes. Luciferase activity was downregulated by miR-279-3p, the effect of which was ablated by a mutation of the putative binding site for CYP325BB1. In DR-strain mosquitoes, the expression of miR-279-3p was increased by microinjection and oral feeding of miR-279-3p agomir (mimic). CYP325BB1 mRNA levels were downregulated, which resulted in a higher mortality of the mosquitoes in miR-279-3p mimic-treated groups. In the DS-strain mosquitoes, microinjection of a miR-279-3p inhibitor decreased miR-279-3p expression, whereas the expression of CYP325BB1 was increased; the mortality of these mosquitoes decreased significantly. In addition, overexpression of pIB/V5-His-CYP325BB1 changed the sensitivity of C6/36 cells to deltamethrin in vitro. Also in DR-strain mosquitoes, downregulation of CYP325BB1 expression by microinjection of si-CYP325BB1 increased mosquito mortality in vivo. Conclusions These findings provide empirical evidence of the involvement of miRNAs in the regulation of insecticide resistance and indicate that miR-279-3p suppresses the expression of CYP325BB1, which in turn decreases deltamethrin resistance, resulting in increased mosquito mortality. Taken together, the results provide important information for use in the development of future mosquito control strategies. Graphical abstract

scholarly journals MiR-4448 Is Involved in Deltamethrin Resistance by Targeting CYP4H31 in Culex Pipiens Pallens

2020 ◽  
Author(s):  
Xixi Li ◽  
Shengli Hu ◽  
Haitao Yin ◽  
Hongbo Zhang ◽  
Dan Zhou ◽  
...  
Keyword(s):  
Mosquito Control ◽  
Resistance Mechanisms ◽  
Oral Feeding ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Functional Verification ◽  
Reverse Transcription Pcr ◽  
Deltamethrin Resistance

Abstract Background: Mosquitoes are vectors of serious diseases affecting human health. Unfortunately, mosquitoes have developed deltamethrin resistance because of its long-term overuse, representing a major challenge to mosquito control. Understanding the molecular regulatory mechanisms of resistance is vital to control mosquitoes. MicroRNAs (miRNAs) are short non‑coding RNAs that have been demonstrated as important regulators of gene expression across a wide variety of organisms, which might function in mosquito deltamethrin resistance. Methods: In the present study, we used quantitative real-time reverse transcription PCR to measure miR-4448 and CYP4H31 (encoding a cytochrome P450) expression levels. The regulatory functions of miR-4448 and CYP4H31 were assessed using Dual-Luciferase reporter assays. Then, oral feeding, RNA interference, and the American Centers for Disease Control and Prevention bottle bioassay were used to determine miR‑4448’s association with deltamethrin resistance by targeting CYP4H31 in vivo. A Cell Counting Kit-8 (CCK-8) was also used to detect the viability of pIB/V5-His-CYP4H31-transfected C6/36 cells after deltamethrin treatment in vitro. Results: MiR-4448 was downregulated in the deltamethrin-resistant strain (DR-strain), whereas CYP4H31 was downregulated in deltamethrin-susceptible strain. negatively regulated CYP4H31 expression was downregulated by miR-4448 recognizing and binding to its 3' untranslated region. Functional verification experiments showed that miR-4448 overexpression resulted in lower expression of CYP4H31. The mortality of miR-4448 mimic-injected DR-strain mosquitoes was higher than that of the controls. CCK-8 assays showed that CYP4H31 decreased cellular resistance to deltamethrin in vitro and the mortality of the DR-strain increased when CYP4H31 was knocked down CYP4H31 in vivo. Conclusions: In mosquitoes, miR-4448 participates in deltamethrin resistance by targeting CYP4H31. The results of the present study increase our understanding of deltamethrin resistance mechanisms.

scholarly journals Transcription factor FTZ-F1 regulates mosquito cuticular protein CPLCG5 conferring resistance to pyrethroids in Culex pipiens pallens

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Yang Xu ◽  
Xiaoshan Yang ◽  
Xiaohong Sun ◽  
Xixi Li ◽  
Zhihan Liu ◽  
...  
Keyword(s):  
Culex Pipiens ◽  
Pyrethroid Resistance ◽  
Luciferase Reporter ◽  
Cuticular Protein ◽  
Culex Pipiens Pallens ◽  
Pyrethroid Insecticides ◽  
Cuticle Thickness ◽  
Pipiens Pallens ◽  
Cuticular Resistance

