Identification and analysis of miRNA targeting CYP450s genes in mosquitoes

doi:10.12140/j.issn.1000-7423.2023.06.004

Abstract

Abstract:

Objective To screen and identify the microRNAs (miRNAs) targeting cytochrome P450 enzymes (CYP450s) genes in mosquitoes, and analyze the differentially expressed miRNAs and the function of their target genes. Methods Ten larvae of each cypermethrin-susceptible (CS) and cypermethrin-resistant (CR) line Culex pipiens pallens mosquitoes and 5 blood-unfed female adult mosquitoes of each line on 3 d post-emergence were collected for extracting total RNA followed by sequencing, to calculate the differential expression between CS and CR lines using DEGseq software. MiRanda and RNAhybrid databases were used to screen the target genes of differentially expressed miRNAs, and gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis was performed. Five differentially expressed miRNAs (miR-11-5p, miR-317_3, miR-278-3p_1, miR-8-5p and miR-305-3p_5) that may regulate the expression of CYP450s genes were selected for real-time quantitative PCR (qRT-PCR) to validate the sequence output. The relative expression level of differentially expressed miRNAs target genes (CYP6a8, CYP6BB1v2, CYP6N22, CYP6N26P, CYP9b2) were detected by qRT-PCR. Results A total of 196 miRNAs were differentially expressed between CS and CR line Culex pipiens pallens. Among them, 16.3% (32/196) were differentially expressed in age Ⅲ larvae, 20.4% (40/196) in age Ⅳ larvae and 26.0% (51/196) in female adult mosquitoes exclusively; 12.2% (24/196) were differentially expressed in all stages. 45 were up-regulated and 46 were down-regulated of 91 miRNAs differentially expressed at age Ⅲ larvae. 59 were up-regulated and 45 were down-regulated of 104 miRNAs differentially expressed at age Ⅳ larvae. 44 were up-regulated and 54 were down-regulated of 98 miRNAs differentially expressed at female adult mosquitoes. The results of the GO and KEGG enrichment analysis showed that the target genes of differentially expressed miRNAs were mainly related to cellular, metabolic and single-cell biological pathways. The molecular functions of target genes were mainly involve with binding, catalytic activity and transporter activity. The target genes were mainly enriched in the pathways related to signal transduction, infectious diseases, cancers and parasitic diseases. The qRT-PCR results showed that the trends of 5 differentially expressed miRNAs were basically consistent with the sequencing results; The expression profiles of 5 miRNAs were opposite to the target genes (CYP6a8, CYP6BB1v2, CYP6N22, CYP6N26P and CYP9b2). Conclusion The five differentially expressed miRNAs screened in this study (miR-11-5p, miR-317_3, miR-278-3p_1, miR-8-5p and miR-305-3p_5) could regulate the insecticide resistance of Culex pipiens pallens by modulating the expression of CYP450s.

Key words: Culex pipiens pallens, Cypermethrin, miRNA, Insecticide resistance, High-throughput sequencing

CLC Number:

R384.1

WU Jiahui, SONG Xiao, CHENG Peng, LIU Hongmei, GUO Xiuxia, WANG Haifang, GONG Maoqing. Identification and analysis of miRNA targeting CYP450s genes in mosquitoes[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2023, 41(6): 683-690.

