The role of miR-2∼13∼71 cluster in resistance to deltamethrin in Culex pipiens pallens

Excessive and continuous application of deltamethrin has resulted in the development of deltamethrin resistance among mosquitoes, which becomes a major obstacle for mosquito control. In a previous study, differentially expressed miRNAs between deltamethrin-susceptible (DS) strain and deltamethrin-resistant (DR) strain using illumina sequencing in Culex pipiens pallens were identified. In this study, we applied RNAi and the Centers for Disease Control and Prevention (CDC) bottle bioassay to investigate the relationship between miR-2∼13∼71 cluster (miR-2, miR-13 and miR-71) and deltamethrin resistance. We used quantitative real-time PCR (qRT-PCR) to measure expression levels of miR-2∼13∼71 clusters. MiR-2∼13∼71 cluster was down regulated in adult female mosquitoes from the DR strain and played important roles in deltamethrin resistance through regulating target genes, CYP9J35 and CYP325BG3. Knocking down CYP9J35 and CYP325BG3 resulted in decreased mortality of DR mosquitoes. This study provides the first evidence that miRNA clusters are associated with deltamethrin resistance in mosquitoes. Moreover, we investigated the regulatory networks formed between miR-2∼13∼71 cluster and its target genes, which provide a better understanding of the mechanism involved in deltamethrin resistance.     

Insecticide resistance,associated mechanisms and fitness aspects in two Brazilian Stegomyia aegypti (= Aedes aegypti) populations

In Brazil, insecticide resistance in Stegomyia aegypti (= Aedes aegypti) (Diptera: Culicidae) populations to pyrethroids and to the organophosphate (OP) temephos is disseminated. Currently, insect growth regulators (IGRs) and the OP malathion are employed against larvae and adults, respectively. Bioassays with mosquitoes from two northeast municipalities, Crato and Aracaju, revealed, in both populations, susceptibility to IGRs and malathion (RR₉₅ ≤ 2.0), confirming the effectiveness of these compounds. By contrast, temephos and deltamethrin (pyrethroid) resistance levels were high (RR₉₅ > 10), which is consistent with the use of intense chemical control. In Crato, RR₉₅ values were > 50 for both compounds. Knock‐down‐resistant (kdr) mutants in the voltage‐gated sodium channel, the pyrethroid target site, were found in 43 and 32%, respectively, of Aracaju and Crato mosquitoes. Biochemical assays revealed higher metabolic resistance activity (esterases, mixed function oxidases and glutathione‐S‐transferases) at Aracaju. With respect to fitness aspects, mating effectiveness was equivalently impaired in both populations, but Aracaju mosquitoes showed more damaging effects in terms of longer larval development, decreased bloodmeal acceptance, reduced engorgement and lower numbers of eggs laid per female. Compared with mosquitoes in Crato, Aracaju mosquitoes exhibited lower OP and pyrethroid RR₉₅, increased activity of detoxifying enzymes and greater effect on fitness. The potential relationship between insecticide resistance mechanisms and mosquito viability is discussed.     

Characterization of mosquito CYP6P7 and CYP6AA3: differences in substrate preference and kinetic properties

