• ISSN 1000-0615
  • CN 31-1283/S
Volume 9 Issue 11
Nov.  2021
Article Contents
Turn off MathJax

Citation:

Targeted regulation of miR-305-5p on Macrobrachium nipponense MnCHT3A gene in vivo

  • Corresponding author: NING Qianji, nqjnqj1964@163.com
  • Received Date: 2020-09-30
    Accepted Date: 2021-03-19
    Available Online: 2021-10-09
  • In order to explore the regulatory effect of microRNA (miRNA) on Macrobrachium nipponense chitinase 3A(MnCHT3A) gene, bioinformatics approach was firstly used to predict and screen the miRNA--miR-305-5p bound specifically to MnCHT3A. Using qRT-PCR, biochemical and histological methods, the regulation of miR-305-5p on target gene MnCHT3A was studied in vivo. The results showed that the expression change of miR-305-5p was negatively correlated with MnCHT3A during the molting cycle of M. nipponense. The level of miR-305-5p peaked at stage C and was the lowest at stage A, while the expression trend of MnCHT3A mRNA was opposite. After injection of miR-305-5p mimics or miR-305-5p inhibitor, the transcription level of MnCHT3A was decreased by 60% or increased by 166%, and the activity of MnCHT enzyme, meanwhile, was decreased by 39.53% or increased by 133%, respectively compared with the control group. Histological results showed that a three-layer cuticle structure of carapace in stage C was observed by means of H-E staining, namely, epicuticle, exocuticle and endocuticle from outside to inside. The images of chitin fluorescence staining showed the presence of chitin in the exocuticle and endocuticle. Results from scanning electron microscopy clearly showed the lamellar structure of the exocuticle and endocuticle. Compared with the control group, a thickening trend in the endocuticle with the lamellae as well as the blue fluorescence chitin stripe was observed in miR-305-5p mimics group. But in miR-305-5p inhibitor group, the culticular structure was disordered. Correspondingly, the blue fluorescence chitin stripe was not uniform and weakened in some areas. The results obtained above indicate that the target gene of miR-305-5p is MnCHT3A, and miR-305-5p can specifically inhibit the transcription of MnCHT3A in vivo.
  • 加载中
  • [1] 叶成凯, 卢志杰, Sarath B V, 等. 罗氏沼虾几丁质酶3B基因的克隆及其在蜕皮周期中的表达[J]. 水产学报, 2019, 43(4): 751-762.Ye C K, Lu Z J, Sarath B V, et al. Cloning and expression analysis of chitinase-3B from giant freshwater prawn (Macrobrachium rosenbergii) during molting cycle[J]. Journal of Fisheries of China, 2019, 43(4): 751-762 (in Chinese).
    [2] Guan J Y, Lv Y J, Zhang Y, et al. A shortening effect of KK-42 on the moult cycle of juvenile Macrobrachium nipponense (De Haan, 1849) (Decapoda, Palaemonidae)[J]. Crustaceana, 2016, 89(1): 85-95. doi: 10.1163/15685403-00003509
    [3] Zhang S Y, Jiang S F, Xiong Y W, et al. Six chitinases from oriental river prawn Macrobrachium nipponense: cDNA characterization, classification and mRNA expression during post-embryonic development and moulting cycle[J]. Comparative Biochemistry and Physiology-Part B: Biochemistry and Molecular Biology, 2014, 167: 30-40. doi: 10.1016/j.cbpb.2013.09.009
    [4] 张凤, 吕建建, 刘萍, 等. 三疣梭子蟹(Portunus trituberculatus)几丁质酶PtCht3基因克隆鉴定及表达分析[J]. 渔业科学进展, 2017, 38(2): 167-176. doi: 10.11758/yykxjz.20151111002Zhang F, Lü J J, Liu P, et al. Cloning and expression analysis of the cDNA of PtCht3 in Portunus trituberculatus[J]. Progress in Fishery Sciences, 2017, 38(2): 167-176 (in Chinese). doi: 10.11758/yykxjz.20151111002
    [5] Huang Q S, Yan J H, Tang J Y, et al. Cloning and tissue expressions of seven chitinase family genes in Litopenaeus vannamei[J]. Fish & Shellfish Immunology, 2010, 29(1): 75-81.
