牦牛lncRNAENSBGRT00000000387.1慢病毒载体构建及其对卵泡GCs凋亡的影响

doi:10.11843/j.issn.0366-6964.2023.03.019

Abstract

Abstract: The purpose of this study was to construct yak lncRNA ENSBGRT00000000387.1 overexpression lentivirus vector and infect yak follicular granulosa cells (GCs) to understand its effect on cell apoptosis. The yak follicular RNA was extracted, and the full-length sequence of lncRNA ENSBGRT00000000387.1 was amplified by RT-PCR, and then its recombinant plasmid was constructed. After it was packaged with lentivirus packaging system (pVSVG: pMDL: pRev), the infective titer on 293T cells was determined by qPCR, and then the isolated and cultured yak GCs were infected. The expression level of lncRNA ENSBGRT00000000387.1 in GCs was detected by qPCR, and the apoptosis rate of GCs was detected by flow cytometry. Western blot and qPCR were used to detect the mRNA and protein expression levels of pro-apoptotic gene CASPASE3, BAX and anti-apoptotic gene BCL-2, respectively. The full-length sequence of 2 566 bp of lncRNA ENSBGRT00000000387.1 was successfully amplified from yak follicles, and it was homologously recombined with the fragment of the homologous region of LV-EF1a-EGFP-2A vector. The restriction enzyme digestion identification and sequencing verification showed that the recombinant plasmid was successfully constructed. After it was packaged and concentrated in the lentivirus packaging system, the infection efficiency in 293T cells reached (75.77±0.850) %. The qPCR test results showed that the titer of the lentivirus was 7.32×10⁸ TU·mL^-1, indicating that the target lncRNA overexpression lentivirus vector was successfully constructed. The infection efficiency of yak follicular GCs infected with lentivirus was (52.93±1.168)%. The qPCR results showed that the expression level of target lncRNA in infected GCs was significantly increased (P<0.01). Flow cytometry showed that the apoptosis rate of cells in lentivirus infection group decreased significantly (P<0.01), the expression of pro-apoptotic genes CASPASE 3 and BAX at mRNA and protein levels decreased significantly (P<0.01), and the expression of anti-apoptotic gene BCL-2 increased significantly (P<0.01). In this experiment, lncRNA ENSBGRT00000000387.1 lentivirus vector was successfully constructed. After successfully infecting yak follicular GCs, it was found to have the effect of inhibiting apoptosis, indicating that it may play a role in yak follicular function.

Key words: lncRNA, lentivirus vector, granulosa cells, apoptosis, yak

CLC Number:

S823.85

MENG Zhaoyi, WANG Yunlu, XU Yefen, NIU Jiaqiang, SUOLANG Sizhu, GUO Min, XI Guangyin. Construction of Yak lncRNA ENSBGRT00000000387.1 Lentivirus Vector and Its Effect on Apoptosis of Yak Follicular Granulosa Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(3): 1058-1070.

