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On-line Access: 2016-02-01

Received: 2015-04-06

Revision Accepted: 2015-11-07

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Journal of Zhejiang University SCIENCE B 2016 Vol.17 No.2 P.127-135

http://doi.org/10.1631/jzus.B1500078


bmo-miR-0001 and bmo-miR-0015 down-regulate expression of Bombyx mori fibroin light chain gene in vitro


Author(s):  Chen Chen, Yang-yang Fan, Xin Wang, Fei Song, Tao Jiang, Ping Qian, Shun-Ming Tang, Xing-Jia Shen

Affiliation(s):  Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212003, China; more

Corresponding email(s):   shenxjsri@163.com

Key Words:  Bombyx mori, MicroRNA, bmo-miR-0001, bmo-miR-0015, BmFib-L, Regulation of expression


Chen Chen, Yang-yang Fan, Xin Wang, Fei Song, Tao Jiang, Ping Qian, Shun-Ming Tang, Xing-Jia Shen. bmo-miR-0001 and bmo-miR-0015 down-regulate expression of Bombyx mori fibroin light chain gene in vitro[J]. Journal of Zhejiang University Science B, 2016, 17(2): 127-135.

@article{title="bmo-miR-0001 and bmo-miR-0015 down-regulate expression of Bombyx mori fibroin light chain gene in vitro",
author="Chen Chen, Yang-yang Fan, Xin Wang, Fei Song, Tao Jiang, Ping Qian, Shun-Ming Tang, Xing-Jia Shen",
journal="Journal of Zhejiang University Science B",
volume="17",
number="2",
pages="127-135",
year="2016",
publisher="Zhejiang University Press & Springer",
doi="10.1631/jzus.B1500078"
}

%0 Journal Article
%T bmo-miR-0001 and bmo-miR-0015 down-regulate expression of Bombyx mori fibroin light chain gene in vitro
%A Chen Chen
%A Yang-yang Fan
%A Xin Wang
%A Fei Song
%A Tao Jiang
%A Ping Qian
%A Shun-Ming Tang
%A Xing-Jia Shen
%J Journal of Zhejiang University SCIENCE B
%V 17
%N 2
%P 127-135
%@ 1673-1581
%D 2016
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B1500078

TY - JOUR
T1 - bmo-miR-0001 and bmo-miR-0015 down-regulate expression of Bombyx mori fibroin light chain gene in vitro
A1 - Chen Chen
A1 - Yang-yang Fan
A1 - Xin Wang
A1 - Fei Song
A1 - Tao Jiang
A1 - Ping Qian
A1 - Shun-Ming Tang
A1 - Xing-Jia Shen
J0 - Journal of Zhejiang University Science B
VL - 17
IS - 2
SP - 127
EP - 135
%@ 1673-1581
Y1 - 2016
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.B1500078


Abstract: 
Based on bioinformatic analysis, we selected two novel microRNAs (miRNAs), bmo-miR-0001 and bmo-miR-0015, from high-throughput sequencing of the Bombyx mori larval posterior silk gland (PSG). Firstly, we examined the expression of bmo-miR-0001 and bmo-miR 12 different tissues of the 5th instar Day-3 larvae of the silkworm. The results showed that the expression levels of both bmo-miR-0001 and bmo-miR-0015 were obviously higher in the PSG than in other tissues, implying there is a spatio-temporal condition for bmo-miR-0001 and bmo-miR-0015 to regulate the expression of BmFib-L. To test this hypothesis, we constructed pri-bmo-miR-0001 expressing the plasmid pcDNA3.0 and pri-bmo-miR-0015 expressing the plasmid pcDNA3.0 [ie1-egfp-pri-bmo-miR-0015-SV40]. Finally, the BmN cells were harvested and luciferase activity was detected. The results showed that luciferase activity was reduced significantly (P<0.05) in BmN cells co-transfected by pcDNA3.0 [ie1-egfp-pri-bmo-miR-0001-SV40] or pcDNA3.0 with pGL3.0 [A3-luc-Fib-L-3'UTR-SV40], suggesting that both bmo-miR-0001 and bmo-miR-0015 can down-regulate the expression of BmFib-L in vitro.

