Gentamicin Inhibits Ca2+ Channel TPRV5 and Induces Calciuresis Independent of the Calcium-Sensing Receptor-Claudin-14 Pathway

2022 ◽  
pp. ASN.2021030392
Author(s):  
Wouter van Megen ◽  
Megan Beggs ◽  
Sung-Wan An ◽  
Patrícia Ferreira ◽  
Justin Lee ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Hek293 Cells ◽  
Luciferase Reporter ◽  
Thick Ascending Limb ◽  
Wild Type ◽  
Distal Nephron ◽  
Proximal Tubular ◽  
Claudin 14

Background Treatment with the aminoglycoside antibiotic gentamicin can be associated with severe adverse effects, including renal calcium wasting. The underlying mechanism is unknown but it has been proposed to involve activation of the Ca2+-sensing receptor (CaSR) in the thick ascending limb, which would increase expression of claudin-14 (CLDN14) and limit Ca2+ reabsorption. However, no direct evidence for this hypothesis has been presented. Methods We studied the effect of gentamicin in vivo using mouse models with impaired Ca2+ reabsorption in the proximal tubule and the thick ascending limb. We used a Cldn14 promoter luciferase-reporter assay to study CaSR activation and investigated the effect of gentamicin on activity of the distal nephron Ca2+ channel transient potential receptor vanilloid 5 (TPRV5), as determined by patch-clamp in HEK293 cells. Results Gentamicin increased urinary Ca2+ excretion in wild-type mice following acute and chronic administration. This calciuretic effect was unaltered in mice with genetic CaSR overactivation and was present in furosemide-treated animals, whereas the calciuretic effect in Cldn14-/-mice and mice with impaired proximal tubular Ca2+ reabsorption (claudin-2 [CLDN2]-deficient Cldn2-/- mice) was equivalent to that of wild-type mice. In vitro, gentamicin failed to activate the CaSR. In contrast, patch-clamp analysis revealed that gentamicin strongly inhibited rabbit and human TRPV5 activity and that chronic gentamicin administration downregulated distal nephron Ca2+ transporters. Conclusions Gentamicin does not cause hypercalciuria via activation of the CaSR-CLDN14 pathway or by interfering with proximal tubular CLDN2-dependent Ca2+ reabsorption. Instead, gentamicin blocks distal Ca2+ reabsorption by direct inhibition of the Ca2+ channel TRPV5. These findings offer new insights into calcium wasting in patients treated with gentamicin.

scholarly journals Protective Signatures of Roselle (Hibiscus sabdariffa L.) Calyx Fractions against Staphylococcus aureus in Drosophila Infection Model

2020 ◽  
Vol 27 (4) ◽  
pp. 306
Author(s):  
Firzan Nainu ◽  
M. Natsir Djide ◽  
Subehan Subehan ◽  
Sartini Sartini ◽  
Tri Puspita Roska ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Early Death ◽  
Luciferase Reporter ◽  
Infection Model ◽  
Hibiscus Sabdariffa ◽  
Immune Stimulation ◽  
Wild Type ◽  
Toll Pathway

The rise of antibiotic-resistant Staphylococcus aureus-related clinical cases is an alarming chronicle for global communities. This research was conducted to examine the antistaphylococcal effect of roselle (Hibiscus sabdariffa L.) calyx fractions in the Drosophila model. In the infection experiment, wild-type and immunodeficient Drosophila were pricked with S. aureus and subsequently subjected to fly survivorship and colony-forming assays, in the presence or absence of roselle calyx fractions. The Involvement of immune stimulation in the host antibacterial protection was assessed in vitro using cell-based luciferase reporter assay and in vivo using RT-qPCR analysis on adult flies. A declining rate of fly survivorship and augmentation of bacterial growth were observable in S. aureus-infected wild-type flies but subject to improvement in the presence of roselle calyx fractions. Cell-based analysis revealed the absence of host immune stimulation via Drosophila Toll pathway and roselle calyx fractions-treated immune-deficient flies lacking for components in the Toll pathway were protected from infection-induced early death phenotype and harbored reduced number of S. aureus colonies. Overall, our data confirmed the in vivo anti-staphylococcal activity of roselle calyx fractions in Drosophila infection model and such protective signature was devoid of host immune stimulation.