Abstract Background Culex pipiens pallens poses a serious threat to human health because of its widespread distribution, high carrier capacity for several arboviruses, frequent human-biting, and growth in urban environments. Pyrethroid insecticides have been mainly used to control adult Cx. pipiens pallens during outbreaks of mosquito-borne diseases. Unfortunately, mosquitoes have developed resistance, rendering the insecticides ineffective. Cuticular resistance is the primary mechanism of pyrethroid resistance. Previously, we revealed that cuticular protein of low complexity CPLCG5 is a major cuticular protein associated with deltamethrin resistance in Cx. pipiens pallens, which is enriched in the cuticle of mosquitoes’ legs and participates in pyrethroid resistance by forming a rigid matrix. However, the regulatory mechanisms of its transcription remain unknown. Results First, qRT-PCR analysis revealed that the expression of FTZ-F1 (encoding Fushi tarazu-Factor 1) was ~ 1.8-fold higher in the deltamethrin-resistant (DR) than deltamethrin-susceptible (DS) strains at 24 h post-eclosion (PE) and ~ 2.2-fold higher in the DR strain than in the DS strain at 48 h PE. CPLCG5 and FTZ-F1 were co-expressed in the legs, indicating that they might play an essential role in the legs. Dual luciferase reporter assays and EMSA (electrophoretic mobility shift experiments) revealed that FTZ-F1 regulates the transcription of CPLCG5 by binding to the FTZ-F1 response element (− 870/− 864). Lastly, knockdown of FTZ-F1 not only affected CPLCG5 expression but also altered the cuticle thickness and structure of the legs, increasing the susceptibility of the mosquitoes to deltamethrin in vivo. Conclusions The results revealed that FTZ-F1 regulates the expression of CPLCG5 by binding to the CPLCG5 promoter region, altering cuticle thickness and structure, and increasing the susceptibility of mosquitoes to deltamethrin in vivo. This study revealed part of the mechanism of cuticular resistance, providing a deeper understanding of insecticide resistance.

scholarly journals A novel phosphoramide compound, DCZ0805, shows potent anti-myeloma activity via the NF-κB pathway

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Xuejie Gao ◽  
Bo Li ◽  
Anqi Ye ◽  
Houcai Wang ◽  
Yongsheng Xie ◽  
...  
Keyword(s):  
Mouse Model ◽  
Tumor Burden ◽  
High Rate ◽  
Cell Counting ◽  
Tumor Xenograft ◽  
Luciferase Reporter ◽  
Therapeutic Effects ◽  
Xenograft Mouse Model

Abstract Background Multiple myeloma (MM) is a highly aggressive and incurable clonal plasma cell disease with a high rate of recurrence. Thus, the development of new therapies is urgently needed. DCZ0805, a novel compound synthesized from osalmide and pterostilbene, has few observed side effects. In the current study, we intend to investigate the therapeutic effects of DCZ0805 in MM cells and elucidate the molecular mechanism underlying its anti-myeloma activity. Methods We used the Cell Counting Kit-8 assay, immunofluorescence staining, cell cycle assessment, apoptosis assay, western blot analysis, dual-luciferase reporter assay and a tumor xenograft mouse model to investigate the effect of DCZ0805 treatment both in vivo and in vitro. Results The results showed that DCZ0805 treatment arrested the cell at the G0/G1 phase and suppressed MM cells survival by inducing apoptosis via extrinsic and intrinsic pathways. DCZ0805 suppressed the NF-κB signaling pathway activation, which may have contributed to the inhibition of cell proliferation. DCZ0805 treatment remarkably reduced the tumor burden in the immunocompromised xenograft mouse model, with no obvious toxicity observed. Conclusion The findings of this study indicate that DCZ0805 can serve as a novel therapeutic agent for the treatment of MM.

scholarly journals LncRNA SNHG15 contributes to doxorubicin resistance of osteosarcoma cells through targeting the miR-381-3p/GFRA1 axis

2020 ◽  
Vol 15 (1) ◽  
pp. 871-883
Author(s):  
Jinshan Zhang ◽  
Dan Rao ◽  
Haibo Ma ◽  
Defeng Kong ◽  
Xiaoming Xu ◽  
...  
Keyword(s):  
Cell Lines ◽  
Noncoding Rna ◽  
Xenograft Model ◽  
Inhibitory Effect ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Osteosarcoma Cell ◽  
Osteosarcoma Cells

