水牛miR-16b过表达腺病毒载体构建及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2020.08.004

Abstract

Abstract: This study was aimed to clone and construct the adenovirus vector of pri-miR-16b gene in buffaloes,meanwhile the bioinformatics analysis of miR-16b and its predicted target genes were carried out to provide a basis for studying its role in oocyte maturation of buffaloes.pri-miR-16b gene was amplified from the ovary genome in buffaloes by RT-PCR,and then the pDC316-mCMV-EGFP-pri-miR-16b plasmid was constructed by homologous recombination.The homology and phylogenetic tree were analyzed and constructed by BLAST program and Mega 7.0,respectively,and the secondary structure of pre-miR-16 were predicted by ViennaRNA Web Services.The constructed vector was packaged with the adenovirus backbone plasmid pBHGloxdelE13cre to obtain the adenoviral particles named Ad-miR-16b,then three times of amplifications were performed and the virus titer was also determined.Ad-miR-16b adenovirus particles infected buffalo cumulus cells,and the expression of miR-16b in cumulus cells was detected by Real-time quantitative PCR.The target genes of miR-16b were predicted by TargetScan,and the KEGG pathway was enriched by the DAVID website for the predicted target genes.The results showed that the pri-miR-16b sequence in buffaloes was successfully cloned,and the sequence similarity with the bovine was found to be 100%.The high homology of pre-miR-16b in buffaloes with other species and the closest relationship with Bos taurus.The ViennaRNA Web Services prediction results showed that the secondary structure of pre-miR-16b had a typical single stem-loop structure,Ad-miR-16b adenoviral particles were obtained with a virus titer of 3.367×10¹⁰ GFU/mL.After the cumulus cells were infected,the expression of miR-16b was extremely significantly increased by Real-time quantitative PCR (P<0.01).The KEGG pathway enrichment analysis was conducted on 1 394 target genes predicted by miR-16b,and it was found that miR-16b might play a role in oocyte maturation mainly by regulating 74 signaling pathways such as MAPK,TGF-β and PI3K/AKT in cumulus cells.

Key words: miR-16b; adenovirus; cumulus cells; bioinformatics analysis

CLC Number:

WANG Lulu, SHEN Penglei, WU Fei, YE Sheng, CHEN Weili, ZHAO Xin, FENG Yun, DENG Yanfei, JIANG Jianrong, SHI Deshun, LU Fenghua. Construction of Overexpression Adenovirus Vector and Bioinformatics Analysis of miR-16b in Buffaloes[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(8): 2368-2377.

