水牛miR-302s真核表达载体的构建及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2019.09.001

Abstract

Abstract: The aim of this study was to clone and construct a buffalo miR-302s (bbu-miR-302s) lentivirus expression vector,analyze its bioinformatics,and try to apply the vector to buffalo somatic cell reprogramming.The bbu-miR-302s precursor sequence was amplified using buffalo genomic DNA as template,after the TA clone was sequenced,it was ligated into pLVX-IRES-ZsGreen1 lentiviral expression vector.After the vector was identified by enzyme digestion,it was transferred into HEK-293T cells by lipofection to package the lentivirus,and bbu-miR-302s lontivirus infect the HEK-293T and fibroblast cells of pig and buffalo.After buffalo fetal fibroblasts (BFFs) were infected by bbu-miR-302s lentivirus,the production of buffalo induced pluripotent stem cells (iPSC) was detected by induction culture.The location analysis of the miR-302s family in the genome was performed using the CoGeMiR database and SnapGene Viewer,its conservative was analyzed using ClustalX 1.83 software;TargetScan and miRWalk were used to predict the major target genes,and DAVID program was used to enrich the signal pathway of target genes.The sequencing results showed that the amplified sequence was buffalo miR-302s family,the titer of the packaged lentivirus was 7.2×10⁶ TU/mL,which could effectively infect the somatic cells of three species.After BFFs were infected by bbu-miR-302s lentivirus,the cells changed morphologically and gathered to form clones.Alkaline phosphatase was positive,but the results of pluripotency gene and surface antigen were negative,which indicated that single factor miR-302s was not enough to completely reprogram BFF into iPSC,it promoted the reprogramming process.The CoGeMiR database showed that the miR-302s family was mainly located in the intron region of LARP7 gene,SnapGene Viewer software analysis result showed that bbu-miR-302s was located in the intron region of LARP7 gene in buffalo chromosome 7.The miR-302s clusters of different species and members of miR-302s family were highly conserved.A total of 255 major target genes of buffalo miR-302s were predicted,and these target genes were mainly concentrated in 33 pathways,among which PGK signal pathway,glucagon signal transduction and regulation of stem cell signal pathway were the most significant.The results laid a foundation for further research on the role of the miR-302s cluster in somatic cell reprogramming.

Key words: bbu-miR-302s; lentivirus eukaryotic vector; bioinformatics analysis

CLC Number:

QIAO Shuye, HUANG Shihai, DENG Yanfei, DENG Haiying, LUO Chan, SHI Deshun, LI Xiangping. Construction of Eukaryotic Expression Vector of Buffalo miR-302s and Its Bioinformatics Analysis[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(9): 2497-2506.