References 46

[1]	BOCCI F,JOLLY M K,LEVINE H,et al.Quantitative characteristic of ncRNA regulation in gene regulatory networks[M]//LAI X,GUPTA S K,VERA J.Computational Biology of Non-Coding RNA.New York:Humana,2019:341-366.
[2]	FEHLMANN T,BACKES C,PIRRITANO M,et al.The sncRNA Zoo:a repository for circulating small noncoding RNAs in animals[J].Nucleic Acids Res,2019,47(9):4431-4441.
[3]	KOSINSKA-SELBI B,MIELCZAREK M,SZYDA J.Review:long non-coding RNA in livestock[J].Animal,2020, 14(10):2003-2013.
[4]	QIAN X Y,ZHAO J Y,YEUNG P Y,et al.Revealing lncRNA structures and interactions by sequencing-based approaches[J]. Trends Biochem Sci,2019,44(1):33-52.
[5]	BRIDGES M C,DAULAGALA A C,KOURTIDIS A.LNCcation:lncRNA localization and function[J].J Cell Biol,2021, 220(2):e202009045.
[6]	KOPP F,MENDELL J T.Functional classification and experimental dissection of long noncoding RNAs[J].Cell,2018, 172(3):393-407.
[7]	MARUO T,LAOAG-FERNANDEZ J B,TAKEKIDA S,et al.Regulation of granulosa cell proliferation and apoptosis during follicular development[J].Gynecol Endocrinol,1999,13(6):410-419.
[8]	ELMORE S.Apoptosis:a review of programmed cell death[J].Toxicol Pathol,2007,35(4):495-516.
[9]	MATSUDA F,INOUE N,MANABE N,et al.Follicular growth and atresia in mammalian ovaries:regulation by survival and death of granulosa cells[J].J Reprod Dev,2012,58(1):44-50.
[10]	ZHANG T J,BASANG W D,CHANG W H,et al.Dynamics of apoptosis-related gene expression during follicular development in yak[J].J Anim Physiol Anim Nutr (Berl),2021,105(6):1002-1013.
[11]	KNET M,WARTALSKI K,HOJA-LUKOWICZ D,et al.Analysis of porcine granulosa cell death signaling pathways induced by vinclozolin[J].Theriogenology,2015,84(6):927-939.
[12]	D'ARCY M S.Cell death:a review of the major forms of apoptosis,necrosis and autophagy[J].Cell Biol Int,2019,43(6):582-592.
[13]	ROSSI M N,ANTONANGELI F.LncRNAs:new players in apoptosis control[J].Int J Cell Biol,2014,2014:473857.
[14]	ZHEN J R,LI J L,LI X,et al.Downregulating lncRNA NEAT1 induces proliferation and represses apoptosis of ovarian granulosa cells in polycystic ovary syndrome via microRNA-381/IGF1 axis[J].J Biomed Sci,2021,28(1):53.
[15]	HU H Y,FU Y F,ZHOU B,et al.Long non-coding RNA TCONS_00814106 regulates porcine granulosa cell proliferation and apoptosis by sponging miR-1343[J].Mol Cell Endocrinol,2021,520:111064.
[16]	YAO X L,GAO X X,BAO Y J,et al.lncRNA FDNCR promotes apoptosis of granulosa cells by targeting the miR-543-3p/DCN/TGF-β signaling pathway in Hu sheep[J].Mol Ther Nucleic Acids,2021,24:223-240.
[17]	SUN L,ZHANG P J,LU W F.lncRNA MALAT1 regulates mouse granulosa cell apoptosis and 17β-estradiol synthesis via regulating miR-205/CREB1 axis[J].Biomed Res Int,2021,2021:6671814.
[18]	WANG L Q,HE T,ZHANG X,et al.Global transcriptomic analysis reveals Lnc-ADAMTS9 exerting an essential role in myogenesis through modulating the ERK signaling pathway[J].J Anim Sci Biotechnol,2021,12(1):4.
[19]	TSAGAKIS I,DOUKA K,BIRDS I,et al.Long non-coding RNAs in development and disease:conservation to mechanisms[J].J Pathol,2020,250(5):480-495.
[20]	JOSHI M,RAJENDER S.Long non-coding RNAs (lncRNAs) in spermatogenesis and male infertility[J].Reprod Biol Endocrinol,2020,18(1):103.
[21]	OTSUKA K,MATSUBARA S,SHIRAISHI A,et al.A testis-specific long noncoding RNA,Start,is a regulator of steroidogenesis in mouse leydig cells[J].Front Endocrinol (Lausanne),2021,12:665874.
[22]	IYYAPPAN R,ALESHKINA D,ZHU L K,et al.Oocyte specific lncRNA variant Rose influences oocyte and embryo development[J]. Noncoding RNA Res,2021,6(2):107-113.
[23]	ZHAO Z J,CHEN L,CAO M S,et al.Comparison of lncRNA expression in the uterus between periods of embryo implantation and labor in mice[J].Animals (Basel),2022,12(3):399.
[24]	LI G,WANG Y L,WANG J Y,et al.Long non-coding RNA placenta-specific protein 2 regulates micorRNA-19a/tumor necrosis factor α to participate in polycystic ovary syndrome[J].Bioengineered,2022,13(1):856-862.