AbstractBackgroundOsteosarcoma is a common primary malignant bone cancer. Long noncoding RNA small nucleolar RNA host gene 15 (SNHG15) has been reported to play an oncogenic role in many cancers. Nevertheless, the role of SNHG15 in the doxorubicin (DXR) resistance of osteosarcoma cells has not been fully addressed.MethodsCell Counting Kit-8 assay was conducted to measure the half-maximal inhibitory concentration value of DXR in osteosarcoma cells. Western blotting was carried out to examine the levels of autophagy-related proteins and GDNF family receptor alpha-1 (GFRA1). Quantitative reverse transcription-polymerase chain reaction was performed to determine the levels of SNHG15, miR-381-3p, and GFRA1. The proliferation of osteosarcoma cells was measured by MTT assay. The binding sites between miR-381-3p and SNHG15 or GFRA1 were predicted by Starbase bioinformatics software, and the interaction was confirmed by dual-luciferase reporter assay. Murine xenograft model was established to validate the function of SNHG15 in vivo.ResultsAutophagy inhibitor 3-methyladenine sensitized DXR-resistant osteosarcoma cell lines to DXR. SNHG15 was upregulated in DXR-resistant osteosarcoma tissues and cell lines. SNHG15 knockdown inhibited the proliferation, DXR resistance, and autophagy of osteosarcoma cells. MiR-381-3p was a direct target of SNHG15, and GFRA1 bound to miR-381-3p in osteosarcoma cells. SNHG15 contributed to DXR resistance through the miR-381-3p/GFRA1 axis in vitro. SNHG15 depletion contributed to the inhibitory effect of DXR on osteosarcoma tumor growth through the miR-381-3p/GFRA1 axis in vivo.ConclusionsSNHG15 enhanced the DXR resistance of osteosarcoma cells through elevating the autophagy via targeting the miR-381-3p/GFRA1 axis. Restoration of miR-381-3p expression might be an underlying therapeutic strategy to overcome the DXR resistance of osteosarcoma.

scholarly journals Ribose-phosphate pyrophosphokinase 1 (PRPS1) associated with deltamethrin resistance in Culex pipiens pallens

2012 ◽  
Vol 112 (2) ◽  
pp. 847-854 ◽  
Author(s):  
Shanchao Hong ◽  
Dan Zhou ◽  
Chen Chen ◽  
Weijie Wang ◽  
Yuan Lv ◽  
...  
Keyword(s):  
Culex Pipiens ◽  
Culex Pipiens Pallens ◽  
Ribose Phosphate ◽  
Deltamethrin Resistance ◽  
Pipiens Pallens

scholarly journals Downregulation of circ-PSMB6 suppresses NSCLC progression and metastasis through sponging miR-532-5p and regulating EZH1 expression

2021 ◽  
Author(s):  
Mingming Jin ◽  
Junqian Zhang ◽  
Yue Wu ◽  
Yitian Dai ◽  
Gang Huang
Keyword(s):  
Gene Expression ◽  
High Throughput Sequencing ◽  
Stem Cell Differentiation ◽  
Nsclc Cell ◽  
Cell Counting ◽  
Regulatory Mechanisms ◽  
Luciferase Reporter ◽  
Circular Rnas

Abstract Background: Accumulating reports showed how circular RNAs (circRNAs) act importantly during tumor progression via regulating gene expression, but regulatory mechanisms remain largely unknown. Current investigation clarified circRNA regulatory mechanisms in non-small cell lung cancer (NSCLC).Methods: High-throughput sequencing and quantitative reverse transcription polymerase chain reaction (RT-qPCR) detection were utilized to explore circRNA expression in NSCLC tissues and cells. Our lab did statistical analyses and luciferase reporter analysis to validate correlations between circRNA, miRNA and gene expression. We transfected NSCLC cells with different vectors, and transwell migration, Cell Counting Kit-8 (CCK-8) proliferation along with colony formation assays were performed. In vivo tumorigenesis and metastasis assays were utilized to validate the circRNA role in NSCLC.Results: Data illustrated that hsa_circ_0041595 (circ-PSMB6) incremented in NSCLC cell lines and tissues, while circ-PSMB6 downregulation suppressed NSCLC cell proliferation and invasion in vitro and in vivo. Bioinformatics analysis and luciferase reporter data verified that miR-532-5p and Enhancer Of Zeste 1 Polycomb Repressive Complex 2 Subunit (EZH1) were circ-PSMB6 downstream targets in NSCLC cells. Overexpression of EZH1 or miR-532-5p inhibition reversed NSCLC cell invasion and proliferation after silencing circ-PSMB6. Further experiments discovered that circ-PSMB6 can influence cancer stem cell differentiation by regulating miR-532-5p/EZH1.Conclusions: Taken together, we found that circ-PSMB6 suppressed NSCLC metastasis and progression via sponging miR-532-5p and regulating EZH1 expression.