Cytochrome P450 monooxygenases are involved in insecticide resistance in insects. We previously observed an increase in CYP6P7 and CYP6AA3 mRNA expression in Anopheles minimus mosquitoes during the selection for deltamethrin resistance in the laboratory. CYP6AA3 has been shown to metabolize deltamethrin, while no information is known for CYP6P7. In this study, CYP6P7 was heterologously expressed in the Spodoptera frugiperda (Sf9) insect cells via baculovirus‐mediated expression system. The expressed CYP6P7 protein was used for exploitation of its enzymatic activity against insecticides after reconstitution with the An. minimus NADPH‐cytochrome P450 reductase enzyme in vitro. The ability of CYP6P7 to metabolize pyrethroids and insecticides in the organophosphate and carbamate groups was compared with CYP6AA3. The results revealed that both CYP6P7 and CYP6AA3 proteins could metabolize permethrin, cypermethrin, and deltamethrin pyrethroid insecticides, but showed the absence of activity against bioallethrin (pyrethroid), chlorpyrifos (organophosphate), and propoxur (carbamate). CYP6P7 had limited capacity in metabolizing λ‐cyhalothrin (pyrethroid), while CYP6AA3 displayed activity toward λ‐cyhalothrin. Kinetic properties suggested that CYP6AA3 had higher efficiency in metabolizing type I than type II pyrethroids, while catalytic efficiency of CYP6P7 toward both types was not significantly different. Their kinetic parameters in insecticide metabolism and preliminary inhibition studies by test compounds in the flavonoid, furanocoumarin, and methylenedioxyphenyl groups elucidated that CYP6P7 had different enzyme properties compared with CYP6AA3. © 2011 Wiley Periodicals, Inc.     

The grapevine (Vitis vinifera) LysM receptor kinases VvLYK1‐1 and VvLYK1‐2 mediate chitooligosaccharide‐triggered immunity

Chitin, a major component of fungal cell walls, is a well‐known pathogen‐associated molecular pattern (PAMP) that triggers defense responses in several mammal and plant species. Here, we show that two chitooligosaccharides, chitin and chitosan, act as PAMPs in grapevine (Vitis vinifera) as they elicit immune signalling events, defense gene expression and resistance against fungal diseases. To identify their cognate receptors, the grapevine family of LysM receptor kinases (LysM‐RKs) was annotated and their gene expression profiles were characterized. Phylogenetic analysis clearly distinguished three V. vinifera LysM‐RKs (VvLYKs) located in the same clade as the Arabidopsis CHITIN ELICITOR RECEPTOR KINASE1 (AtCERK1), which mediates chitin‐induced immune responses. The Arabidopsis mutant Atcerk1, impaired in chitin perception, was transformed with these three putative orthologous genes encoding VvLYK1‐1, ‐2, or ‐3 to determine if they would complement the loss of AtCERK1 function. Our results provide evidence that VvLYK1‐1 and VvLYK1‐2, but not VvLYK1‐3, functionally complement the Atcerk1 mutant by restoring chitooligosaccharide‐induced MAPK activation and immune gene expression. Moreover, expression of VvLYK1‐1 in Atcerk1 restored penetration resistance to the non‐adapted grapevine powdery mildew (Erysiphe necator). On the whole, our results indicate that the grapevine VvLYK1‐1 and VvLYK1‐2 participate in chitin‐ and chitosan‐triggered immunity and that VvLYK1‐1 plays an important role in basal resistance against E. necator.     

A TIR–NBS protein encoded by Arabidopsis Chilling Sensitive 1 (CHS1) limits chloroplast damage and cell death at low temperature

Survival of plants at low temperature depends on mechanisms for limiting physiological damage and maintaining growth. We mapped the chs1‐1 (chilling sensitive1‐1) mutation in Arabidopsis accession Columbia to the TIR‐NBS gene At1g17610. In chs1‐1, a single amino acid exchange at the CHS1 N‐terminus close to the conserved TIR domain creates a stable mutant protein that fails to protect leaves against chilling stress. The sequence of another TIR‐NBS gene (At5g40090) named CHL1 (CHS1‐like 1) is related to that of CHS1. Over‐expression of CHS1 or CHL1 alleviates chilling damage and enhances plant growth at moderate (24°C) and chilling (13°C) temperatures, suggesting a role for both proteins in growth homeostasis. chs1‐1 mutants show induced salicylic acid production and defense gene expression at 13°C, indicative of autoimmunity. Genetic analysis of chs1‐1 in combination with defense pathway mutants shows that chs1‐1 chilling sensitivity requires the TIR‐NBS‐LRR and basal resistance regulators encoded by EDS1 and PAD4 but not salicylic acid. By following the timing of metabolic, physiological and chloroplast ultrastructural changes in chs1‐1 leaves during chilling, we have established that alterations in photosynthetic complexes and thylakoid membrane integrity precede leaf cell death measured by ion leakage. At 24°C, the chs1‐1 mutant appears normal but produces a massive necrotic response to virulent Pseudomonas syringae pv. tomato infection, although this does not affect bacterial proliferation. Our results suggest that CHS1 acts at an intersection between temperature sensing and biotic stress pathway activation to maintain plant performance over a range of conditions.     