The cathepsin L gene is a direct target of FOXO1 in skeletal muscle

2010 ◽  
Vol 427 (1) ◽  
pp. 171-178 ◽  
Author(s):  
Yoshihiro Yamazaki ◽  
Yasutomi Kamei ◽  
Satoshi Sugita ◽  
Fumiko Akaike ◽  
Sayaka Kanai ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Transgenic Mice ◽  
Binding Sites ◽  
Cathepsin L ◽  
Skeletal Muscle Atrophy ◽  
Luciferase Reporter ◽  
Wild Type ◽  
Direct Target

FOXO1 (forkhead box O1), a forkhead-type transcription factor whose gene expression is up-regulated in the skeletal muscle during starvation, appears to be a key molecule of energy metabolism and skeletal muscle atrophy. Cathepsin L, a lysosomal proteinase whose expression is also up-regulated in the skeletal muscle during starvation, is induced in transgenic mice overexpressing FOXO1 relative to wild-type littermates. In the present study, we conducted in vivo and in vitro experiments focusing on FOXO1 regulation of Ctsl (cathepsin L gene; CTSL1 in humans) expression in the skeletal muscle. During fasting and refeeding of C57BL/6 mice, Ctsl was regulated in parallel with FOXO1 in the skeletal muscle. Fasting-induced Ctsl expression was attenuated in transgenic mice overexpressing a dominant-negative form of FOXO1 or in skeletal-muscle-specific Foxo1-knockout mice relative to respective wild-type controls. Using C2C12 mouse myoblasts overexpressing a constitutively active form of FOXO1, we showed that FOXO1 induces Ctsl expression. Moreover, we found FOXO1-binding sites in both the mouse Ctsl and human CTSL1 promoters. The luciferase reporter analysis revealed that the mouse Ctsl and human CTSL1 promoters are activated by FOXO1, which is abolished by mutations in the consensus FOXO1-binding sites. Gel mobility-shift and chromatin immunoprecipiation assays showed that FOXO1 is recruited and binds to the Ctsl promoter. The present study provides in vivo and in vitro evidence that Ctsl is a direct target of FOXO1 in the skeletal muscle, thereby suggesting a role for the FOXO1/cathepsin L pathway in fasting-induced skeletal muscle metabolic change and atrophy.

scholarly journals The Calcium-Sensing Receptor Increases Activity of the Renal NCC through the WNK4-SPAK Pathway

2018 ◽  
Vol 29 (7) ◽  
pp. 1838-1848 ◽  
Author(s):  
Silvana Bazúa-Valenti ◽  
Lorena Rojas-Vega ◽  
María Castañeda-Bueno ◽  
Jonatan Barrera-Chimal ◽  
Rocío Bautista ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Oral Administration ◽  
Apical Membrane ◽  
Hek293 Cells ◽  
Thick Ascending Limb ◽  
Dependent Manner ◽  
Calcium Sensing Receptor ◽  
Calcium Sensing

Background Hypercalciuria can result from activation of the basolateral calcium-sensing receptor (CaSR), which in the thick ascending limb of Henle’s loop controls Ca2+ excretion and NaCl reabsorption in response to extracellular Ca2+. However, the function of CaSR in the regulation of NaCl reabsorption in the distal convoluted tubule (DCT) is unknown. We hypothesized that CaSR in this location is involved in activating the thiazide-sensitive NaCl cotransporter (NCC) to prevent NaCl loss.Methods We used a combination of in vitro and in vivo models to examine the effects of CaSR on NCC activity. Because the KLHL3-WNK4-SPAK pathway is involved in regulating NaCl reabsorption in the DCT, we assessed the involvement of this pathway as well.Results Thiazide-sensitive 22Na+ uptake assays in Xenopus laevis oocytes revealed that NCC activity increased in a WNK4-dependent manner upon activation of CaSR with Gd3+. In HEK293 cells, treatment with the calcimimetic R-568 stimulated SPAK phosphorylation only in the presence of WNK4. The WNK4 inhibitor WNK463 also prevented this effect. Furthermore, CaSR activation in HEK293 cells led to phosphorylation of KLHL3 and WNK4 and increased WNK4 abundance and activity. Finally, acute oral administration of R-568 in mice led to the phosphorylation of NCC.Conclusions Activation of CaSR can increase NCC activity via the WNK4-SPAK pathway. It is possible that activation of CaSR by Ca2+ in the apical membrane of the DCT increases NaCl reabsorption by NCC, with the consequent, well known decrease of Ca2+ reabsorption, further promoting hypercalciuria.

