Exploring the correlation between deltamethrin stress and Keap1-Nrf2-ARE pathway from Drosophila melanogaster RNASeq data.

Deltamethrin (DM) is widely used in a variety of pest control, resulting in serious drug resistance. Keap1-Nrf2-ARE is the antioxidant stress pathway. We identified 268 genes differentially expressed (DEGs) in Drosophila Kc cells treated with DM, including up-regulated 180 genes and down-regulated 88 genes compared with the control group (fold-change≥2, qValue≤0.001) by RNA-seq, they are mainly linked to metabolic process, stimulation response, immune system process. When the cells are treated with DM in the case of overexpression of the Keap1 gene, the cytochrome P450 family genes were significantly down-regulated, and some diseases-related genes and non-coding genes also changed. Our data shown that Keap1-Nrf2-ARE pathway may play an important role in DM stress, which will provide a new direction for studying the mechanism of insect resistance.

The Nrf2-Keap1 pathway: A secret weapon against pesticide persecution in Drosophila Kc cells.

Nrf2-Keap1 pathway defends organisms against the detrimental effects of oxidative stress, and play pivotal roles in preventing xenobiotic-related toxicity. We designed experiments to explore and verify its role and function under deltamethrin (DM) stress. In experiments, DM was selected as the inducer, and Drosophila Kc cells were treated as the objects. The result showed the oxidative stress of cells proliferated in a very short time after DM treatment, reaching the maximum after one hour of treatment. The experimental data showed Nrf2 could be up-regulated and activated by DM which were manifested by the increase of Nrf2 mRNA, Nrf2 protein in the nucleus and the expression of detoxification enzyme genes. We further tested the activity of all groups, and found the survival rate of cells was basically proportional to the expression of Nrf2. Based on the above experimental results, Keap1 overexpression (K+), Nrf2 overexpression (N+) or interference (N-) cells were used to verified the relationship between Nrf2, cell survival and detoxification enzymes expression. We found the cell survival rate of N+ group was significantly higher than that of other groups and the expression of detoxification enzymes were increased compared to the control group. These results demonstrated that Nrf2 is related to cell detoxification and associated with the tolerance to DM. Our evidence suggested Nrf2 is a potential therapeutic target for oxidative stress and a potential molecular target gene of resistance control.

IDENTIFICATION OF PROTEASOME ALPHA6 SUBUNIT ASSOCIATED WITH DELTAMETHRIN RESISTANCE IN Drosophila melanogaster Kc CELLS.

Differential expression of the proteasome alpha6 (prosalpha6) was previously reported between Plutella xylostella strains that are resistant or susceptible to the pesticide deltamethrin (DM). This finding indicated that the prosalpha6 may be involved in DM resistance. In this article, qPCR analysis revealed that the prosalpha6 was also significantly upregulated in Drosophila Kc cells treated with DM. To better understand the contribution of prosalpha6 in DM resistance, RNA interference, heterologous expression, and a proteasome inhibitor (MG-132) were used. MG-132 was used to suppress proteasomal activity, and the dsRNA was designed to block the function of prosalpha6. The results indicated that both MG-132 and prosalpha6 knockdown decreased the cellular viability following DM treatment. Prosalpha6 was cloned and transfected into Drosophila Kc cells. The result showed that overexpression of prosalpha6 in Drosophila Kc cells conferred some protection against DM. Taken together, our results indicate that prosalpha6 is involved in Drosophila cells DM resistance.

PROTEOMIC ANALYSIS OF UBIQUITINATED PROTEINS FROM DELTAMETHRIN-RESISTANT AND SUSCEPTIBLE STRAINS OF THE DIAMONDBACK MOTH, Plutella Xylostella L.

Ubiquitin, a small protein consisting of 76 amino acids, acts in protein degradation, DNA repair, signal transduction, transcriptional regulation, and receptor control through endocytosis. Using proteomics, we compared the differentially ubiquitinated proteins between a deltamethrin-resistant (DR) strain and a deltamethrin-sensitive (DS) strain in third-instar larvae of the diamondback moth. We used polyubiquitin affinity beads to enrich ubiquitinated proteins and then performed one-dimensional SDS-PAGE separation and mass spectrometric identification. In the DR strain, We found 17 proteins that were upregulated (relative to the DS strain), including carbonic anhydrase family members, ADP ribosylation factor 102F CG11027-PA, protein kinase 61C, phospholipase A2 , dihydrolipoamide dehydrogenase, tyrosine hydroxylase, and heat shock proteins, and five proteins that were downregulated in the DS strain, including carboxylesterase and DNA cytosine-5 methyltransferase. These results were also verified by qPCR. The differentially ubiquitinated proteins/enzymes were mainly responsible for protein binding, catalytic activity, and molecular transducer activity. These results improve our understanding of the relationship between protein ubiquitination and the deltamethrin stress response.