家蚕miR-0001和miR-0015体外下调BmFib-L基因的表达

目的:探究新发现的两个miRNA对家蚕丝素轻链基因BmFib-L的调控作用。
创新点:在家蚕后部丝腺中发现两个新的miRNA,并首次证明它们对家蚕丝素蛋白轻链基因BmFib-L有负调控作用。
方法:本实验通过生物信息学分析,从家蚕后部丝腺miRNA高通量测序获得的新miRNA中,筛选出两个可能对家蚕丝素蛋白轻链基因BmFib-L有调控作用的miRNA,即bmo-miR-0001和bmo-miR-0015。设计茎环引物,采用反转录聚合酶链反应(RT-PCR)方法对家蚕5龄3 d头部、表皮、脂肪体、马氏管、精巢/卵巢、丝腺(前、中、后)、气管、中肠和血淋巴细胞等12个不同组织的bmo-miR-0001和bmo-miR-00015进行半定量表达分析。并采用双荧光报告基因检测系统进一步在细胞水平上验证bmo-miR-0001和bmo-miR-0015对BmFib-L表达的调控作用。
结论:本实验中RT-PCR结果显示,这两个新miRNA在家蚕后部丝腺中表达量最高(图4)。双荧光报告基因检测结果显示,报告基因荧光素酶活性明显低于阳性对照组(图7),转染bmo-miR-0001和bmo-miR-0015表达载体的细胞,报告基因和荧光素酶活性分别只及对照组60%和69%。t检验分析结果显示两个实验组与对照组之间差异都达到显著水平(P<0.05)。由此可见,bmo-miR-0001和bmo-miR-0015在体外对BmFib-L的表达具有显著的抑制作用。

关键词:家蚕;miRNA;bmo-miR-0001;bmo-miR-0015;BmFib-L;功能验证

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article

Reference

[1]Bushati, N., Cohen, S.M., 2007. MicroRNA functions. Annu. Rev. Cell Dev. Biol., 23(1):175-205.

[2]Cai, Y., Yu, X., Zhou, Q., et al., 2010. Novel microRNAs in silkworm (Bombyx mori). Funct. Integr. Genomics, 10(3):405-415.

[3]Cao, J., Tong, C., Wu, X., et al., 2008. Identification of conserved microRNAs in Bombyx mori (silkworm) and regulation of fibroin L chain production by microRNAs in heterologous system. Insect Biochem. Mol. Biol., 38(12):1066-1071.

[4]Chen, A., Xia, D., Qiu, Z., et al., 2013. Expression of a vitelline membrane protein, BmVMP 23, is repressed by bmo-miR-3p in silkworm, Bombyx mori. FEBS Lett., 587(7):970-975.

[5]Couble, P., Moine, A., Garel, A., et al., 1983. Developmental variations of a nonfibroin mRNA of Bombyx mori silkgland, encoding for a low-molecular-weight silk protein. Dev. Biol., 97(2):398-407.

[6]da Sacco, L., Masotti, A., 2012. Recent insights and novel bioinformatics tools to understand the role of microRNAs binding to 5' untranslated region. Int. J. Mol. Sci., 14(1):480-495.

[7]Dai, Z., Wu, R., Zhao, Y., et al., 2014. Early lethality of shRNA-transgenic pigs due to saturation of microRNA pathways. J. Zhejiang Univ.-Sci. B (Biomed. & Biotechnol.), 15(5):466-473.

[8]Ebert, M.S., Sharp, P.A., 2010. MicroRNA sponges: progress and possibilities. RNA, 16(11):2043-2050.