scholarly journals N-Glycosylation Increases the Circulatory Half-Life of Human Growth Hormone

Endocrinology ◽  
2010 ◽  
Vol 151 (11) ◽  
pp. 5326-5336 ◽  
Author(s):  
Thomas V. Flintegaard ◽  
Peter Thygesen ◽  
Henrik Rahbek-Nielsen ◽  
Steven B. Levery ◽  
Claus Kristensen ◽  
...  
Keyword(s):  
Anion Exchange ◽  
Half Life ◽  
Glycosylation Site ◽  
Exchange Chromatography ◽  
Site Directed Mutagenesis ◽  
Hek293 Cells ◽  
Wild Type ◽  
Pharmacodynamic Effect

Therapeutic use of recombinant GH typically involves daily sc injections. We examined the possibilities for prolonging the in vivo circulation of GH by introducing N-glycans. Human GH variants with a single potential N-glycosylation site (N-X-S/T) introduced by site-directed mutagenesis were expressed in HEK293 cells. In a scan of 15 different positions for N-glycosylation sites, four positions (amino acids 93, 98, 99, and 101) were efficiently utilized and did not influence GH in vitro activity. A GH variant (3N-GH) with all these sites was produced in CHOK1SV cells and contained up to three N-glycans. Two pools of 3N-GH were purified and separated according to their charge by anion-exchange chromatography. Anion-exchange HPLC revealed that the N-glycans in the two pools were very similar except for the extent of sialylation. Both 3N-GH pools circulated longer in rats than wild-type GH. The terminal half-life of 3N-GH after iv injection was 24-fold prolonged compared with wild-type GH for the pool with the most pronounced sialylation, 13-fold prolonged for the less sialylated pool, and similar to the wild-type for desialylated 3N-GH. The less sialylated 3N-GH pool exhibited a profound pharmacodynamic effect in GH-deficient rats. Over a 4-d period, a single injection of 3N-GH induced a stronger IGF-I response and a larger increase in body weight than daily injections with wild-type GH. Thus, N-glycans can prolong the in vivo circulation and enhance the pharmacodynamic effect of GH. Sialic acids seem to play a pivotal role for the properties of glycosylated GH.

scholarly journals TrxR, a New CovR-Repressed Response Regulator That Activates the Mga Virulence Regulon in Group A Streptococcus

2008 ◽  
Vol 76 (10) ◽  
pp. 4659-4668 ◽  
Author(s):  
Temekka V. Leday ◽  
Kathryn M. Gold ◽  
Traci L. Kinkel ◽  
Samantha A. Roberts ◽  
June R. Scott ◽  
...  
Keyword(s):  
Group A Streptococcus ◽  
Response Regulator ◽  
Luciferase Reporter ◽  
Infection Model ◽  
Insertion Mutation ◽  
Wild Type ◽  
Virulence Attenuation ◽  
Group A

ABSTRACT Coordinate regulation of virulence factors by the group A streptococcus (GAS) Streptococcus pyogenes is important in this pathogen's ability to cause disease. To further elucidate the regulatory network in this human pathogen, the CovR-repressed two-component system (TCS) trxSR was chosen for further analysis based on its homology to a virulence-related TCS in Streptococcus pneumoniae. In a murine skin infection model, an insertion mutation in the response regulator gene, trxR, led to a significant reduction in lesion size, lesion severity, and lethality. Curing the trxR mutation restored virulence comparable to the wild-type strain. The trxSR operon was defined in vivo, and CovR was found to directly repress its promoter in vitro. DNA microarray analysis established that TrxR activates transcription of Mga-regulated virulence genes, which may explain the virulence attenuation of the trxR mutant. This regulation appears to occur by activation of the mga promoter, Pmga, as demonstrated by analysis of a luciferase reporter fusion. Complementation of the trxR mutant with trxR on a plasmid restored expression of Mga regulon genes and restored virulence in the mouse model to wild-type levels. TrxR is the first TCS shown to regulate Mga expression. Because it is CovR repressed, TrxR defines a new pathway by which CovR can influence Mga to affect pathogenesis in the GAS.