Figures/Tables 8

Table 1

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Fig. 7

References 26

[1]	Meenambigai K, Kokila R, Chandhirasekar K, et al. Green synthesis of selenium nanoparticles mediated by nilgirianthus ciliates leaf extracts for antimicrobial activity on foodborne pathogenic microbes and pesticidal activity against Aedes aegypti with molecular docking[J]. Biol Trace Elem Res, 2022, 200(6): 2948-2962. doi: 10.1007/s12011-021-02868-y
[2]	Mugenzi LMJ, Akosah-Brempong G, Tchouakui M, et al. Escalating pyrethroid resistance in two major malaria vectors Anopheles funestus and Anopheles gambiae (s.l.) in Atatam, Southern Ghana[J]. BMC Infect Dis, 2022, 22(1): 799. doi: 10.1186/s12879-022-07795-4 pmid: 36284278
[3]	Ghavami MB, Panahi S, Nabati SM, et al. A comprehensive survey of permethrin resistance in human head louse populations from northwest Iran: ex vivo and molecular monitoring of knockdown resistance alleles[J]. Parasit Vectors, 2023, 16(1): 57. doi: 10.1186/s13071-023-05652-0
[4]	Peng H, Wang HY, Guo XX, et al. In vitro and in vivo validation of CYP6A14 and CYP6N6 participation in deltamethrin metabolic resistance in Aedes albopictus[J]. Am J Trop Med Hyg, 2023, 108(3): 609-618. doi: 10.4269/ajtmh.22-0524
[5]	Montgomery M, Harwood JF, Yougang AP, et al. Spatial distribution of insecticide resistant populations of Aedes aegypti and Ae. albopictus and first detection of V410L mutation in Ae. aegypti from Cameroon[J]. Infect Dis Poverty, 2022, 11: 90. doi: 10.1186/s40249-022-01013-8 pmid: 35974351
[6]	Højland DH, Kristensen M. Analysis of differentially expressed genes related to resistance in spinosad- and neonicotinoid-resistant Musca domestica L. (Diptera ∶ Muscidae) strains[J]. PLoS One, 2017, 12(1): e0170935. doi: 10.1371/journal.pone.0170935
[7]	Itokawa K, Komagata O, Kasai S, et al. A single nucleotide change in a core promoter is involved in the progressive overexpression of the duplicated CYP9M10 haplotype lineage in Culex quinquefasciatus[J]. Insect Biochem Mol Biol, 2015, 66: 96-102. doi: 10.1016/j.ibmb.2015.10.006
[8]	Chipman LB, Pasquinelli AE. miRNA targeting: growing beyond the seed[J]. Trends Genet, 2019, 35(3): 215-222. doi: S0168-9525(18)30224-5 pmid: 30638669
[9]	Mellis D, Caporali A. microRNA-based therapeutics in cardiovascular disease: screening and delivery to the target[J]. Biochem Soc Trans, 2018, 46(1): 11-21. doi: 10.1042/BST20170037
[10]	Bartel DP. Metazoan microRNAs[J]. Cell, 2018, 173(1): 20-51. doi: S0092-8674(18)30286-1 pmid: 29570994
[11]	Ofer D, Linial M. Inferring microRNA regulation: a proteome perspective[J]. Front Mol Biosci, 2022, 9: 916639. doi: 10.3389/fmolb.2022.916639
[12]	Prasad A, Sharma N, Muthamilarasan M, et al. Recent advances in small RNA mediated plant: virus interactions[J]. Crit Rev Biotechnol, 2019, 39(4): 587-601. doi: 10.1080/07388551.2019.1597830
[13]	Hoen PA, Ariyurek Y, Thygesen HH, et al. Deep sequencing-based expression analysis shows major advances in robustness, resolution and inter-lab portability over five microarray platforms[J]. Nucl Acid Res, 2008, 36(21): e141. doi: 10.1093/nar/gkn705
[14]	Wang LK, Feng ZX, Wang X, et al. DEGseq: an R package for identifying differentially expressed genes from RNA-seq data[J]. Bioinformatics, 2010, 26(1): 136-138. doi: 10.1093/bioinformatics/btp612 pmid: 19855105
[15]	Xu P, Wang JH, Sun B, et al. Integrated analysis of miRNA and mRNA expression data identifies multiple miRNAs regulatory networks for the tumorigenesis of colorectal cancer[J]. Gene, 2018, 659: 44-51. doi: S0378-1119(18)30288-9 pmid: 29555201
[16]	Galehdari H, Azarshin SZ, Bijanzadeh M, et al. Polymorphism studies on microRNA targetome of thalassemia[J]. Bioinformation, 2018, 14(5): 252-258. doi: 10.6026/97320630014252 pmid: 30108424
[17]	Liu SW, Xie X, Lei HJ, et al. Identification of key circRNAs/lncRNAs/miRNAs/mRNAs and pathways in preeclampsia using bioinformatics analysis[J]. Med Sci Monit, 2019, 25: 1679-1693. doi: 10.12659/MSM.912801
[18]	Song X, Cheng P, Wang HF, et al. Study on insecticide resistance of Culex pipiens pallens in southwest region of Shandong Province[J]. Chin J Schisto Control, 2020, 32(1): 69-72. (in Chinese)
	(宋晓, 程鹏, 王海防, 等. 鲁西南地区淡色库蚊抗药性评价[J]. 中国血吸虫病防治杂志, 2020, 32(1): 69-72.)
[19]	Liu K, Huang HB, Yang GL. miRNA functions in parasite-related immune regulation in hosts[J]. Chin J Parasitol Parasit Dis, 2018, 36(4): 405-408. (in Chinese)
	(刘可, 黄海斌, 杨桂连. miRNA在寄生虫宿主免疫调控中的研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(4): 405-408.)
[20]	Sun XH, Xu N, Xu Y, et al. A novel miRNA, miR-13664, targets CpCYP314A1 to regulate deltamethrin resistance in Culex pipiens pallens[J]. Parasitology, 2019, 146(2): 197-205. doi: 10.1017/S0031182018001002 pmid: 29966536
[21]	Li XX, Hu SL, Yin HT, et al. miR-4448 is involved in deltamethrin resistance by targeting CYP4H31 in Culex pipiens pallens[J]. Parasit Vectors, 2021, 14(1): 159. doi: 10.1186/s13071-021-04665-x
[22]	Lei ZT, Lv Y, Wang WJ, et al. miR-278-3p regulates pyrethroid resistance in Culex pipiens pallens[J]. Parasitol Res, 2015, 114(2): 699-706. doi: 10.1007/s00436-014-4236-7
[23]	Tian MM, Liu BQ, Hu HX, et al. miR-285 targets P450 (CYP6N23) to regulate pyrethroid resistance in Culex pipiens pallens[J]. Parasitol Res, 2016, 115(12): 4511-4517. doi: 10.1007/s00436-016-5238-4
[24]	van Dijk EL, Jaszczyszyn Y, Thermes C. Library preparation methods for next-generation sequencing: tone down the bias[J]. Exp Cell Res, 2014, 322(1): 12-20. doi: 10.1016/j.yexcr.2014.01.008 pmid: 24440557
[25]	Zhong SH, Sun Y, Guo XL, et al. Identification and bioinformatics analysis of differentially expressed miRNAs in splenic lymphocytes in Echinococcus multilocularis infected mice[J]. Chin J Parasitol Parasit Dis, 2022, 40(3): 288-295. (in Chinese)
	(仲顺虎, 孙玥, 郭小腊, 等. 多房棘球蚴感染小鼠脾淋巴细胞中差异表达miRNA的鉴定及其生物信息学分析[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(3): 288-295.)
[26]	Yang J, Xie M, Xu XJ, et al. Research progress of insect miRNAs[J]. Acta Entomol Sin, 2021, 64(2): 259-280. (in Chinese)
	(杨婕, 谢苗, 徐雪娇, 等. 昆虫miRNA研究进展[J]. 昆虫学报, 2021, 64(2): 259-280.)

