家蚕bmo-miR-0031-3p体内下调丝素轻链基因BmFib-L的表达
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摘要
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。
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 m RNA was used as the target for screen of bmo-miRNAs. By using RNAhybrid software, the bmo-miR-0031-3 p(abbreviated as"miR-0031-3 p") was screened out to completely bind the target gene with the seed sequence. A miR-0031-3 p expression plasmid pc DNA3.0[ie1-egfp-pre-miR-0031-3 p-SV40] and a BmFib-L 3′ UTR fused luciferase report plasmid p GL3.0[A3-luc-Fib-L-3′ UTR-SV40] were constructed, respectively. Bm N cells were cotransfected 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-0031-3 p on BmFib-L by assay of dual luciferase activities, as well as artificially synthesized the mimic and inhibitor of miR-0031-3 p. The results revealed that the miR-0031-3 p significantly down-regulated the expression of BmFib-L in the BmN cells. To validate the regulatory function of miR-0031-3 p in vivo, the day-2 5 th 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-0031-3 p 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.
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 m RNA was used as the target for screen of bmo-miRNAs. By using RNAhybrid software, the bmo-miR-0031-3 p(abbreviated as"miR-0031-3 p") was screened out to completely bind the target gene with the seed sequence. A miR-0031-3 p expression plasmid pc DNA3.0[ie1-egfp-pre-miR-0031-3 p-SV40] and a BmFib-L 3′ UTR fused luciferase report plasmid p GL3.0[A3-luc-Fib-L-3′ UTR-SV40] were constructed, respectively. Bm N cells were cotransfected 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-0031-3 p on BmFib-L by assay of dual luciferase activities, as well as artificially synthesized the mimic and inhibitor of miR-0031-3 p. The results revealed that the miR-0031-3 p significantly down-regulated the expression of BmFib-L in the BmN cells. To validate the regulatory function of miR-0031-3 p in vivo, the day-2 5 th 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-0031-3 p 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.
引文
[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-148targets 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-2758targets BmFMBP-1(Lepidoptera:Bombycidae)and suppresses 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 microR NAs 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.
[20]KROL J, LOEDIGE I, FILIPOWICZ W. The widespread regulation of microRNA biogenesis, function and decay.Nature Reviews Genetics, 2010,11(9):597-610.
[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-148targets 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-2758targets BmFMBP-1(Lepidoptera:Bombycidae)and suppresses 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 microR NAs 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.
[20]KROL J, LOEDIGE I, FILIPOWICZ W. The widespread regulation of microRNA biogenesis, function and decay.Nature Reviews Genetics, 2010,11(9):597-610.
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