过表达组蛋白甲基转移酶ASH1L对牛卵丘细胞增殖和凋亡的影响

doi:10.11843/j.issn.0366-6964.2023.08.021

Abstract

Abstract: The aim of this study was to investigate the effects of overexpression of absent, small, or homeotic 1-like (ASH1L) on the proliferation and apoptosis of bovine cumulus cells (CCs). The cDNA fragment of bovine ASH1L SET domain was amplified by PCR using CCs cDNA as templates and cloned into pcDNA3.1 in order to construct a bovine ASH1L overexpression vector. The bovine CCs transfected with pcDNA3.1+ASH1L vector were used as the overexpression group (ASH1L-OE) and the wild CCs were the control group (Control). The levels of ASH1L protein and H3K36me1/2/3 methylation in CCs of ASH1L-OE and the control group were detected by immunofluorescence staining. The apoptosis and proliferation of CCs in both groups were analyzed by flow cytometry. The mRNA expression levels of apoptosis-related and proliferation-related genes were detected by quantitative RT-PCR. A 2 487 bp DNA fragment of bovine ASH1L SET domain was obtained by PCR amplification using bovine CCs cDNA as template and ligated with pcDNA3.1 vector; the overexpression vector pcDNA3.1+ASH1L was successfully constructed after NheⅠ/NotⅠ double digestion and sequencing. Levels of ASH1L mRNA, protein and H3K36me1/2 methylation were significantly increased in CCs transfected with overexpression vector (P<0.05). Moreover, ASH1L overexpression in CCs significantly increased the percentage of live-cells (91.85±1.25)% vs. (87.39±1.71)% and decreased the apoptosis rate (8.08±1.21)% vs.(12.51±1.72)%. The mRNA expression levels of apoptosis-related genes BAX and CASPASE-3 were significantly downregulated (P<0.05) and expression level of anti-apoptotic gene BCL-2 was upregulated in CCs with ASH1L overexpression (P<0.01). The proliferation rate of cells and the expression levels of proliferation-related genes PCNA and CCND2 in CCs with ASH1L overexpression was significantly increased at 24 and 36 h after transfection (P<0.05). These results suggest that ASH1L overexpression inhibits CCs apoptosis, induces cell proliferation and H3K36me1/2 methylation in bovine CCs. The study provides a technical and theoretical basis for revealing the regulation of ASH1L on CCs growth and follicle development.

Key words: ASH1L methyltransferase, bovine cumulus cell, overexpression, cell proliferation, cell apoptosis, H3K36 me1/2

CLC Number:

S823.3

WANG Wanjie, CHEN Nanzhu, ZOU Huiying, ZHOU Xinyi, HAO Haisheng, PANG Yunwei, ZHU Huabin, ZHAO Xueming, YU Dawei, DU Weihua. Effects of Histone Methyltransferase ASH1L Overexpression on Proliferation and Apoptosis of Bovine Cumulus Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3358-3368.