References

[1] GE L,SUI H S,LAN G C,et al.Coculture with cumulus cells improves maturation of mouse oocytes denuded of the cumulus oophorus:Observations of nuclear and cytoplasmic events[J].Fertility and Sterility,2008,90(6):2376-2388.
[2] DALTON C M,SZABADKAI G,CARROLL J.Measurement of ATP in single oocytes:Impact of maturation and cumulus cells on levels and consumption[J].Journal of Cellular Physiology,2014,229(3):353-361.
[3] RUSSELL D L,GILCHRIST R B,BROWN H M,et al.Bidirectional communication between cumulus cells and the oocyte:Old hands and new players?[J].Theriogenology,2016,86(1):62-68.
[4] FERRE-PUJOL P,NGUYEN X K,NAGAHARA T,et al.Removal of cumulus cells around 20 h after the start of in vitro maturation improves the meiotic competence of porcine oocytes via reduction in cAMP and cGMP levels[J].The Journal of Reproduction Development,2019,65(2):177-182.
[5] KOWSAR R,IRANSHAHI V N,SADEGHI N,et al.Urea influences amino acid turnover in bovine cumulus-oocyte complexes,cumulus cells and denuded oocytes,and affects in vitro fertilization outcome[J].Scientific Reports,2018,8(1):12191.
[6] BIASE F H,KIMBLE K M.Functional signaling and gene regulatory networks between the oocyte and the surrounding cumulus cells[J].BMC Genomics,2018,19(1):351.
[7] BERNARDO B C,OOI J Y,LIN R C,et al.miRNA therapeutics:A new class of drugs with potential therapeutic applications in the heart[J].Future Medicinal Chemistry,2015,7(13):1771-1792.
[8] STEPIEN A,KNOP K,DOLATA J,et al.Posttranscriptional coordination of splicing and miRNA biogenesis in plants[J].Wiley Interdisciplinary Reviews:RNA,2017,8(3):e1403.
[9] WANG M,MOISA S,KHAN M J,et al.microRNA expression patterns in the bovine mammary gland are affected by stage of lactation[J].Jouranl of Dairy Science,2012,95(11):6529-6535.
[10] CHEN T,YAO Q,WANG W,et al.Involvement of androgen receptor (AR)/microRNA-21 axis in hypoxia/reoxygenation-induced apoptosis of mouse renal tubular epithelial cells[J].American Journal of Translational Research,2019,11(9):5611-5622.
[11] HARTIG S M,HAMILTON M P,BADER D A,et al.The miRNA interactome in metabolic homeostasis[J].Trends in Endocrinology & Metabolism,2015,26(12):733-745.
[12] MARTIN E C,QURESHI A T,DASA V,et al.microRNA regulation of stem cell differentiation and diseases of the bone and adipose tissue:Perspectives on miRNA biogenesis and cellular transcriptome[J].Biochimie,2016,124:98-111.
[13] SINHA P B,TESFAYE D,RINGS F,et al.micro-RNA-130b is involved in bovine granulosa and cumulus cells function,oocyte maturation and blastocyst formation[J].Journal of Ovarian Research,2017,10(1):37.
[14] CHEN H,LIU C,JIANG H,et al.Regulatory role of miRNA-375 in expression of BMP15/GDF9 receptors and its effect on proliferation and apoptosis of bovine cumulus cells[J].Cellular Physiology and Biochemistry,2017,41(2):439-450.
[15] PAN B,TOMS D,SHEN W,et al.microRNA-378 regulates oocyte maturation via the suppression of aromatase in porcine cumulus cells[J].American Journal of Physiology Endocrinology and Metabolism,2015,308(6):E525-E534.
[16] 沈朋雷,秦希玲,尹倩,等.水牛卵母细胞GV/MⅡ期对应卵丘细胞miRNA差异表达分析[J].畜牧兽医学报,2017,48(5):836-845. SHEN P L,QIN X L,YIN Q,et al.Characterization of the expression of miRNA in cumulus cells during GV/MⅡ stages of buffalo oocyte[J].Acta Veterinaria et Zootechnica Sinica,2017,48(5):836-845.(in Chinese)
[17] RAAD M,BAYAT A,SHARAFSHAH A,et al.Association and in silico investigations of miR-302c insertion/deletion variant as a novel biomarker with susceptibility to gastric cancer[J].Journal of Cellular Biochemistry,2019,120(11):18946-18955.
[18] 鲍春旸,李军锋,张慧娜,等.用于鉴定microRNA靶基因的新型双萤光素酶报告基因系统的构建及应用[J].生物技术通讯,2011,22(5):651-656. BAO C Y,LI J F,ZHANG H N,et al.Construction and application of a novel dual-luciferase reporter system used for identification of microRNA target gene[J].Letters in Biotechnology,2011,22(5):651-656.(in Chinese)
[19] WANG J,YAN X,NESENGANI L T,et al.Integrated analysis of miRNA and mRNA expression profiling in bovine endometrial cells in response to lipopolysaccharide-stimulation[J].Microbial Pathogenesis,2018,114:129-138.
[20] ZHANG J,GUAN Y,SHEN C,et al.microRNA-375 regulates oocyte in vitro maturation by targeting ADAMTS1 and PGR in bovine cumulus cells[J].Biomedicine & Pharmacotherapy,2019,118:109350.
[21] LI X,WANG H,SHENG Y,et al.microRNA-224 delays oocyte maturation through targeting Ptx3 in cumulus cells[J].Mechanisms of Development,2017,143:20-25.
[22] GAGNON J D,KAGEYAMA R,SHEHATA H M,et al.miR-15/16 restrain memory T cell differentiation,cell cycle,and survival[J].Cell Reports,2019,28(8):2169-2181.
[23] WILCZYNSKA A,GIT A,ARGASINSKA J,et al.CPEB and miR-15/16 co-regulate translation of Cyclin E1 mRNA during Xenopus oocyte maturation[J].PLoS One,2016,11(2):e146792.
[24] LOVAT F,FASSAN M,GASPARINI P,et al.miR-15b/16-2 deletion promotes B-cell malignancies[J].Proceedings of the National Academy of Sciences of the United States of America,2015,112(37):11636-11641.
[25] 刘玉玲,钟柏茂.腺病毒载体构建研究进展[J].国际儿科学杂志,2018,45(3):163-166. LIU Y L,ZHONG B M.Progress on the construction of adenovirus vectors[J].International Journal of Pediatrics,2018,45(3):163-166.(in Chinese)
[26] KROON C R,PRECIADO-LLANES L,AZAR S R,et al.A single and un-adjuvanted dose of a chimpanzee adenovirus-vectored vaccine against chikungunya virus fully protects mice from lethal disease[J].Pathogens,2019,8(4):231.
[27] KALMA Y,GRANOT I,GALIANI D,et al.Luteinizing hormone-induced connexin 43 down-regulation:Inhibition of translation[J].Endocrinology,2004,145(4):1617-1624.
[28] YANG J,ZHANG Y,XU X,et al.Transforming growth factor-beta is involved in maintaining oocyte meiotic arrest by promoting natriuretic peptide type C expression in mouse granulosa cells[J].Cell Death & Disease,2019,10(8):558.
[29] PARK H,CHAE S,KIM J,et al.Ganglioside GM3 induces cumulus cell apoptosis through inhibition of epidermal growth factor receptor-mediated PI3K/AKT signaling pathways during in vitro maturation of pig oocytes[J].Molecular Reproduction and Development,2017,84(8):702-711.

Construction of Overexpression Adenovirus Vector and Bioinformatics Analysis of miR-16b in Buffaloes

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