| Animal sciences & veterinary medicine |
|
|
|
|
| Bmo-miR-0031-3p down-regulates the expression of Bombyx mori fibroin light chain gene BmFib-L in vivo |
Yanhua CHEN1,2( ),Tao JIANG1,2,Xuezhen WANG1,2,Ping QIAN1,2,Shunming TANG1,2,Xingjia SHEN2() |
1. Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212018, Jiangsu, China
2. Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture and Rural Affairs, Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang 212018, Jiangsu, China |
|
|
|
Abstract To study the regulatory function of Bombyx mori microRNAs (bmo-miRNAs) on expression of the fibroin light chain gene (BmFib-L), the 3′ untranslated region (3′ UTR) of BmFib-L mRNA was used as the target for screen of bmo-miRNAs. By using RNAhybrid software, the bmo-miR-0031-3p (abbreviated as “miR-0031-3p”) was screened out to completely bind the target gene with the seed sequence. A miR-0031-3p expression plasmid pcDNA3.0[ie1-egfp-pre-miR-0031-3p-SV40] and a BmFib-L 3′ UTR fused luciferase report plasmid pGL3.0[A3-luc-Fib-L-3′ UTR-SV40] were constructed, respectively. BmN cells were co-transfected with the above mentioned plasmids, and the pRL-CMV (contains a Renilla luciferase gene) was served as an intrinsic plasmid to validate the regulatory function of miR-0031-3p on BmFib-L by assay of dual luciferase activities, as well as artificially synthesized the mimic and inhibitor of miR-0031-3p. The results revealed that the miR-0031-3p significantly down-regulated the expression of BmFib-L in the BmN cells. To validate the regulatory function of miR-0031-3p in vivo, the day-2 5th instar larvae were injected with a transfection solution for overexpression and inhibition of endogenous expression analysis, and the BmFib-L expression was analyzed by quantitative reverse transcription polymerase chain reaction (RT-PCR) using total RNAs extracted from silk glands. The results showed that the miR-0031-3p significantly down-regulated the expression of BmFib-L in individuals. These findings are beneficial to clarify the molecular mechanism of miRNAs in regulating B. mori silk protein biosynthesis.
|
|
Received: 26 November 2018
Published: 25 April 2019
|
|
|
|
Corresponding Authors:
Xingjia SHEN
E-mail: 2317965026@qq.com;shenxjsri@163.com
|
家蚕bmo-miR-0031-3p体内下调丝素轻链基因BmFib-L的表达
为了研究家蚕微RNA（microRNA, miRNA）对丝素轻链基因BmFib-L表达的调控作用，以BmFib-L mRNA的3′非翻译区（3′ untranslated region, 3′ UTR）为靶标，通过RNAhybrid软件分析，筛选出种子序列与BmFib-L 3′ UTR完全互补的家蚕miRNA——bmo-miR-0031-3p（简称“miR-0031-3p”）。分别构建miR-0031-3p表达载体pcDNA3.0[ie1-egfp-pre-miR-0031-3p-SV40]和BmFib-L 3′ UTR融合萤光素酶报告基因重组表达质粒pGL3.0[A3-luc-Fib-L-3′ UTR-SV40]，以海肾萤光素酶表达载体pRL-CMV为内参，共转染BmN细胞，通过检测双萤光素酶活性验证miR-0031-3p的功能；人工合成miR-0031-3p的模拟物（mimic）和抑制物（inhibitor），再进一步验证miR-0031-3p对BmFib-L的调控功能。结果显示，在BmN细胞中，miR-0031-3p显著抑制BmFib-L的表达。为了进一步验证miR-0031-3p在家蚕体内对BmFib-L表达的调控作用，在5龄第2天幼虫体腔内注射转染物，分别在体内过表达和抑制内源性miR-0031-3p，荧光定量分析靶基因表达水平。结果显示，miR-0031-3p在幼虫体内能够下调BmFib-L的表达。该研究结果有利于阐明家蚕miRNA功能和蚕丝蛋白表达调控的分子机制。
关键词：
微RNA,
家蚕,
bmo-miR-0031-3p,
丝素轻链基因,
转录后调控
|
|
| [1] |
JUNICHI T , HIROYUKI K , KIYOSHI Y , et al . Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival.Cancer Research, 2004,64(11):3753-3756.
|
|
|
| [2] |
LI H , XIE H , LIU W , et al . A novel microRNA targeting HDAC5 regulates osteoblast differentiation in mice and contributes to primary osteoporosis in humans.Journal of Clinical Investigation, 2009,119(12):3666-3677.