Μελέτη της επίδρασης της ελευρωπαΐνης στην ομοιοστασία των λιπιδίων

2017 ◽  
Author(s):  
Φωτεινή Μάλλιου
Keyword(s):  
Reporter Gene ◽  
In Silico ◽  
Reporter Gene Assay ◽  
Luciferase Reporter ◽  
Wild Type ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Gene Assay

Η Ελευρωπαΐνη, το κύριο πολυφαινολικό συστατικό της ελιάς, παρουσιάζει αντιοξειδωτικές και υπολιπιδαιμικές ιδιότητες. Ο ενεργοποιημένος υποδοχέας επαγωγής του πολλαπλασιασμού των υπεροξεισωμάτων τύπου α (PPARα), διαδραματίζει καίριο ρόλο στον έλεγχο του μεταβολισμού των λιπιδίων και στην ενεργειακή ομοιαστασία του κυττάρου. Η συγκεκριμένη έρευνα εστιάζει στους μηχανισμούς της υπολιπιδαιμικής δράσης της Ελευρωπαΐνης με έμφαση στο ρόλο της Ελευρωπαΐνης στην ενεργοποίηση του PPARα. Για τον σκοπό αυτό, έγινε αξιολόγηση in silico της ικανότητας της Ελευρωπαΐνης να προσδένεται στον PPARα. Θεωρητικά μοντέλα πρόσδεσης στην κρυσταλλική δομή του PPARα με τη χρήση Μοριακής Προσομοίωσης Πρόσδεσης, επιβεβαιώνουν την υπόθεσή μας ότι η Ελευρωπαΐνη είναι αγωνιστής του PPARα. Επιπλέον, διερευνήθηκε in vitro με το Luciferase reporter gene assay η ικανότητα της Ελευρωπαΐνης να προσδένεται στον υποδοχέα PPARα και να τον ενεργοποιεί. Τα αποτελέσματα από την in silico και την in vitro μελέτη δείχνουν σαφώς ότι η ελευρωπαΐνη ενεργοποιεί τον PPARα. Στη συνέχεια έγινε in vivo διερεύνηση της ενεργοποίησης του PPARα από την Ελευρωπαΐνη και αξιολόγηση της επίδρασής της στο λιπιδαιμικό προφίλ των πειραματόζωων. Αγωγή αρσενικών μυών άγριου τύπου (SV129 Wild Type) με Ελευρωπαΐνη σε δοσολογία 100mg/kg, p.o, τα οποία ακολούθησαν τυπική δίαιτα για τρωκτικά, για 6 εβδομάδες, είχε ως αποτέλεσμα επαγωγή του Pparα και των γονιδίων-στόχων του στο ήπαρ, πιθανώς μέσω ενεργοποίησης του PI3K/AKT/p70S6K σηματοδοτικού μονοπατιού. Αυτή η επίδραση της Ελευρωπαΐνης φαίνεται να σχετίζεται άμεσα με σημαντική μείωση των επιπέδων των TGs του ορού και της ολικής χοληστερόλης, γιατί η Ελευρωπαΐνη δεν είχε καμία επίδραση σε αυτούς τους λιπιδαιμικούς δείκτες σε διαγονιδιακούς Pparα null μύες. Στην κατεύθυνση της διερεύνησης της επίδρασης της Ελευρωπαΐνης σε ηπατικούς παράγοντες και σε παράγοντες, που εκφράζονται στο λευκό λιπώδη, κρίσιμους για την ομοιοστασία των Τριγλυκεριδίων, διαπιστώθηκαν τα ακόλουθα: 1) Ενεργοποίηση της ορμονο-ευαίσθητης λιπάσης (HSL) στο λευκό λιπώδη ιστό (W.A.T.) των άγριου τύπου μυών, 2) επαγωγή ποικίλλων ηπατικών παραγόντων, που συμμετέχουν στη σύνθεση, τη μεταφορά, τον μεταβολισμό και την απέκκριση των τριγλυκεριδίων. Αυτή η ενεργοποίηση της HSL στον W.A.T. και των ηπατικών παραγόντων, που συμμετέχουν στην ομοιοστασία των λιπιδίων, είναι πιθανόν να ενισχύει την επαγόμενη από την Ελευρωπαΐνη μείωση των επιπέδων των τριγλυκεριδίων και της χοληστερόλης στον ορό. Συνοψίζοντας, φαίνεται ότι η Ελευρωπαΐνη μειώνει τα τριγλυκερίδια και την ολική χοληστερόλη του ορού σε μύες μέσω ενεργοποίησης του PPARα. Τα δεδομένα αυτής της μελέτης δείχνουν επίσης, ότι στην υπολιπιδαιμική δράση της Ελευρωπαΐνης συμμετέχει και η ενεργοποίηση της HSL στο λευκό λιπώδη ιστό καθώς και η επαγωγή ηπατικών γονιδίων, που διαδραματίζουν βασικό ρόλο την ομοιοστασία των τριγλυκεριδίων, δηλαδή τη σύνθεση, την μεταφορά, τον μεταβολισμό και την κάθαρσή τους. Συμπερασματικά, η μελέτη έδειξε τις υπολιπιδαιμικές δυνατότητες της Ελευρωπαΐνης σε μύες και αποσαφήνισε σε σημαντικό βαθμό τους μηχανισμούς της υπολιπιδαιμικής δράσης της, φωτίζοντας κυρίως τον ρόλο του PPARα. Επειδή η διατήρηση της ομοιοστασίας των λιπιδίων είναι μια πολύπλοκη διαδικασία, που ρυθμίζεται από πολλούς παράγοντες, πιστεύουμε ότι πιθανώς εμπλέκονται και άλλοι μηχανισμοί στην δράση της Ελευρωπαΐνης, η διερεύνηση των οποίων είναι αντικείμενο μελλοντικών μελετών.