The functional link between TCTP and Drsl1 in deltamethrin-stressed Drosophila Kc cells.

Translationally controlled tumor protein (TCTP) is a growth and development related protein found in almost all eukaryotes and can be involved in a variety of cellular life activities. Our previous studies found that TCTP is involved in the response of Drosophila Kc cells to deltamethrin (DM) stimulation, and it may be a candidate gene related to DM stress. Therefore, we would further investigate the functions of TCTP and its mechanism under DM stress. The qPCR results showed that the expression level of Drsl1 increasing first and then decreasing with the change of DM concentration and treatment duration. The optimal concentration was 20 ppm and the optimal time was 24 h. qPCR and WB results together showed that the expression levels of TCTP and Drsl1 were positively correlated. The flow cytometry showed the expression levels of TCTP and Drsl1 in deltamethrin-stressed cells are related to apoptosis. The apoptosis rate reached the highest level in the cells with simultaneous interference of both genes. Taken together, our data mainly suggest that TCTP interacts with Drsl1 in response to DM stress in Drosophila Kc cells, which helps to investigate the mechanisms of DM toxicity and the mechanisms by which insects develop resistance to it.

Ran participates in deltamethrin stress through regulating the nuclear import of Nrf2.

The small GTPase Ran has a variety of biological functions, one of the most prominent of which is to regulate nucleocytoplasmic transport. In our previous study, it was suggested that Ran is involved in the deltamethrin (DM) stress. In addition, Keap1-Nrf2-ARE pathway was also confirmed to be associated with DM stress. We report here that under DM stress, interfering Ran or nuclear transport factor Ntf2 by RNAi could suppress the nuclear import of nuclear transcription factor Nrf2 which then down-regulates the expressions of detoxification enzyme genes (Cyp4d20, Cyp4ae1, GstD5, Sod3, etc.), ultimately resulting in a significant apoptosis of Drosophila Kc cells. In contrast, after overexpressing Ran in Kc cells, Nrf2 has a higher concentration in the nucleus, and the expressions of detoxification enzyme genes are up-regulated, while the DM-induced apoptosis is significantly lower than that of the control group. Additionally, we preliminary found silencing Ntf2 or Ran could prevent the nuclear import of transcription factor Dif under DM stress, subsequently decreased expressions of antimicrobial peptide genes (Drsl1). In summary, our data mainly indicates that Ran may participate in DM stress through regulating the nuclear import of Nrf2, which could help to study the mechanism of deltamethrin resistance.

Analysis of UB and L40, related to deltamethrin stress in the diamondback moth, Plutella xylostella (L.).

Pests have been subjected to heavy selection by insecticide pressure, producing greater and more serious resistance. It is extremely valuable to identify the resistance genes that are relevant for pest control. In our previous studies, we reported that UBL40 is the deltamethrin resistance-associated gene. UBL40 is cleaved by specific endopeptidases to release UB and L40. Therefore, further we compared the expression of UB and L40 in different tissues from larvae of the diamondback moth using a deltamethrin-resistant (DR) strain and a deltamethrin-sensitive (DS) strain. The results showed that both UB and L40 knockdown significantly reduced the level of resistance to deltamethrin and clearly decreased the survival rate in P. xylostella after 72 h. These results provide evidence that UB and L40 both play roles in the regulation of deltamethrin resistance, which could help elucidate the resistance mechanisms and identify new target genes associated with deltamethrin resistance in P. xylostella.

Analysis of translation control tumor protein related to deltamethrin stress in Drosophila kc cells.