    [6] 姚琴琴, 杨志刚, 王瑶, 等. 中华绒螯蟹几丁质酶基因HXchit全长cDNA克隆及其在蜕皮过程中的表达分析[J]. 中国水产科学, 2015, 22(2): 185-195.Yao Q Q, Yang Z G, Wang Y, et al. Full length cDNA cloning of the chitinase gene (HXchit) and analysis of expression during the molting cycle of the Chinese mitten crab, Eriocheir sinensis[J]. Journal of Fishery Sciences of China, 2015, 22(2): 185-195 (in Chinese).
    [7] 张世勇. 青虾几丁质酶基因全长cDNA序列的克隆及时空表达分析[D]. 南京: 南京农业大学, 2014.Zhang S Y. Six chitinases cDNA cloning and expression analysis during post-embryonic development and moulting cycle in Macrobrachum nipponense[D]. Nanjing: Nanjing Agricultural University, 2014 (in Chinese).
    [8] Zhu Q S, Arakane Y, Banerjee D, et al. Domain organization and phylogenetic analysis of the chitinase-like family of proteins in three species of insects[J]. Insect Biochemistry and Molecular Biology, 2008, 38(4): 452-466. doi: 10.1016/j.ibmb.2007.06.010
    [9] Yang M L, Wang Y L, Jiang F, et al. miR-71 and miR-263 jointly regulate target genes Chitin synthase and Chitinase to control locust molting[J]. PLoS Genetics, 2016, 12(8): e1006257. doi: 10.1371/journal.pgen.1006257
    [10] Agrawal N, Sachdev B, Rodrigues J, et al. Development associated profiling of chitinase and microRNA of Helicoverpa armigera identified chitinase repressive microRNA[J]. Scientific Reports, 2013, 3: 2292. doi: 10.1038/srep02292
    [11] Zhang Y L, Huang Q X, Yin G H, et al. Identification of microRNAs by small RNA deep sequencing for synthetic microRNA mimics to control Spodoptera exigua[J]. Gene, 2015, 557(2): 215-221. doi: 10.1016/j.gene.2014.12.038
    [12] Li R, Weng J Y, Ren L Q, et al. A novel microRNA and its pfk target control growth length in the freshwater shrimp Neocaridina heteropoda[J]. Journal of Experimental Biology, 2020, 223(13): 223529.
    [13] Kirirat P, Promwikorn W, Thaweethamsewee P. Index of molt staging in the black tiger shrimp (Penaeus monodon)[J]. Songklanakarin Journal of Science and Technology, 2004, 26(5): 765-772.
    [14] 范文涛, 钟杨生, 陈芳艳, 等. miRNA在动物胚胎形成、发育中的研究进展[J]. 生物学杂志, 2016, 33(2): 91-94. doi: 10.3969/j.issn.2095-1736.2016.02.091Fan W T, Zhong Y S, Chen Y F, et al. Research progress of miRNA regulation in animal embryo formation and development[J]. Journal of Biology, 2016, 33(2): 91-94 (in Chinese). doi: 10.3969/j.issn.2095-1736.2016.02.091
    [15] Flynt A S, Lai E C. Biological principles of microRNA-mediated regulation: shared themes amid diversity[J]. Nature Reviews Genetics, 2008, 9(11): 831-842. doi: 10.1038/nrg2455
    [16] 马圣运, 白玉, 韩凝, 等. miRNA*生物合成及其功能研究的新发现[J]. 遗传, 2012, 34(4): 383-388. doi: 10.3724/SP.J.1005.2012.00383Ma S Y, Bai Y, Han N, et al. Recent research progress of biogenesis and functions of miRNA*[J]. Hereditas (Beijing), 2012, 34(4): 383-388 (in Chinese). doi: 10.3724/SP.J.1005.2012.00383
    [17] Filipowicz W, Jaskiewicz L, Kolb F A, et al. Post-transcriptional gene silencing by siRNAs and miRNAs[J]. Current Opinion in Structural Biology, 2005, 15(3): 331-341. doi: 10.1016/j.sbi.2005.05.006
    [18] 朱文奇, 陈宽维, 李慧芳, 等. 动物miRNA的最新研究进展[J]. 中国畜牧兽医, 2009, 36(11): 66-69.Zhu W Q, Chen K W, Li H F, et al. The latest research progress of animal miRNA[J]. China Animal Husbandry & Veterinary Medicine, 2009, 36(11): 66-69 (in Chinese).
    [19] 李都悦, 黄伊子, 刘伟, 等. miRNA分析研究进展[J]. 湖南农业科学, 2014(16): 14-17. doi: 10.3969/j.issn.1006-060X.2014.16.007Li D Y, Hang Y Z, Liu W, et al. Progress in miRNA research[J]. Hunan Agricultural Sciences, 2014(16): 14-17 (in Chinese). doi: 10.3969/j.issn.1006-060X.2014.16.007
    [20] Wang Y L, Yang M L, Jiang F, et al. MicroRNA-dependent development revealed by RNA interference-mediated gene silencing of LmDicer1 in the migratory locust[J]. Insect Science, 2013, 20(1): 53-60. doi: 10.1111/j.1744-7917.2012.01542.x
    [21] Wienholds E, Plasterk R H A. MicroRNA function in animal development[J]. FEBS Letters, 2005, 579(26): 5911-5922. doi: 10.1016/j.febslet.2005.07.070
  • Relative Articles