Splice variant specific increase in Ca2+/calmodulin‐dependent protein kinase 1‐gamma mRNA expression in response to acute pyrethroid exposure

In mammals, pyrethroids are neurotoxicants that interfere with ion channel function in excitable neuronal membranes. Previous work demonstrated increases in the expression of Ca²⁺/calmodulin‐dependent protein kinase 1‐gamma (Camk1g) mRNA following acute deltamethrin and permethrin exposure. In the rat, this gene is expressed as two distinct splice variants, Camk1g1 and Camk1g2. The present study tests the hypothesis that changes in Camk1g mRNA expression in the rat following acute pyrethroid exposure are due to a specific increase in the Camk1g1 splice variant and not the Camk1g2 splice variant. Long‐Evans rats were acutely exposed to permethrin, deltamethrin, or corn oil vehicle. Frontal cortex was collected at 6 h postdosing. In addition, rats were exposed to permethrin (100 mg/kg) or deltamethrin (3 mg/kg), and frontal cortex was collected at 1, 3, 6, 9, 12, or 24 h along with time‐matched vehicle controls. Expression of Camk1g1 and Camk1g2 mRNA was measured by quantitative real‐time RT‐PCR and quantified using the 2^{‐Δ Δ C}T method. Dose‐dependent increases in Camk1g1 mRNA expression were observed for both pyrethroids at 6 h. In addition, a dose‐dependent increase in Camk1g2 was observed at 6 h although it was very small in magnitude. The increases in Camk1g1 expression for deltamethrin and permethrin peak between 3 and 6 h postexposure and returns to control levels by 9 h. There was no increase in CAMK1G1 protein as measured with Western blots. The present data demonstrate that pyrethroid‐induced changes in Camk1g are driven mainly by increased expression of the Camk1g1 splice variant. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:174–186, 2010; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20324     

Down‐regulation of a chitin synthase a gene by RNA interference enhances pathogenicity of Beauveria bassiana ANU1 against Spodoptera exigua (HÜBNER)

Chitin synthase (CHS) is an important enzymatic component, which is required for chitin formation in the cuticles and cuticular linings of other tissues in insects. CHSs have been divided into two classes, classes A and B, based on their amino acid sequence similarities and functions. Class A CHS (CHS‐A) is specifically expressed in the epidermis and related ectodermal cells such as tracheal cells, while class B CHS (CHS‐B) is expressed in gut epithelial cells that produce peritrophic matrices. In this study, we cloned the CHS‐A gene from the beet armyworm, Spodoptera exigua (SeCHS‐A). The SeCHS‐A contains an open reading frame of 4,698 nucleotides, encoding a protein of 1,565 amino acids with a predicted molecular mass of approximately 177.8 kDa. The SeCHS‐A mRNA was expressed in all developmental stages and specifically in the epidermis and tracheae tissue by quantitative real‐time‐PCR analysis. Expression of SeCHS‐A gene was suppressed by feeding double‐stranded RNA (dsCHS‐A, 400 ng/larva) in the third instar larvae of S. exigua. Suppression of the SeCHS‐A gene expression significantly increased 35% of mortality on pupation of S. exigua. Also, the third instar larvae fed with dsCHS‐A significantly increased susceptibility to entomopathogenic fungi, Beauveria bassiana ANU1 at 3 days after treatment. These results suggest that the SeCHS‐A gene plays an important role in development of S. exigua and RNA interference may apply to effective pest control with B. bassiana.     