The translation control tumor protein (TCTP) is a kind of conservative, common and important molecule, several functions (such as regulating cell cycle, apoptosis and calcium binding) have been reported. However, few academic researches for role of TCTP in insecticides stress were made so far. In this research, Drosophila kc cells treated with different doses of deltamethrin at different times, indicated that the expression of TCTP reached the highest level when the cells were treated with 20 ppm of deltamethrin at 24 h. The results showed that TCTP expression is associated with deltamethrin stress. To investigate the functional relationship between this gene and deltamethrin resistance, RNA interference (RNAi) and cell transfection were utilized. TCTP knockdown significantly reduced the level of resistance of RNAi-treated cells, and the overexpressions of TCTP in Drosophila kc cells conferred a degree of protection against deltamethrin. Flow cytometry data showed increased apoptosis rate of RNAi-treated cells and decreased apoptosis following cell transfection. These results represent the first evidence that TCTP plays an important role in the regulation of deltamethrin resistance. Therefore, this study could help us to elucidate the environmental toxicity of deltamethrin and new target genes associated with resistance.

Analysis of Ran related to pesticide resistance in Drosophila Kc cells.

Pesticides have been used extensively for pest control, resulting in serious pesticide resistance. It is extremely valuable to identify the resistance genes related to pest control. In our previous studies, we reported that Ran is the deltamethrin (DM) resistance-associated gene. To clarify whether Ran is also related to the resistance of other pesticides, we selected four kinds of pesticides, including parathion, DDT, carbaryl and pleocidin, for further study. The results showed that Ran could be up-regulated by all pesticides. We further verified the relationship between Ran and resistance to the 4 pesticides by Ran RNAi, Ran overexpression and cell apoptosis. We found that the dsRNA of Ran induced more cell apoptosis than the control. Ran overexpression can significantly improve cell tolerance to various pesticides. These results demonstrate that Ran is associated with the resistance to and tolerance of multiple pesticides. Our evidence suggests the Ran is a potential molecular target gene of resistance control.

[The effect of Angelica sinensis on adhesion, invasion, migration and metastasis of melanoma cells].

OBJECTIVE: To study the effect of Angelica sinensis on invasion, adhesion, migration and metastasis of B16-BL6 metastatic mouse melanoma cells and discuss its functional mechanism. METHODS: The proliferation, adhesion, invasion and migration capacity of B16-BL6 metastatic cells was evaluated by MTT assay, adhesion assay and reconstituted basement membrane invasion and migration assay in vitro respectively. Mouse spontaneous melanoma model was used to study the effect of Angelica sinensis on metastasis in vivo. RESULTS: The extract of Angelica sinensis inhibited the proliferation of B16-BL6 metastatic cells and its migration capacity significantly. It regulated bidirectionally the adhesion of B16-BL6 metastatic cells to the basement component laminin while it had no effect on the invasion capacity. In the mouse spotaneous melanoma model, the lung metastatic nodes number and its volume were significantly decreased after continuously treated with the extract of Angelica sinensis at the concentration of 3.67 mg/kg. CONCLUSION: The extract of Angelica sinensis can inhibit the metastasis of of B16-BL6 metastatic mouse melanoma cells and its mechanism is maybe that Angelica sinensis can inhibit the B16-BL6 cells adhering to the ECM and reduce the migration of B16-BL6 cells.

Up-regulated expression of Ran reveals its potential role to deltamethrin stress in Kc cells.

The GTP-binding nuclear protein Ran has mostly been reported to be an essential player in nuclear transport, chromosome alignment, microtubule dynamics, centrosome duplication, kinetochore attachment of microtubules, nuclear-envelope dynamics, and phagocytosis. However, until now, there has been no report showing the involvement of Ran in DM stress. In this paper, two-dimensional electrophoresis analysis showed that the expression level of Ran in Kc cells in response to DM was higher than that in the control group. In addition, quantitative analysis using real-time PCR revealed that the expression of Ran was obviously up-regulated at various concentrations of DM. Western blot analysis showed that Ran was up-regulated 2.27-fold over the control at 48h. Because we still could not pinpoint whether Ran was actually involved in DM stress reaction, to further verify the role of Ran in stress reaction, RNA interference and cell transfection were utilized. Overexpression of Ran in cells conferred a degree of protection against DM after 72h. Furthermore, interference with Ran significantly decrease cell viability. All of the above findings strongly imply that Ran may participate in the development of stress reaction to DM. Therefore, investigating the possible role of Ran in DM stress will broaden our limited knowledge regarding DM stress inducible genes.

Analysis of UB and L40 resistance related to deltamethrin in Drosophila kc cells.