    [1] LÜ Yanjie, GUAN Jianyi, DU Juan, NING Qianji. Molecular cloning of N-acetyl-β-D-glucosaminidase (NAGase) gene and the effect of KK-42 on NAGase gene in Macrobrachium nipponense. Journal of fisheries of china, 2018, 42(5): 646-652.  doi: 10.11964/jfc.20170110689
    [2] JIANG Zhenting, LIU Bo, GE Xianping, ZHOU Qunlan, SUN Cunxin. Effects of dietary n-3/n-6 fatty acid ratio on growth performance, body composition, serum antioxidant capacity and related genes expression of oriental river prawn (Macrobrachium nipponense). Journal of fisheries of china, 2019, 43(10): 2109-2122.  doi: 10.11964/jfc.20190811929
    [3] SUN Shengming, FU Hongtuo, XUAN Fujun, GE Xianping, ZHU Jian, WU Xugan. Molecular cloning, prokaryotic expression and localization analysis of C-type lectin 3 (MnLec3) cDNA from Macrobrachium nipponense. Journal of fisheries of china, 2019, 43(11): 2317-2326.  doi: 10.11964/jfc.20180611327
    [4] JIANG Hongxia, LIN Xinhui, LI Shukai, WANG Jiechen, LI Xuejun. Molecular cloning and characterization of serum amyloid A gene and the analysis of its immune function in Macrobrachium nipponense. Journal of fisheries of china, 2020, 44(2): 300-313.  doi: 10.11964/jfc.20181211581
    [5] ZHANG Junfang, DU Juan, CHEN Ke, YANG Hong, NING Qianji. Effect of KK-42 on the carapace ultrastructure in Macrobrachium nipponense during postmolt. Journal of fisheries of china, 2020, 44(4): 575-580.  doi: 10.11964/jfc.20190311677
  • 加载中
通讯作者: 陈斌, bchen63@163.com
  • 1. 

    沈阳化工大学材料科学与工程学院 沈阳 110142

  1. 本站搜索
  2. 百度学术搜索
  3. 万方数据库搜索
  4. CNKI搜索

Figures(6)

Article views(440) PDF downloads(3) Cited by()

Related
Proportional views

Targeted regulation of miR-305-5p on Macrobrachium nipponense MnCHT3A gene in vivo

    Corresponding author: NING Qianji, nqjnqj1964@163.com
  • College of Life Sciences, Henan Normal University, Xinxiang    453007, China