[9]Fabian, M.R., Sonenberg, N., Filipowicz, W., 2010. Regulation of mRNA translation and stability by microRNAs. Annu. Rev. Biochem., 79(1):351-379.

[10]He, P.A., Nie, Z., Chen, J., et al., 2008. Identification and characteristics of microRNAs from Bombyx mori. BMC Genomics, 9(1):248.

[11]Huang, Y., Zou, Q., Tang, S.M., et al., 2010. Computational identification and characteristics of novel microRNAs from the silkworm (Bombyx mori L.). Mol. Biol. Rep., 37(7):3171-3176.

[12]Huang, Y., Zou, Q., Wang, S.P., et al., 2011. Construction and detection of expression vectors of microRNA in BmN cells. J. Zhejiang Univ.-Sci. B (Biomed. & Biotechnol.), 12(7):527-533.

[13]Huang, Y., Zou, Q., Song, F., et al., 2012. The regulation of silkworm fibroin L chain production by miRNA-965 and miRNA insect cells. Russ. J. Bioorg. Chem., 38(4):417-421.

[14]Inoue, S., Tanaka, K., Arisaka, F., et al., 2000. Silk fibroin of Bombyx mori is secreted, assembling a high molecular mass elementary unit consisting of H-chain, L-chain, and P25, with a 6:6:1 molar ratio. J. Biol. Chem., 275(51):40517-40528.

[15]Johnston, R.J., Hobert, O., 2003. A microRNA controlling left/right neuronal asymmetry in Caenorhabditis elegans. Nature, 426(6968):845-849.

[16]Jopling, C.L., Yi, M., Lancaster, A.M., et al., 2005. Modulation of hepatitis C virus RNA abundance by a liver-specific microRNA. Science, 309(5740):1577-1581.

[17]Khan, A.A., Betel, D., Miller, M.L., et al., 2009. Transfection of small RNAs globally perturbs gene regulation by endogenous microRNAs. Nat. Biotechnol., 27(6):549-555.

[18]Kramer, M.F., 2011. Stem-loop RT-qPCR for miRNAs. Curr. Protoc. Mol. Biol., 15:15.10.1-15.10.15.

[19]Lee, Y., Kim, M., Han, J., et al., 2004. MicroRNA genes are transcribed by RNA polymerase II. EMBO J., 23(20):4051-4060.

[20]Li, J., Cai, Y., Ye, L., et al., 2014. MicroRNA expression profiling of the fifth-instar posterior silk gland of Bombyx mori. BMC Genomics, 15(1):410.

[21]Lim, L.P., Glasner, M.E., Yekta, S., et al., 2003. Vertebrate microRNA genes. Science, 299(5612):1540.

[22]Liu, S., Gao, S., Zhang, D., et al., 2010. MicroRNAs show diverse and dynamic expression patterns in multiple tissues of Bombyx mori. BMC Genomics, 11(1):85.

[23]Liu, S., da Cunha, A.P., Rezende, R.M., et al., 2016. The host shapes the gut microbiota via fecal microRNA. Cell Host Microbe, 19(1):32-43.

[24]Lu, Y.L., Jing, W., Feng, L.S., et al., 2014. Effects of hypoxic exercise training on microRNA expression and lipid metabolism in obese rat livers. J. Zhejiang Univ.-Sci. B (Biomed. & Biotechnol.), 15(9):820-829.

[25]Mallory, A.C., Vaucheret, H., 2006. Functions of microRNAs and related small RNAs in plants. Nat. Genet., 38(7):850-850.

[26]Poy, M.N., Eliasson, L., Krutzfeldt, J., et al., 2004. A pancreatic islet-specific microRNA regulates insulin secretion. Nature, 432(7014):226-230.

[27]Sen, R., Ghosal, S., Das, S., et al., 2014. Competing endogenous RNA: the key to posttranscriptional regulation. Sci. World J., 2014:896206.

[28]Shalgi, R., Lieber, D., Oren, M., et al., 2007. Global and local architecture of the mammalian microRNA-transcription factor regulatory network. PLoS Comput. Biol., 3(7):e131.