References 40

[1]	WANG C, ZHOU B, XIA G L.Mechanisms controlling germline cyst breakdown and primordial follicle formation[J].Cell Mol Life Sci, 2017, 74(14):2547-2566.
[2]	DUMESIC D A, MELDRUM D R, KATZ-JAFFE M G, et al.Oocyte environment:follicular fluid and cumulus cells are critical for oocyte health[J].Fertil Steril, 2015, 103(2):303-316.
[3]	TIAN C L, LIU L L, YE X Y, et al.Functional oocytes derived from granulosa cells[J].Cell Rep, 2019, 29(13):4256-4267.e9.
[4]	ELGEBALY M M, HAZAA A B M, AMER H A, et al.L-Cysteine improves bovine oocyte developmental competence in vitro via activation of oocyte-derived growth factors BMP-15 and GDF-9[J].Reprod Domest Anim, 2022, 57(7):734-742.
[5]	SUN L C, ZHANG H F, GAO P.Metabolic reprogramming and epigenetic modifications on the path to cancer[J].Protein Cell, 2022, 13(12):877-919.
[6]	SHA Q Q, ZHANG J, FAN H Y.Function and regulation of histone H3 lysine-4 methylation during oocyte meiosis and maternal-to-zygotic transition[J].Front Cell Dev Biol, 2020, 8:597498.
[7]	SHA Q Q, ZHU Y Z, XIANG Y L, et al.Role of CxxC-finger protein 1 in establishing mouse oocyte epigenetic landscapes[J].Nucleic Acids Res, 2021, 49(5):2569-2582.
[8]	SHEARN A, HERSPERGER E, HERSPERGER G.Genetic studies of mutations at two loci of Drosophila melanogaster which cause a wide variety of homeotic transformations[J].Rouxs Arch Dev Biol, 1987, 196(4):231-242.
[9]	MIYAZAKI H, HIGASHIMOTO K, YADA Y, et al.Ash1l methylates Lys36 of histone H3 independently of transcriptional elongation to counteract polycomb silencing[J].PLoS Genet, 2013, 9(11):e1003897.
[10]	ROGAWSKI D S, NDOJ J, CHO H J, et al.Two loops undergoing concerted dynamics regulate the activity of the ASH1L histone methyltransferase[J].Biochemistry, 2015, 54(35):5401-5413.
[11]	JONES M, CHASE J, BRINKMEIER M, et al.Ash1l controls quiescence and self-renewal potential in hematopoietic stem cells[J].J Clin Invest, 2015, 125(5):2007-2020.
[12]	STEWART K R, VESELOVSKA L, KIM J, et al.Dynamic changes in histone modifications precede de novo DNA methylation in oocytes[J].Genes Dev, 2015, 29(23):2449-2462.
[13]	BRINKMEIER M L, GEISTER K A, JONES M, et al.The histone methyltransferase gene Absent, Small, or homeotic discs-1 like is required for normal Hox gene expression and fertility in mice[J].Biol Reprod, 2015, 93(5):121.
[14]	CUI L X, TIAN Y Q, HAO H S, et al.Knockdown of ASH1L methyltransferase induced apoptosis inhibiting proliferation and H3K36 methylation in bovine cumulus cells[J].Theriogenology, 2021, 161:65-73.
[15]	ZHANG W, CHEN E M, CHEN M, et al.IGFBP7 regulates the osteogenic differentiation of bone marrow-derived mesenchymal stem cells via Wnt/β-catenin signaling pathway[J].FASEB J, 2018, 32(4):2280-2291.
[16]	ZHANG T, REN T H, LIN H, et al.ASH1L contributes to oocyte apoptosis by regulating DNA damage[J].Am J Physiol Cell Physiol, 2022, 323(4):C1264-C1273.
[17]	XIA M, LIU J, WU X H, et al.Histone methyltransferase Ash1l suppresses interleukin-6 production and inflammatory autoimmune diseases by inducing the ubiquitin-editing enzyme A20[J].Immunity, 2013, 39(3):470-481.
[18]	WANG W J, ZOU H Y, CHEN N Z, et al.Knockdown of CLAUDIN-6 inhibited apoptosis and induced proliferation of bovine cumulus cells[J].Int J Mol Sci, 2022, 23(21):13222.