scholarly journals In vitro and in vivo assessment of the protective effect of sufentanil in acute lung injury

2021 ◽  
Vol 49 (2) ◽  
pp. 030006052098635
Author(s):  
Qi Gao ◽  
Ningqing Chang ◽  
Donglian Liu
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Protective Effect ◽  
High Mobility ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Inflammatory Factors ◽  
Luciferase Reporter ◽  
Hmgb1 Protein

Objectives To investigate the mechanisms underlying the protective effect of sufentanil against acute lung injury (ALI). Material and Methods Rats were administered lipopolysaccharide (LPS) by endotracheal instillation to establish a model of ALI. LPS was used to stimulate BEAS-2B cells. The targets and promoter activities of IκB were assessed using a luciferase reporter assay. Apoptosis of BEAS-2B cells was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling. Results Sufentanil treatment markedly reduced pathological changes in lung tissue, pulmonary edema and secretion of inflammatory factors associated with ALI in vivo and in vitro. In addition, sufentanil suppressed apoptosis induced by LPS and activated NF-κB both in vivo and in vitro. Furthermore, upregulation of high mobility group box protein 1 (HMGB1) protein levels and downregulation of miR-129-5p levels were observed in vivo and in vitro following sufentanil treatment. miR-129-5p targeted the 3ʹ untranslated region and its inhibition decreased promoter activities of IκB-α. miR-129-5p inhibition significantly weakened the protective effect of sufentanil on LPS-treated BEAS-2B cells. Conclusion Sufentanil regulated the miR-129-5p/HMGB1 axis to enhance IκB-α expression, suggesting that sufentanil represents a candidate drug for ALI protection and providing avenues for clinical treatment.

scholarly journals Long noncoding RNA SNHG14 promotes hepatocellular carcinoma progression by regulating miR-876-5p/SSR2 axis

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Zhibin Liao ◽  
Hongwei Zhang ◽  
Chen Su ◽  
Furong Liu ◽  
Yachong Liu ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Animal Experiments ◽  
Luciferase Reporter ◽  
Western Blot Assay ◽  
Downstream Target ◽  
Protein Levels ◽  
Elevated Expression ◽  
Rna Immunoprecipitation