Abstract: In order to explore the regulatory effect of microRNA (miRNA) on Macrobrachium nipponense chitinase 3A(MnCHT3A) gene, bioinformatics approach was firstly used to predict and screen the miRNA--miR-305-5p bound specifically to MnCHT3A. Using qRT-PCR, biochemical and histological methods, the regulation of miR-305-5p on target gene MnCHT3A was studied in vivo. The results showed that the expression change of miR-305-5p was negatively correlated with MnCHT3A during the molting cycle of M. nipponense. The level of miR-305-5p peaked at stage C and was the lowest at stage A, while the expression trend of MnCHT3A mRNA was opposite. After injection of miR-305-5p mimics or miR-305-5p inhibitor, the transcription level of MnCHT3A was decreased by 60% or increased by 166%, and the activity of MnCHT enzyme, meanwhile, was decreased by 39.53% or increased by 133%, respectively compared with the control group. Histological results showed that a three-layer cuticle structure of carapace in stage C was observed by means of H-E staining, namely, epicuticle, exocuticle and endocuticle from outside to inside. The images of chitin fluorescence staining showed the presence of chitin in the exocuticle and endocuticle. Results from scanning electron microscopy clearly showed the lamellar structure of the exocuticle and endocuticle. Compared with the control group, a thickening trend in the endocuticle with the lamellae as well as the blue fluorescence chitin stripe was observed in miR-305-5p mimics group. But in miR-305-5p inhibitor group, the culticular structure was disordered. Correspondingly, the blue fluorescence chitin stripe was not uniform and weakened in some areas. The results obtained above indicate that the target gene of miR-305-5p is MnCHT3A, and miR-305-5p can specifically inhibit the transcription of MnCHT3A in vivo.

  • 甲壳动物体表覆盖的坚硬表皮,又称“外骨骼”,限制其生长,必须通过周期性蜕皮完成生长发育,蜕皮周期的调控机制一直是相关领域的研究热点。几丁质是构成表皮的主要成分之一,在蜕皮过程中,几丁质酶通过水解β-1,4糖苷键将几丁质聚合物或者寡聚物降解成几丁质单体[1],是参与旧表皮降解的关键酶,其基因表达的变化与蜕皮周期密切相关[2-3]。目前,已经从三疣梭子蟹(Portunus trituberculatus)[4]、凡纳滨对虾(Litopenaeus vannamei)[5]和中华绒螯蟹(Eriocheir sinensis)[6]等甲壳动物中克隆出多种几丁质酶cDNA全长序列,根据基因的碱基序列以及结构等方面的差异,将几丁质酶基因分为6类(GroupⅠ~Ⅵ),每一类又分为多个亚类,分别用A、B等表示[7-8]。日本沼虾几丁质酶3A (Macrobrachium nipponense chitinase 3A,MnCHT3A)基因属于Group3,初步研究显示,该基因可能参与动物的胚后发育[7]

    microRNA (miRNA)是一类广泛存在于真核生物,长度约为 20~24 个核苷酸的内源性非编码RNA,在转录后水平上具有多重的调控作用。在节肢动物中,涉及miRNA参与调控生长发育的研究报道以昆虫为多,甲壳动物相对较少。miR-263直接抑制飞蝗(Locusta migratoria)几丁质酶10基因,蝗虫的蜕皮呈缺陷表型,影响其正常的蜕皮发育[9]。在棉铃虫(Helicoverpa armigera)中,miR-24过表达可降低几丁质酶表达,阻碍幼虫的蜕皮[10]。在甜菜夜蛾(Spodoptera exigua)中,miR-4924通过下调几丁质酶1的表达影响其幼虫的蜕皮发育行为[11]。黄金米虾(Neocaridina heteropoda)注射miR-26 agomirs,可导致磷酸果糖激酶基因表达降低,动物的蜕皮周期延长及个体变小[12]

    本实验以日本沼虾为材料,首先采用生物信息学方法,预测筛选出了靶基因MnCHT3A对应的miRNA(miR-305-5p);在分子及组织学水平研究了注射miR-305-5p mimics和miR-305-5p inhibitor 后,头胸甲表皮组织MnCHT3A转录水平、酶活性、表皮结构的变化,旨在揭示miR-305-5p对其靶基因MnCHT3A的调控作用,为阐明甲壳动物蜕皮周期的调控机制积累资料。

1.   材料与方法
  • 选取体长(3.0±0.5) cm的健康日本沼虾幼虾(捕捞于河南省卫辉市)饲养在实验室玻璃水族箱,水温 (25±1) °C,早晚各投喂1次,1周后用于实验。蜕皮周期的鉴定参照Kirirat等[13]

  • 在获得MnCHT3A全长和Small RNA测序(miRNA-Seq)的基础上,筛选头胸甲表皮组织中特异表达的miRNA。使用NCBI GEO中miRNA测序数据库对micRNA分析,得到的micRNA作为靶向micRNA;使用miRanda和RNAhybrid对MnCHT3A的3′UTR靶向预测miRNA,根据mRNA和miRNA的序列匹配程度和形成复合结构的自由能,筛选二者相同结果的miRNA。