Experimental hut comparisons of nets treated with carbamate or pyrethroid insecticides, washed or unwashed, against pyrethroid-resistant mosquitoes

Abstract. The efficacy against mosquitoes (Diptera: Culicidae) of a bednet treated with carbamate insecticide [carbosulfan capsule suspension (CS) 200 mg/m²] was compared with four types of pyrethroid‐treated nets in veranda‐trap huts at Yaokoffikro near Bouaké, Côte d'Ivoire, where the malaria vector Anopheles gambiae Giles carries the kdr gene (conferring pyrethroid resistance) at high frequency and Culex quinquefasciatus Say is also pyrethroid resistant. Pyrethroids compared were lambdacyhalothrin CS 18 mg/m², alphacypermethrin water dispersible granules (WG) 20 mg/m², deltamethrin 50 mg/m² (Permanet?) and permethrin emulsifiable concentrate (EC) 500 mg/m². Insecticidal power and personal protection from mosquito bites were assessed before and after the nets were used for 8 months and hand washed five times in cold soapy water. Before washing, all treatments except permethrin significantly reduced blood‐feeding and all had significant insecticidal activity against An. gambiae. The carbosulfan net gave significantly higher killing of An. gambiae than all pyrethroid treatments except the Permanet. Against Culex spp., carbosulfan was more insecticidal and gave a significantly better protective effect than any of the pyrethroid treatments. After washing, treated nets retained various degrees of efficacy against both mosquito genera – but least for the carbosulfan net. Washed nets with three types of pyrethroid treatment (alphacypermethrin, lambdacyhalothrin, permethrin) gave significantly higher mortality rates of Culex than in huts with the same pyrethroid‐treated nets before washing. After five washes, the Permanet?, which is sold as a long‐lasting insecticidal product, performed no better than the other nets in our experimental conditions.     

Differential mRNA expression levels and gene sequences of carboxylesterase in both deltamethrin resistant and susceptible strains of the cotton aphid, Aphis gossypii

Extensive use of insecticides on cotton has prompted resistance development in the cotton aphid, Aphis gossypii (Glover) in China. A deltamethrin‐selected population of cotton aphids from Xinjiang Uygur Autonomous Region, China with 228.59‐fold higher resistance to deltamethrin was used to examine how carboxylesterase conferred resistance to this pyrethroid insecticide. The carboxylesterase activity in the deltamethrin‐resistant strain was 3.67‐, 2.02‐ and 1.16‐fold of the susceptible strain when using α‐naphthyl acetate (α‐NA), β‐naphthyl acetate (β‐NA) and α‐naphthyl butyrate (α‐NB) as substrates, respectively. Carboxylesterase cDNA was cloned and sequenced from both deltamethrin‐resistant and susceptible strains. The cDNA contained 1581 bp open reading frames (ORFs) coding a 526 amino acid protein. Only one amino acid substitution (Val⁸⁷‐Ala) was observed between deltamethrin‐resistant and susceptible strains but it is not genetically linked to resistance by the catalytic triad and signature motif analysis. The real‐time polymerase chain reaction analysis indicated that the resistant strain had a 6.61‐fold higher level of carboxylesterase mRNA than the susceptible strain. The results revealed that up‐regulation of the carboxylesterase gene, not modified gene structure, may be responsible for the development of resistance in cotton aphids to deltamethrin.     

Pyrethroid resistance reduces the efficacy of space sprays for dengue control on the island of Martinique (Caribbean)

Background

Dengue fever is reemerging on the island of Martinique and is a serious threat for the human population. During dengue epidemics, adult Aedes aegypti control with pyrethroid space sprays is implemented in order to rapidly reduce transmission. Unfortunately, vector control programs are facing operational challenges with the emergence of pyrethroid resistant Ae. aegypti populations.