[29]Song, F., Huang, Y., Wang, X., et al., 2013. Bmo-miR down regulates the expression of Bm-ase gene in vitro. Russ. J. Bioorg. Chem., 39(2):170-175.

[30]Song, F., Wang, X., Qian, P., et al., 2014. Expressional regulation of Bombyx mori fibroin heavy-chain gene by Bmo-miR-2739. Sci. Seric., 40(3):0404-0408.

[31]Song, F., Wang, X., Chen, C., et al., 2015. Characterization and profiling of microRNAs in posterior silk gland of the silkworm (Bombyx mori). Genes Genom., 37(8):703-712.

[32]Sprague, K.U., 1975. Bombyx mori silk proteins: characterization of large polypeptides. Biochemistry, 14(5):925-931.

[33]Sun, X., Zhu, C., Wang, S., 2009. Quantitative analysis of fibroin genes’ expression in silkworm, Bombyx mori. Sci. Seric., 35(1):30-35 (in Chinese).

[34]Wang, G., Li, J., Xu, X., et al., 2011. Changes of fibroin gene expression during the different development period form silkworm. North Seric., 32(4):17-20 (in Chinese).

[35]Wang, S., Lu, C., 2006. Leaked expression of cloned fibroin heavy chain promoter sequence in silkworm, Bombyx mori, and insect cultured cells. Sci. Seric., 32(4):491-494 (in Chinese).

[36]Xia, Q., Zhou, Z., Lu, C., et al., 2004. A draft sequence for the genome of the domesticated silkworm (Bombyx mori). Science, 306(5703):1937-1940.

[37]Xia, Q., Guo, Y., Zhang, Z., et al., 2009. Complete resequencing of 40 genomes reveals domestication events and genes in silkworm (Bombyx). Science, 326(5951):433-436.

[38]Yamaguchi, K., Kikuchi, Y., Takagi, T., et al., 1989. Primary structure of the silk fibroin light chain determined by cDNA sequencing and peptide analysis. J. Mol. Biol., 210(1):127-139.

[39]Yang, L., Lu, X., Liu, Y., et al., 2012. Expression analysis of miRNAs in BmN cells. Gene, 505(2):240-245.

[40]Yu, X., Zhou, Q., Li, S.C., et al., 2008. The silkworm (Bombyx mori) microRNAs and their expressions in multiple developmental stages. PLoS ONE, 3(8):e2997.

[41]Yu, X., Zhou, Q., Cai, Y., et al., 2009. A discovery of novel microRNAs in the silkworm (Bombyx mori) genome. Genomics, 94(6):438-444.

[42]Yuan, Z.M., Yang, Z.L., Zheng, Q., 2014. Deregulation of microRNA expression in thyroid tumors. J. Zhejiang Univ.-Sci. B (Biomed. & Biotechnol.), 15(3):212-224.

[43]Zeng, F., Xie, H., Nie, Z., et al., 2009. Characterization of the gene BmEm4, a homologue of Drosophila E(spl)m4, from the silkworm, Bombyx mori. Comp. Funct. Genomics, 2009:947490.

[44]Zhang, Y., Zhou, X., Ge, X., et al., 2009. Insect-specific microRNA involved in the development of the silkworm Bombyx mori. PLoS ONE, 4(3):e4677.

[45]Zhao, Q.L., Shen, X.J., Zhu, L.J., et al., 2007. Characterization of CIb1 gene promoter from silkworm, Bombyx mori. Z. Naturforsch. C, 62(11-12):875-880.

[46]Zhou, Y.J., Xiao, Q.L., Zhang, Z.F., et al., 2002. Foreign insect hormone stimulating the transcription of ie-1 promoter of Bombyx mori nuclear polyhedrosis virus in vivo and in vitro. Biosci. Biotechnol., Biochem., 66(7):1488-1494.

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