References

[1] FABIAN M R,SONENBERG N.The mechanics of miRNA-mediated gene silencing:A look under the hood of miRISC[J].Nature Structural & Molecular Biology,2012,19(6):586-593.
[2] HUSEBEKK A,MOFFETT A,DE MESTRE A M,et al.IFPA Meeting 2011 workshop report Ⅲ:Placental immunology;Epigenetic and microRNA-dependent gene regulation;Comparative placentation;Trophoblast differentiation;Stem cells[J].Placenta,2012,33(Suppl A):S15-S22.
[3] HOUBAVIY H B,MURRAY M F,SHARP P A.Embryonic stem cell-specific microRNAs[J].Developmental Cell,2003,5(2):351-358.
[4] BAR M,WYMAN S K,FRITZ B R,et al.microRNA discovery and profiling in human embryonic stem cells by deep sequencing of small RNA libraries[J].Stem Cells,2008,26(10):2496-2505.
[5] ROSA A,BRIVANLOU A H.A regulatory circuitry comprised of miR-302 and the transcription factors OCT4 and NR2F2 regulates human embryonic stem cell differentiation[J].The EMBO Journal,2011,30(2):237-248.
[6] LI H,WEI J,FAN L,et al.miR-302 regulates pluripotency,teratoma formation and differentiation in stem cells via an AKT1/OCT4-dependent manner[J].Cell Death and Disease,2016,7(1):e2078.
[7] MARSON A,LEVINE S S,COLE M F,et al.Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells[J].Cell,2008,134(3):521-533.
[8] GREER CARD D A,HEBBAR P B,LI L,et al.Oct4/Sox2-regulated miR-302 targets cyclin D1 in human embryonic stem cells[J].Molecular and Cellular Biology,2008,28(20):6426-6438.
[9] LIPCHINA I,ELKABETZ Y,HAFNER M,et al.Genome-wide identification of microRNA targets in human ES cells reveals a role for miR-302 in modulating BMP response[J].Genes & Development,2011,25(20):2173-2186.
[10] ZHANG Z,HONG Y,XIANG D,et al.microRNA-302/367 cluster governs hESC self-renewal by dually regulating cell cycle and apoptosis pathways[J].Stem Cell Reports,2015,4(4):645-657.
[11] ANOKYE-DANSO F,TRIVEDI C M,JUHR D,et al.Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency[J].Cell Stem Cell,2011,8(4):376-388.
[12] MIYOSHI N,ISHⅡ H,NAGANO H,et al.Reprogramming of mouse and human cells to pluripotency using mature microRNAs[J].Cell Stem Cell,2011,8(6):633-638.
[13] 朱海鲸.CD49f和miR-302对奶山羊雄性生殖干细胞体外培养生物学特性的影响[D].杨凌:西北农林科技大学,2014. ZHU H Q.Influences of CD49f and miR-302 on biological properties of dairy goat male germline stem cells cultured in vitro[D].Yangling:Northwest A & F University,2014.(in Chinese)
[14] 崔亚茹,张坤山,罗玉萍,等.12个物种miR-302基因簇的生物信息学分析[J].基因组学与应用生物学,2011,30(3):379-384. CUI Y R,ZHANG K S,LUO Y P,et al.Bioinformatics analysis of miR-302 gene cluster from 12 different species[J].Genomics and Applied Biology,2011,30(3):379-384.(in Chinese)
[15] 王龙.小鼠Dazl和Ddx4基因真核表达载体构建和miR302/367簇诱导重编程的初步研究[D].杨凌:西北农林科技大学,2013. WANG L.Construction of Dazl-,Ddx4-expression vectors and preliminary study of miR302/367-induced reprogramming[D].Yangling:Northwest A & F University,2013.(in Chinese)
[16] YING S Y,FANG W,LIN S L.The miR-302-mediated induction of pluripotent stem cells (iPSC):Multiple synergistic reprogramming mechanisms[J].Methods in Molecular Biology,2018,1733:283-304.
[17] 张耀林.表达pri-miR-302/367基因的慢病毒颗粒的制备及其感染人脐血单个核细胞的实验究[D].银川:宁夏医科大学,2014. ZHANG Y L.Generation of lentivirus expressing pri-miR-302/367 gene and its infecting human cord blood mononuclear cells[D].Yinchuan:Ningxia Medical University,2014.(in Chinese)
[18] 杨桦,倪良荣,万颖寒,等.诱导表达型miR-302s敲入小鼠的构建及表型分析[J].中国细胞生物学学报,2016,38(8):997-1003. YANG H,NI L R,WAN Y H,et al.Establishment and characterization of inducible miR-302s knock-in mice[J].Chinese Journal of Cell Biology,2016,38(8):997-1003.(in Chinese)
[19] DE CHIARA L,ANDREWS D,WATSON A,et al.miR-302 regulates SNAI1 expression to control mesangial cell plasticity[J].Scientific Reports,2017,7(1):42407.
[20] LIN S L,CHANG D C,CHANG-LIN S,et al.miR-302 reprograms human skin cancer cells into a pluripotent ES-cell-like state[J].RNA,2008,14(10):2115-2124.
[21] MA K,SONG G,AN X,et al.miRNAs promote generation of porcine-induced pluripotent stem cells[J].Molecular and Cellular Biochemistry,2014,389(1-2):209-218.
[22] SANDMAIER S E S,NANDAL A,POWELL A,et al.Generation of induced pluripotent stem cells from domestic goats[J].Molecular Reproduction and Development,2015,82(9):709-721.
[23] CHUNG T,BRENA R M,KOLLE G,et al.Vitamin C promotes widespread yet specific DNA demethylation of the epigenome in human embryonic stem cells[J].Stem Cells,2010,28(10):1848-1855.
[24] SHI Y,ZHAO Y,DENG H.Powering reprogramming with vitamin C[J].Cell Stem Cell,2010,6(1):1-2.
[25] RAMEZANKHANI B,TAHA M F,JAVERI A.Vitamin C counteracts miR-302/367-induced reprogramming of human breast cancer cells and restores their invasive and proliferative capacity[J].Journal of Cellular Physiology,2019,234(3):2672-2682.

Construction of Eukaryotic Expression Vector of Buffalo miR-302s and Its Bioinformatics Analysis

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