Methodology/Principal Findings

To assess the impact of pyrethroid resistance on the efficacy of treatments, applications of deltamethrin and natural pyrethrins were performed with vehicle-mounted thermal foggers in 9 localities of Martinique, where Ae. aegypti populations are strongly resistant to pyrethroids. Efficacy was assessed by monitoring mortality rates of naturally resistant and laboratory susceptible mosquitoes placed in sentinel cages. Before, during and after spraying, larval and adult densities were estimated. Results showed high mortality rates of susceptible sentinel mosquitoes treated with deltamethrin while resistant mosquitoes exhibited very low mortality. There was no reduction of either larval or adult Ae. aegypti population densities after treatments.

Conclusions/Significance

This is the first documented evidence that pyrethroid resistance impedes dengue vector control using pyrethroid-based treatments. These results emphasize the need for alternative tools and strategies for dengue control programs.     

CHITIN SYNTHASE B: A MIDGUT‐SPECIFIC GENE INDUCED BY INSECT HORMONES AND INVOLVED IN FOOD INTAKE IN Bombyx mori LARVAE

Chitin synthase (CHS) is the key regulatory enzyme in chitin synthesis and excretion in insects, and a specific target of insecticides. We cloned a CHS B gene of Bombyx mori (BmChsB) and showed it to be midgut specific, highly expressed during the feeding process in the larva. Knockdown of BmChsB expression in the third‐instar larvae increased the number of nonmolting and abnormally molting larvae. Exposure to nikkomycin Z, a CHS inhibitor, reduced the amount of chitin in the peritrophic membrane of molted larvae, whereas abnormally elevated BmChsB mRNA levels were readily detected from the end of molting and in the newly molted larvae. Exogenous 20‐hydroxyecdysone (20E) and methoprene, a juvenile hormone analogue, significantly upregulated the expression of BmChsB when the levels of endogenous molting hormone (MH) were low and the levels of endogenous juvenile hormone (JH) were high immediately after molting. When levels of endogenous MH were high and those of endogenous JH were low during the molting stage, exogenous 20E did not upregulate BmChsB expression and exogenous methoprene upregulated it negligibly. When the endogenous hormone levels were low during the mulberry‐leaf intake process, BmChsB expression was upregulated by exogenous methoprene. We conclude that the expression of BmChsB is regulated by insect hormones, and directly affects the chitin‐synthesis‐dependent form of the peritrophic membrane and protects the food intake and molting process of silkworm larvae.     

Organophosphate and pyrethroid resistances in the tomato leaf miner Tuta absoluta (Lepidoptera: Gelechiidae) from Iran

The tomato leafminer, Tuta absoluta (Meyrich) (Lepidoptera: Gelechiidae), is a serious pest of tomato crops worldwide. The intensive use of chemical pesticides to control it has led to the selection of resistant populations. This study investigated the resistance of T. absoluta populations to pyrethroid and the organophosphate insecticides from ten regions of Iran. The resistance ratios at LC₅₀ for chlorpyrifos and diazinon varied among populations from 4.3 to 12 and from 1.4 to 9.0, respectively. The resistance ratios of the pyrethroids cypermethrin, deltamethrin and permethrin varied from 1.3 to 3.7, 2.7 to 13 and 1.2 to 4.3, respectively. Inclusion of synergists in toxicological bioassays and the variation observed in the activity of esterases, glutathione S‐transferase and cytochrome P450‐dependent monooxygenase suggest the existence of metabolically based resistance. Esterase and P450 biochemical assays were positively correlated with deltamethrin, and cypermethrin tolerance and diazinon tolerance correlated with esterase activity. The genes encoding the organophosphate and pyrethroid target sites acetylcholinesterase (ace1) and sodium channel (kdr) were partly sequenced. The genotyping revealed mutations in high frequencies in all populations leading to an A201S substitution in ace1 and three substitutions in the sodium channel gene L1014F, M918T, T929I. In summary, our results indicate the presence of organophosphate and pyrethroid resistance in Iranian T. absoluta populations with involvement of both detoxification enzymes and target site alterations. Most likely the populations of T. absoluta imported to Iran were resistant upon arrival.     