  • 选取处于C期、D0-2期、D3-4期和A期的虾各3只,分别取头胸甲表皮,利用Mini BEST Universal RNA Extraction Kit (TaKaRa)试剂盒提取总RNA,琼脂糖凝胶中电泳检测RNA完整,Nano Drop ONE(赛默飞世尔科技公司)超微量紫外-分光光度计检测RNA纯度。

  • 使用HiScript QRT Super Mix for qPCR (南京诺唯赞生物科技股份有限公司)反转录试剂盒,总RNA反转录合成cDNA。每一蜕皮周期与内参分别设置3个重复。再以cDNA为模板,正向引物:5′-GTCATTGCTTGCACCTTCTACC-3′,反向引物:5′- GACCTTGATCGC ATAATCAGCA-3′,使用AceQ qPCR SYBR Green Master Mix(南京诺唯赞生物科技股份有限公司)试剂盒实时荧光定量PCR (qRT-PCR)检测MnCHT3A的表达量。

    使用miRNA第一链cDNA合成试剂盒 [生工生物工程(上海)股份有限公司]合成miRNA的cDNA第一链。再以产物为模板,正向引物:5′-ACACTCCAGCTGGGTCAAAATCGTGAAGCG-3′,反向引物:5′- CTCAACTGGTGTCGTGGAGTCGGCAA-3′。每一蜕皮周期的不同阶段设置3个重复,用miRNA荧光定量PCR试剂盒[生工生物工程(上海)股份有限公司]检测miRNA的表达量。

  • 根据miRNA的序列或者互补序列,合成miR-305-5p模拟物(mimics)和抑制物(inhibitor)(武汉金开瑞生物有限公司)。

    选取C期的日本沼虾,微量进样器从围心腔分别注射DEPE水(对照组)、4 μg/g miR-305-5p mimics (miR-305-5p mimics组)和4 μg/g miR-305-5p inhibitor (miR-305-5p inhibitor组)。48 h后,对照组、miR-305-5p mimics组再注射相同剂量,miR-305-5p inhibitor组剂量加倍。168 h后,取头胸甲表皮组织,分别用于MnCHT3A的mRNA水平测定、几丁质酶活性分析(南京建成科技有限公司)以及显微、亚显微结构观察。

  • 取头胸甲表皮(3只/组),常规石蜡包埋及切片,分别进行苏木精-伊红(H.E)染色、几丁质荧光染色(FB28、碘化丙啶试剂)观察表皮的显微结构,扫描电镜(JSM-7800F,JROL)观察亚显微结构。

  • 实验所得数据用SPSS 13.0软件数据分析,采用单因素方差分析(One-Way ANOVA),显著、极显著差异分别用P<0.05和P<0.01表示。所有的数据结果均以平均值±标准差(mean±SE)表示。

2.   结果
  • 通过miRanda和RNAhybrid软件对miRNA测序数据库与MnCHT3A的3′UTR的分析,miR-305-5p的5′端2~8位种子区碱基互补配对(图1),结合自由能较小(–32.9 kcal/mol),判定miR-305-5p的潜在靶基因为MnCHT3A

    Figure 1.  Predicted binding site of miR-305-5p in 3′UTR of MnCHT3A

  • MnCHT3A表达量最低为C期,至A期达到最高;而miR-305-5p的表达量变化则与之相反(图2)。

    Figure 2.  Expression analysis of miR-305-5p and MnCHT3A in different molting stages of M. nipponense

  • qRT-PCR结果显示,与对照组相比,miR-305-5p mimic组MnCHT3A表达量降低60%(P<0.01),而miR-305-5p inhibitor组MnCHT3A的表达量增加了166%(P<0.01)(图3-a)。结果表明,miR-305-5p对其靶基因的转录具有抑制效应。

    Figure 3.  Effects of miR-305-5p mimic and miR-305-5p inhibitor on MnCHT3A expression (a) and MnCHT enzyme activity (b)

  • miR-305-5p mimic组MnCHT酶活性极显著降低,仅为对照组的39.53% (P<0.01);而miR-305-5p inhibitor组酶活性显著升高,为对照组的133% (P<0.01)(图3-b)。