[25]	贺小云,狄冉,胡文萍,等.动物繁殖相关lncRNA的研究新进展[J].畜牧兽医学报,2016,47(9):1749-1756.HE X Y,DI R,HU W P,et al.New research progress in animal reproduction-related long noncoding RNAs[J].Acta Veterinaria et Zootechnica Sinica,2016,47(9):1749-1756.(in Chinese)
[26]	ZHENG L P,LUO R C,SU T,et al.Differentially expressed lncRNAs after the activation of primordial follicles in mouse[J].Reprod Sci,2019,26(8):1094-1104.
[27]	ZHAO Z F,ZOU X,LU T T,et al.Identification of mRNAs and lncRNAs involved in the regulation of follicle development in goat[J].Front Genet,2020,11:589076.
[28]	MU L S,SUN X T,TU M X,et al.Non-coding RNAs in polycystic ovary syndrome:a systematic review and meta-analysis[J].Reprod Biol Endocrinol,2021,19(1):10.
[29]	ZHU D M,XIAO Z,WANG Z M,et al.MEG3/MIR-376B-3P/HMGA2 axis is involved in pituitary tumor invasiveness[J].J Neurosurg,2021,134(2):499-511.
[30]	SHEYBANI N,BAKHTIARIZADEH M R,SALEHI A.An integrated analysis of mRNAs,lncRNAs,and miRNAs based on weighted gene co-expression network analysis involved in bovine endometritis[J].Sci Rep,2021,11(1):18050.
[31]	ASSELIN E,XIAO C W,WANG Y F,et al.Mammalian follicular development and atresia:role of apoptosis[J].Biol Signals Recept,2000,9(2):87-95.
[32]	MOREIRA A S,CAVACO D G,FARIA T Q,et al.Advances in lentivirus purification[J].Biotechnol J,2021,16(1):e2000019.
[33]	PERRY C,RAYAT A C M E.Lentiviral vector bioprocessing[J].Viruses,2021,13(2):268.
[34]	QIN J Y,ZHANG L,CLIFT K L,et al.Systematic comparison of constitutive promoters and the doxycycline-inducible promoter[J].PLoS One,2010,5(5):e10611.
[35]	NALDINI L,BLÖMER U,GALLAY P,et al.In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector[J].Science,1996,272(5259):263-267.
[36]	OBENG E.Apoptosis (programmed cell death) and its signals-a review[J].Braz J Biol,2021,81(4):1133-1143.
[37]	HSU S Y,LAI R J,FINEGOLD M,et al.Targeted overexpression of Bcl-2 in ovaries of transgenic mice leads to decreased follicle apoptosis,enhanced folliculogenesis,and increased germ cell tumorigenesis[J].Endocrinology,1996,137(11):4837-4843.
[38]	GVRSOY E,ERGIN K,BAŞALOǦU H,et al.Expression and localisation of Bcl-2 and Bax proteins in developing rat ovary[J].Res Vet Sci,2008,84(1):56-61.
[39]	VALDEZ K E,CUNEO S P,TURZILLO A M.Regulation of apoptosis in the atresia of dominant bovine follicles of the first follicular wave following ovulation[J].Reproduction,2005,130(1):71-81.
[40]	CAROU M C,CRUZANS P R,MARURI A,et al.Apoptosis of bovine granulosa cells:Intracellular pathways and differentiation[J].Acta Histochem,2017,119(5):462-470.
[41]	JIANG J Y,CHEUNG C K M,WANG Y F,et al.Regulation of cell death and cell survival gene expression during ovarian follicular development and atresia[J].Front Biosci,2003,8:d222-237.
[42]	ASADI M,TAGHIZADEH S,KAVIANI E,et al.Caspase-3:structure,function,and biotechnological aspects[J].Biotechnol Appl Biochem,2022,69(4):1633-1645.
[43]	BRUCKHEIMER E M,CHO S H,SARKISS M,et al.The Bcl-2 gene family and apoptosis[M]//AL-RUBEAI M.Apoptosis. Berlin:Springer,1998:75-105.
[44]	GENG X Y,ZHAO J,HUANG J Y,et al.lnc-MAP3K13-7:1 inhibits ovarian GC proliferation in PCOS via DNMT1 downregulation-mediated CDKN1A promoter hypomethylation[J].Mol Ther,2021,29(3):1279-1293.
[45]	YAO W,PAN Z X,DU X,et al.NORHA,a novel follicular atresia-related lncRNA,promotes porcine granulosa cell apoptosis via the miR-183-96-182 cluster and FoxO1 axis[J].J Anim Sci Biotechnol,2021,12(1):103.
[46]	KIMURA A P,YONEDA R,KURIHARA M,et al.A long noncoding RNA,lncRNA-Amhr2,plays a role in Amhr2 gene activation in mouse ovarian granulosa cells[J]. Endocrinology,2017,158(11):4105-4121.

Construction of Yak lncRNA ENSBGRT00000000387.1 Lentivirus Vector and Its Effect on Apoptosis of Yak Follicular Granulosa Cells

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