The cytochrome P450 CYP6P4 is responsible for the high pyrethroid resistance in knockdown resistance-free Anopheles arabiensis

Pyrethroid insecticides are the front line vector control tools used in bed nets to reduce malaria transmission and its burden. However, resistance in major vectors such as Anopheles arabiensis is posing a serious challenge to the success of malaria control.Herein, we elucidated the molecular and biochemical basis of pyrethroid resistance in a knockdown resistance-free Anopheles arabiensis population from Chad, Central Africa. Using heterologous expression of P450s in Escherichia coli coupled with metabolism assays we established that the over-expressed P450 CYP6P4, located in the major pyrethroid resistance (rp1) quantitative trait locus (QTL), is responsible for resistance to Type I and Type II pyrethroid insecticides, with the exception of deltamethrin, in correlation with field resistance profile. However, CYP6P4 exhibited no metabolic activity towards non-pyrethroid insecticides, including DDT, bendiocarb, propoxur and malathion. Combining fluorescent probes inhibition assays with molecular docking simulation, we established that CYP6P4 can bind deltamethrin but cannot metabolise it. This is possibly due to steric hindrance because of the large vdW radius of bromine atoms of the dihalovinyl group of deltamethrin which docks into the heme catalytic centre.The establishment of CYP6P4 as a partial pyrethroid resistance gene explained the observed field resistance to permethrin, and its inability to metabolise deltamethrin probably explained the high mortality from deltamethrin exposure in the field populations of this Sudano-Sahelian An. arabiensis. These findings describe the heterogeneity in resistance towards insecticides, even from the same class, highlighting the need to thoroughly understand the molecular basis of resistance before implementing resistance management/control tools.     

AP2α is essential for MUC8 gene expression in human airway epithelial cells

Mucins are high molecular weight proteins that make up the major components of mucus. Hypersecretion of mucus is a feature of several chronic inflammatory airway diseases. MUC8 is an important component of airway mucus, and its gene expression is upregulated in nasal polyp epithelium. Little is known about the molecular mechanisms of MUC8 gene expression. We first observed overexpression of activator protein‐2alpha (AP2α) in human nasal polyp epithelium. We hypothesized that AP2α overexpression in nasal polyp epithelium correlates closely with MUC8 gene expression. We demonstrated that phorbol 12‐myristate 13‐acetate (PMA) treatment of the airway epithelial cell line NCI‐H292 increases MUC8 gene and AP2α expression. In this study, we sought to determine which signal pathway is involved in PMA‐induced MUC8 gene expression. The results show that the protein kinase C and mitogen‐activating protein/ERK kinase (MAPK) pathways modulate MUC8 gene expression. PD98059 or ERK1/2 siRNA and RO‐31‐8220 or PKC siRNA significantly suppress AP2α as well as MUC8 gene expression in PMA‐treated cells. To verify the role of AP2α, we specifically knocked down AP2α expression with siRNA. A significant AP2α knock‐down inhibited PMA‐induced MUC8 gene expression. While dominant negative AP2α decreased PMA‐induced MUC8 gene expression, overexpressing wildtype AP2α increased MUC8 gene expression. Furthermore, using lentiviral vectors for RNA interference in human nasal polyp epithelial cells, we confirmed an essential role for AP2α in MUC8 gene expression. From these results, we concluded that PMA induces MUC8 gene expression through a mechanism involving PKC, ERK1/2, and AP2α activation in human airway epithelial cells. J. Cell. Biochem. 110: 1386–1398, 2010. © 2010 Wiley‐Liss, Inc.     