scholarly journals Mutant NPM1-regulated lncRNA HOTAIRM1 promotes leukemia cell autophagy and proliferation by targeting EGR1 and ULK3

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Yipei Jing ◽  
Xueke Jiang ◽  
Li Lei ◽  
Meixi Peng ◽  
Jun Ren ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Animal Studies ◽  
Leukemia Cell ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Promising Therapeutic Target ◽  
Rna Immunoprecipitation ◽  
Incorporation Assay

Abstract Background Acute myeloid leukemia (AML) with mutated nucleophosmin (NPM1), which displays a distinct long noncoding RNA (lncRNA) expression profile, has been defined as a unique subgroup in the new classification of myeloid neoplasms. However, the biological roles of key lncRNAs in the development of NPM1-mutated AML are currently unclear. Here, we aimed to investigate the functional and mechanistic roles of the lncRNA HOTAIRM1 in NPM1-mutated AML. Methods The expression of HOTAIRM1 was analyzed with a public database and further determined by qRT-PCR in NPM1-mutated AML samples and cell lines. The cause of upregulated HOTAIRM1 expression was investigated by luciferase reporter, chromatin immunoprecipitation and ubiquitination assays. The functional role of HOTAIRM1 in autophagy and proliferation was evaluated using western blot analysis, immunofluorescence staining, a Cell Counting Kit-8 (CCK-8) assay, a 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay, flow cytometric analyses and animal studies. The action mechanism of HOTAIRM1 was explored through RNA fluorescence in situ hybridization, RNA pulldown and RNA immunoprecipitation assays. Results HOTAIRM1 was highly expressed in NPM1-mutated AML. High HOTAIRM1 expression was induced in part by mutant NPM1 via KLF5-dependent transcriptional regulation. Importantly, HOTAIRM1 promoted autophagy and proliferation both in vitro and in vivo. Mechanistic investigations demonstrated that nuclear HOTAIRM1 promoted EGR1 degradation by serving as a scaffold to facilitate MDM2-EGR1 complex formation, while cytoplasmic HOTAIRM1 acted as a sponge for miR-152-3p to increase ULK3 expression. Conclusions Taken together, our findings identify two oncogenic regulatory axes in NPM1-mutated AML centered on HOTAIRM1: one involving EGR1 and MDM2 in the nucleus and the other involving the miR-152-3p/ULK3 axis in the cytoplasm. Our study indicates that HOTAIRM1 may be a promising therapeutic target for this distinct leukemia subtype.

scholarly journals Diverse sensitivities of TRPA1 from different mosquito species to thermal and chemical stimuli

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Tianbang Li ◽  
Claire T. Saito ◽  
Tomoyuki Hikitsuchi ◽  
Yoshihiro Inoguchi ◽  
Honami Mitsuishi ◽  
...  
Keyword(s):  
Culex Pipiens ◽  
Transient Receptor Potential ◽  
Mosquito Control ◽  
Splice Variants ◽  
Mosquito Species ◽  
Temperature Threshold ◽  
Culex Pipiens Pallens ◽  
Chemical Stimuli ◽  
Pipiens Pallens

AbstractTemperature and odors profoundly affect the behavior of animals. Transient receptor potential channel, subfamily A, member 1 (TRPA1) functions as a polymodal nociceptor for sensing both vital environmental cues in insects. Mosquitoes are recognized as disease vectors, and many efforts have been devoted to investigations of their host-seeking behaviors and repellents. However, the physiological characteristics of mosquito TRPA1 have not been systematically studied. We identified multiple alternative splice variants of the TrpA1 gene from Anopheles gambiae, Anopheles stephensi, Aedes aegypti and Culex pipiens pallens mosquitoes. And we performed comparative analyses of the responses of mosquito TRPA1s to heat or chemical stimuli with calcium-imaging and whole-cell patch-clamp methods. Comparison of TRPA1 among four mosquito species from different thermal niches revealed that TRPA1 of Culex pipiens pallens inhabiting the temperate zone had a lower temperature threshold for heat-evoked activation, which was supported by the in vivo heat-avoidance test. Notably, the chemosensitivity of mosquito TRPA1 channels revealed differences not only between variants but also among species. Moreover, we discovered three novel mosquito TRPA1 agonists. Thermal niches selection and evolutionary trajectories significantly affect the functional properties of mosquito TRPA1, which represents a hallmark of the behaviors that may permit the design of improved mosquito control methods.

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