Pests have been subjected to heavy selection pressure, and the development of resistance to pyrethroid has been recorded. It is extremely valuable to identify the resistance genes that are relevant for pest control. In our previous studies, we reported that UBL40 is the deltamethrin resistance-associated gene. UBL40 is cleaved by specific endopeptidases to release UB and L40. Whether UB or L40 participates in deltamethrin resistance requires further study. In this study, quantitative real-time PCR was applied to reveal that UB and L40 were both overexpressed in Drosophila kc cells after deltamethrin stimulation. To investigate the roles of UB and L40 further, RNA interferences (RNAi) and cell transfections were utilized. UB and L40 knockdown both significantly reduced the level of resistance of RNAi-treated cells after 48 h, and the overexpressions of UB and L40 in Drosophila kc cells conferred a degree of protection against deltamethrin. These results represent the first evidence that UB and L40 both play roles in the regulation of deltamethrin resistance and that this study could help us to elucidate the resistance mechanisms and identify new target genes associated with resistance.

Identification of proteasome subunit beta type 2 associated with deltamethrin detoxification in Drosophila Kc cells by cDNA microarray analysis and bioassay analyses.

Insecticide deltamethrin resistance has presented a difficult obstacle for pest control and the resistance development is complex and associated with many genes. To better understand the possible molecular mechanisms involved in DM stress, in this study, cDNA microarray analysis was employed. 448 differentially expressed genes with at least a 2-fold expression difference were identified in Drosophila cells after DM exposure. Moreover, some genes were confirmed with qPCR, which yielded results consistent with the microarray analysis. Three members of the ubiquitin-proteasome system were significantly elevated in DM-stressed cells, suggesting that the ubiquitin-proteasome pathway may play an important role in DM detoxification. The proteasome beta2 subunit (Prosbeta2) is a member of 20S proteasome subunit family, which forms the proteolytic core of 26S proteasome. Whether Prosbeta2 participates in DM detoxification requires further study. RNAi and heterologous expression were conducted to investigate the contribution of Prosbeta2 in DM detoxification. The results revealed Prosbeta2 knockdown significantly reduce the level of DM detoxification in RNAi-treated cells after 48 h. Overexpression of Prosbeta2 increased cellular viability. These detoxification results represent the first evidence that Prosbeta2 plays a role in the detoxification of DM, which may provide new idea and target for studying the molecular mechanisms of insect resistance.

The identification and characterisation of a new deltamethrin resistance-associated gene, UBL40, in the diamondback moth, Plutella xylostella (L.).

Differential expression of ubiquitin was previously reported between Plutella xylostella strains that are resistant or susceptible to the pesticide deltamethrin (DM). This finding hinted at the potential involvement of ubiquitin in deltamethrin resistance, a theory that demanded further testing. Real-time PCR analyses revealed that one of the ubiquitin genes, UBL40, was overexpressed in the deltamethrin-resistant strain during the fourth instar. To investigate the functional relationship between this gene and deltamethrin resistance, RNA interference (RNAi) and cell transfection were utilised. UBL40 knockdown was observed to significantly reduce the level of resistance in RNAi-treated larvae after 48 h. Conversely, overexpression of UBL40 in Drosophila Kc cells conferred a degree of protection against deltamethrin. These results represent the first evidence that UBL40 plays a role in the regulation of deltamethrin resistance in P. xylostella.

Identification and characterization of Piwi subfamily in insects.

As a subfamily of Argonaute proteins, Piwi is poorly understood compared with Ago subfamily until recent discovery of Piwi protein interacting with piRNA. We did a large scale screening of insect genomes to identify piwi-like genes. Full or partial cDNA sequences were obtained by EST elongation and GENSCAN. We found that the exon numbers were totally different between vertebrates and invertebrates, approximately 20 exons in mammals but only 6-9 exons in insects. This infers either intron insertion or loss occurred during evolution. Characterized PAZ, c-terminal PIWI domains exist in almost all predicted Piwi-like proteins. We found six conserved motifs, which contain active catalytic triad "Asp-Asp-His/Lys" required for slicer activity. The expression of siwi1 and siwi2 in Bombyx mori were verified with RT-PCR. Phylogenetic tree inferred by Bayesian algorithm indicates invertebrate Piwi-like proteins are classified into three clades, of which Ago3 clade is closer to mammalian Piwi proteins.