  • H.E染色结果显示,C期表皮分为3层,由外向内依次为上表皮(Ep)、外表皮(Ex)和内表皮(En)(图版Ⅰ-1)。miR-305-5p mimic组表皮结构正常且内表皮厚度增加(图版Ⅰ-2),miR-305-5p inhibitor组表皮结构紊乱(图版Ⅰ-3)。

    Figure 图版Ⅰ.  H.E staining cuticle after miR-305-5p mimic or miR-305-5p inhibitor administration

    通过FB28 和PI 两种染色剂染色表皮,在荧光显微镜下含有几丁质成分的组织成蓝色阳性,表皮的其他细胞成分被PI染成红色(图版Ⅱ-23)。位于外表皮和内表皮的几丁质成分被FB28染成了蓝色且分布较为均匀(图版Ⅱ-13)。相较于对照组,miR-305-5p mimic组有几丁质分布的表皮层蓝色荧光增厚现象(图版Ⅱ-46),而miR-305-5p inhibitor组几丁质荧光信号分布不均且部分区域减弱(图版Ⅱ-79)。

    Figure 图版Ⅱ.  Fluorescence staining of epidermal chitin after injection of miR-305-5p mimic or miR-305-5p inhibitor

    扫描电镜可以清晰看到表皮各层的结构(图版Ⅲ-1),miR-305-5p mimic组内表皮较对照组出现增厚现象(图版Ⅲ-2),与图版Ⅰ -2结果一致;而miR-305-5p inhibitor组表皮结构紊乱(图版Ⅲ-3)。

    Figure 图版Ⅲ.  Epidermal scanning electron microscopic results after injection of miR-305-5p mimic and miR-305-5p inhibitor

3.   讨论
  • miRNA具有高度组织特异性、保守性和时序性[14-16],与靶基因结合引起的调控效应与二者的匹配程度有关[17-18],研究表明,如果miRNA与mRNA的序列不完全匹配,但只要位于3′UTR的种子序列与mRNA匹配,也能实现对靶基因的调控[19]。根据匹配程度和形成复合结构自由能的大小,本实验筛选出了能靶向结合MnCHT3A的miR-305-5p。MnCHT3A与miR-305-5p在蜕皮周期中的表达趋势呈负相关(图2),初步印证了miR-305-5p对靶基因表达的抑制效应,作用机理可能是miRNA与靶基因结合后直接抑制mRNA的翻译导致转录水平下降[20],也可能是通过影响mRNA的稳定性导致其降解[21],有待进一步研究。关于MnCHT3A的功能目前尚无确切的结论,有学者认为该基因可能在降解围食膜等方面发挥作用[2,7],而本实验结果显示,MnCHT3A在蜕皮周期中呈波动性变化,D3-4期表达量较C期显著升高(图2),推测该基因参与旧表皮的降解。

    理论上,处于C期的甲壳动物表皮结构相对稳定,几丁质酶水平变化相对较小[2],所以,本实验选择C期的日本沼虾用于研究在体条件下miR-305-5p对靶基因的调控。注射miR-305-5p mimic后,MnCHT3A的转录水平显著下降(图3-a),MnCHT酶活性相应地降低(图3-b),进一步验证了上述推测。组织学观察结果也支持上述推断,MnCHT酶活性的降低可减慢表皮,尤其是内表皮中几丁质降解的速率,所以内表皮厚度相应增加(图版Ⅰ -2图版Ⅲ-2),这与飞蝗表皮中几丁质酶10受到干扰时的结果一致[9]。为了进一步说明MnCHT3A和miR-305-5p的靶向调控关系,合成了miR-305-5p inhibitor,得到了与miR-305-5p mimic组相反的结果,由于miR-305-5p inhibitor抑制了与靶基因的结合,故MnCHT3A转录水平及MnCHT酶活性升高(图3),加快了表皮中几丁质的降解速率,表皮结构出现紊乱(图版Ⅰ -3图版Ⅱ-9图版Ⅲ-3)。以上结果表明,MnCHT3A是miR-305-5p的靶基因,在体条件下miR-305-5p对MnCHT3A的表达具有抑制效应。

Reference (21)

Catalog

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return