引物名称 Primer name	引物序列（5'→3'） Primer sequence (5'→3')
miR-11-5p-F	GCGAGAACTCCGGCTGTGA
miR-317_3-F	CGTGAACACAGCTGGTGGTAT
miR-278-3p_1-F	CGTCGGTGGGACTTTCGT
miR-8-5p-F	CGCATCTTACCGGGCAGC
miR-305-3p_5-F	CGCGGCACATGTTGGAGTA
miR-UP-R	AGTGCAGGGTCCGAGGTATT
U6-F	GCTTCGGCTGGACATATACTAAAAT
U6-R	GAACGCTTCACGATTTTGCG
CYP6a8-F	CGGATGATACGAAGAGCGACGATG
CYP6a8-R	ACCCAGCCAAATAGAACACGAACG
CYP6BB1v2-F	AGACCTGGAGCTGCGAGAAGTG
CYP6BB1v2-R	TGTTGCCCATCTTTCGGAACTCT
CYP6N22-F	TTGAACGAGATTGCCGCACAGG
CYP6N22-R	ACTTCGAGAACATTCTGCCTTGCC
CYP6N26P-F	CAGCACTTCCACGACCGTTCC
CYP6N26P-R	CTCAGGTTCCGCCACTTGTTCC
CYP9b2-F	GTTGGTGCCTGCTGCGATCTAC
CYP9b2-R	CGAACGGAACTCCACGCTTCTC
β-actin-F	AGGACTCGTACGTCGGTGAC
β-actin-R	TGGTGCCAGATCTTCTCTCCAT