|
|
|
| [3] |
KARA M , YUMRUTAS O , OZCAN O , et al . Differential expressions of cancer-associated genes and their regulatory miRNAs in colorectal carcinoma. Gene, 2015,567(1):81-86.
|
|
|
| [4] |
FRANKEL L B , CHRISTOFFERSEN N R , JACOBSEN A , et al . Programmed cell death 4 (PDCD4) is an important functional target of the microRNA miR-21 in breast cancer cells. Journal of Biological Chemistry, 2008,283(2):1026-1033.
|
|
|
| [5] |
PILLAI R S , BHATTACHARYYA S N , FILIPOWICZ W . Repression of protein synthesis by miRNAs: how many mechanisms? Trends in Cell Biology , 2007,17(3):118-126.
|
|
|
| [6] |
BARTEL D P . MicroRNAs: target recognition and regulatory functions. Cell, 2009,136(2):215-233.
|
|
|
| [7] |
HUANG Y , ZOU Q , SONG F , et al . The regulation of silkworm fibroin L chain production by miRNA-965 and miRNA-1926 in insect cells. Russian Journal of Bioorganic Chemistry, 2012,38(4):417-421.
|
|
|
| [8] |
YU F Y , YAO H R , ZHU P C , et al . let-7 regulates self renewal and tumorigenicity of breast cancer cells. Cell, 2007,131(6):1109-1123.
|
|
|
| [9] |
FORMAN J J , LEGESSE-MILLER A , COLLER H A . A search for conserved sequences in coding regions reveals that the let-7 microRNA targets Dicer within its coding sequence.Proceedings of the National Academy of Sciences of the USA, 2008,105(39):14879-14884.
|
|
|
| [10] |
DUURSMA A M , KEDDE M , SCHRIER M , et al . miR-148 targets human DNMT3b protein coding region. RNA, 2008,14(5):872-877.
|
|
|
| [11] |
ZHOU H L , RIGOUTSOS I . MiR-103a-3p targets the 5′ UTR of GPRC5A in pancreatic cells. RNA, 2014,20(9):1431-1439.
|
|
|
| [12] |
JULIEN E , BORDEAUX M C , GAREL A , et al . Fork head alternative binding drives stage-specific gene expression in the silk gland of Bombyx mori .Insect Biochemistry & Molecular Biology, 2002,32(4):377-387.
|
|
|
| [13] |
DURAND B , DREVET J , COUBLE P . P25 gene regulation in Bombyx mori silk gland: two promoter-binding factors have distinct tissue and developmental specificities.Molecular & Cellular Biology, 1992,12(12):5768-5777.
|
|
|
| [14] |
范洋洋,陈晨,王欣,等 .Bmo-miR-2755*对丝素蛋白基因BmFib-L和BmP25表达的调控作用.蚕业科学,2015,41(5):847-853.
FAN Y Y , CHEN C , WANG X , et al . Expressional regulation of Bombyx mori fibroin genes Fib-L and P25 by Bmo-miR-2755*. Science of Sericulture, 2015,41(5):847-853. (in Chinese with English abstract)
|
|
|
| [15] |
WANG X , TANG S M , SONG F , et al . Bmo-miR-2758 targets BmFMBP-1 (Lepidoptera: Bombycidae) and supp-resses its expression in BmN cells. Journal of Insect Science, 2016,16(1):28.
|
|
|
| [16] |
SONG F , WANG X , CHEN C , et al . Characterization and profiling of microRNAs in posterior silk gland of the silkworm (Bombyx mori). Genes & Genomics, 2015,37(8):703-712.
|
|
|
| [17] |
CAO J , TONG C Z , WU X J , et al . Identification of conserved microRNAs in Bombyx mori (silkworm) and regulation of fibroin L chain production by microRNAs in heterologous system.Insect Biochemistry and Molecular Biology, 2008,38(12):1066-1071.
|
|
|
| [18] |
汪生鹏,孙霞 .家蚕蚕丝蛋白基因表达调控研究进展.安徽农业科学,2009,37(28):13514-13518.
WANG S P , SUN X . Research progress of gene expression regulation of silkworm silk protein. Journal of Anhui Agricultural Sciences, 2009,37(28):13514-13518. (in Chinese with English abstract)
|
|
|
| [19] |
SHALGI R , LIEBER D , OREN M , et al . Global and local architecture of the mammalian microRNA-transcription factor regulatory network.PLoS Computational Biology, 2007,3(7):e131.
|
|
|
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