Abstract Background Aberrant expressions of long noncoding RNAs (lncRNAs) have been demonstrated to be related to the progress of HCC. The mechanisms that SNHG14 has participated in the development of HCC are obscure. Methods Quantitative real-time PCR (qRT-PCR) was used to measure the lncRNA, microRNA and mRNA expression level. Cell migration, invasion and proliferation ability were evaluated by transwell and CCK8 assays. The ceRNA regulatory mechanism of SNHG14 was evaluated by RNA immunoprecipitation (RIP) and dual luciferase reporter assay. Tumorigenesis mouse model was used to explore the roles of miR-876-5p in vivo. The protein levels of SSR2 were measured by western blot assay. Results In this study, we demonstrated that SNHG14 was highly expressed in HCC tissues, meanwhile, the elevated expression of SNHG14 predicted poor prognosis in patients with HCC. SNHG14 promoted proliferation and metastasis of HCC cells. We further revealed that SNHG14 functioned as a competing endogenous RNA (ceRNA) for miR-876-5p and that SSR2 was a downstream target of miR-876-5p in HCC. Transwell, CCK8 and animal experiments exhibited miR-876-5p inhibited HCC progression in vitro and in vivo. By conducting rescue experiments, we found the overexpression of SSR2 or knocking down the level of miR-876-5p could reverse the suppressive roles of SNHG14 depletion in HCC. Conclusion SNHG14 promotes HCC progress by acting as a sponge of miR-876-5p to regulate the expression of SSR2 in HCC.

scholarly journals Honeysuckle Aqueous Extracts Induced let-7a Suppress EV71 Replication and Pathogenesis In Vitro and In Vivo and Is Predicted to Inhibit SARS-CoV-2

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 308
Author(s):  
Ying-Ray Lee ◽  
Chia-Ming Chang ◽  
Yuan-Chieh Yeh ◽  
Chi-Ying F. Huang ◽  
Feng-Mao Lin ◽  
...  
Keyword(s):  
Protein Expression ◽  
Western Blotting ◽  
Expression Profiles ◽  
Plaque Assay ◽  
Virus Titer ◽  
Enterovirus 71 ◽  
Luciferase Reporter ◽  
Pathogen Invasion

Honeysuckle (Lonicera japonica Thunb) is a traditional Chinese medicine (TCM) with an antipathogenic activity. MicroRNAs (miRNAs) are small non-coding RNA molecules that are ubiquitously expressed in cells. Endogenous miRNA may function as an innate response to block pathogen invasion. The miRNA expression profiles of both mice and humans after the ingestion of honeysuckle were obtained. Fifteen overexpressed miRNAs overlapped and were predicted to be capable of targeting three viruses: dengue virus (DENV), enterovirus 71 (EV71) and SARS-CoV-2. Among them, let-7a was examined to be capable of targeting the EV71 RNA genome by reporter assay and Western blotting. Moreover, honeysuckle-induced let-7a suppression of EV71 RNA and protein expression as well as viral replication were investigated both in vitro and in vivo. We demonstrated that let-7a targeted EV71 at the predicted sequences using luciferase reporter plasmids as well as two infectious replicons (pMP4-y-5 and pTOPO-4643). The suppression of EV71 replication and viral load was demonstrated in two cell lines by luciferase activity, RT-PCR, real-time PCR, Western blotting and plaque assay. Furthermore, EV71-infected suckling mice fed honeysuckle extract or inoculated with let-7a showed decreased clinical scores and a prolonged survival time accompanied with decreased viral RNA, protein expression and virus titer. The ingestion of honeysuckle attenuates EV71 replication and related pathogenesis partially through the upregulation of let-7a expression both in vitro and in vivo. Our previous report and the current findings imply that both honeysuckle and upregulated let-7a can execute a suppressive function against the replication of DENV and EV71. Taken together, this evidence indicates that honeysuckle can induce the expression of let-7a and that this miRNA as well as 11 other miRNAs have great potential to prevent and suppress EV71 replication.

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.

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