Behavioural and insecticidal effects of organophosphate‐, carbamate‐ and pyrethroid‐treated mosquito nets against African malaria vectors

Three insecticides – the pyrethroid deltamethrin, the carbamate carbosulfan and the organophosphate chlorpyrifos‐methyl – were tested on mosquito nets in experimental huts to determine their potential for introduction as malaria control measures. Their behavioural effects and efficacy were examined in Anopheles gambiae Giles s.s. (Diptera: Culicidae) and Anopheles funestus Giles s.s. in Muheza, Tanzania, and in Anopheles arabiensis Patton and Culex quinquefasciatus Say in Moshi, Tanzania. A standardized dosage of 25 mg/m² plus high dosages of carbosulfan (50 mg/m², 100 mg/m² and 200 mg/m²) and chlorpyrifos‐methyl (100 mg/m²) were used to compare the three types of insecticide. At 25 mg/m², the rank order of the insecticides for insecticide‐induced mortality in wild An. gambiae and An. funestus was, respectively, carbosulfan (88%, 86%) > deltamethrin (79%, 78%) > chlorpyrifos‐methyl (35%, 53%). The rank order of the insecticides for blood‐feeding inhibition (reduction in the number of blood‐fed mosquitoes compared with control) in wild An. gambiae and An. funestus was deltamethrin > chlorpyrifos‐methyl > carbosulfan. Carbosulfan was particularly toxic to endophilic anophelines at 200 mg/m², killing 100% of An. gambiae and 98% of An. funestus that entered the huts. It was less effective against the more exophilic An. arabiensis (67% mortality) and carbamate‐resistant Cx quinquefasciatus (36% mortality). Carbosulfan deterred anophelines from entering huts, but did not deter carbamate‐resistant Cx quinquefasciatus. Deltamethrin reduced the proportion of insects engaged in blood‐feeding, probably as a consequence of contact irritancy, whereas carbosulfan seemed to provide personal protection through deterred entry or perhaps a spatial repellent action. Any deployment of carbosulfan as an individual treatment on nets should be carried out on a large scale to reduce the risk of diverting mosquitoes to unprotected individuals. Chlorpyrifos‐methyl was inferior to deltamethrin in terms of mortality and blood‐feeding inhibition and would be better deployed on a net in combination with a pyrethroid to control insecticide‐resistant mosquitoes.     

MiR‐129‐5p promotes docetaxel resistance in prostate cancer by down‐regulating CAMK2N1 expression

This study focuses on the effect of miR‐129‐5p on docetaxel‐resistant (DR) prostate cancer (PCa) cells invasion, migration and apoptosis. In our study, the expression of CAMK2N1 was assessed by qRT‐PCR in PCa patient tissues and cell lines including PC‐3 and PC‐3‐DR. Cells transfected with miR‐129‐5p mimics, inhibitor, CAMK2N1 or negative controls (NC) were used to interrogate their effects on DR cell invasions, migrations and apoptosis during docetaxel (DTX) treatments. The apoptosis rate of the PCa cells was validated by flow cytometry. Relationships between miR‐129‐5p and CAMK2N1 levels were identified by qRT‐PCR and dual‐luciferase reporter assay. CAMK2N1 was found to be down‐expressed in DR PCa tissue sample, and low levels of CAMK2N1 were correlated with high docetaxel resistance and clinical prediction of poor survival. CAMK2N1 levels were decreased in DR PCa cells treated with DXT. We further explored that up‐regulation of miR‐129‐5p could promote DR PCa cells viability, invasion and migration but demote apoptosis. Involved molecular mechanism studies revealed that miR‐129‐5p reduced downstream CAMK2N1 expression to further impact on chemoresistance to docetaxel of PCa cells, indicating its vital role in PCa docetaxel resistance. Our findings revealed that miR‐129‐5p contributed to the resistance of PC‐3‐DR cells to docetaxel through suppressing CAMK2N1 expression, and thus targeting miR‐129‐5p may provide a novel therapeutic approach in sensitizing PCa to future docetaxel treatment.