[19]	TIAN Y Q, CUI L X, WANG W J, et al.Knockdown of bone morphogenetic protein 4 gene induces apoptosis and inhibits proliferation of bovine cumulus cells[J].Theriogenology, 2022, 188:28-36.
[20]	CHENG Q, XIE H, ZHANG X Y, et al.An essential role for PTIP in mediating Hox gene regulation along PcG and trxG pathways[J].FEBS J, 2022, 289(20):6324-6341.
[21]	CASTIGLIONI I, CACCIA R, GARCIA-MANTEIGA J M, et al.The Trithorax protein Ash1L promotes myoblast fusion by activating Cdon expression[J].Nat Commun, 2018, 9(1):5026.
[22]	YIN B, YU F Y, WANG C L, et al.Epigenetic control of mesenchymal stem cell fate decision via histone methyltransferase Ash1l[J].Stem Cells, 2019, 37(1):115-127.
[23]	ZHANG K, HAVERSAT J M, MAGER J. CTR9/PAF1c regulates molecular lineage identity, histone H3K36 trimethylation and genomic imprinting during preimplantation development[J].Dev Biol, 2013, 383(1):15-27.
[24]	YU M M, JIA Y J, MA Z C, et al.Structural insight into ASH1L PHD finger recognizing methylated histone H3K4 and promoting cell growth in prostate cancer[J].Front Oncol, 2022, 12:906807.
[25]	LIU W M, XU L L, ZHANG C, et al.ASH1L may contribute to the risk of Tourette syndrome:combination of family-based analysis and case-control study[J].Brain Behav, 2022, 12(4):e2539.
[26]	ROGAWSKI D S, DENG J, LI H, et al.Discovery of first-in-class inhibitors of ASH1L histone methyltransferase with anti-leukemic activity[J].Nat Commun, 2021, 12(1):2792.
[27]	YUAN W, XU M, HUANG C, et al.H3K36 methylation antagonizes PRC2-mediated H3K27 methylation[J].J Biol Chem, 2011, 286(10):7983-7989.
[28]	ZHU L, LI Q, WONG S H K, et al.ASH1L links histone H3 lysine 36 dimethylation to MLL leukemia[J].Cancer Discov, 2016, 6(7):770-783.
[29]	BALBO POGLIANO C, GATTI M, RVTHEMANN P, et al.ASH1L histone methyltransferase regulates the handoff between damage recognition factors in global-genome nucleotide excision repair[J].Nat Commun, 2017, 8(1):1333.
[30]	PARMAR N, CHANDRAKAR P, KAR S.Leishmania donovani Subverts Host Immune response by epigenetic reprogramming of Macrophage M(Lipopolysaccharides+IFN-γ)/M(IL-10) polarization[J].J Immunol, 2020, 204(10):2762-2778.
[31]	ALJAZI M B, GAO Y E, WU Y, et al.Histone H3K36me2-Specific methyltransferase ASH1L promotes MLL-AF9-Induced leukemogenesis[J].Front Oncol, 2021, 11:754093.
[32]	崔立欣, 田雅晴, 郝海生, 等.ASH1L甲基转移酶在牛卵丘细胞中的表达与功能研究[J].畜牧兽医学报, 2020, 51(8):1866-1877.CUI L X, TIAN Y Q, HAO H S, et al.Expression and function of ASH1L methyltransferase in bovine cumulus cells[J].Acta Veterinaria et Zootechnica Sinica, 2020, 51(8):1866-1877.(in Chinese)
[33]	QIN L Y, WILLIAMS J B, TAN T, et al.Deficiency of autism risk factor ASH1L in prefrontal cortex induces epigenetic aberrations and seizures[J].Nat Commun, 2021, 12(1):6589.
[34]	FUJIMOTO A, FURUTA M, TOTOKI Y, et al.Whole-genome mutational landscape and characterization of noncoding and structural mutations in liver cancer[J].Nat Genet, 2016, 48(6):500-509.
[35]	ABBASTABAR M, KHEYROLLAH M, AZIZIAN K, et al.Multiple functions of p27 in cell cycle, apoptosis, epigenetic modification and transcriptional regulation for the control of cell growth:a double-edged sword protein[J].DNA Repair (Amst), 2018, 69:63-72.
[36]	MU W P, STARMER J, SHIBATA Y, et al.EZH1 in germ cells safeguards the function of PRC2 during spermatogenesis[J].Dev Biol, 2017, 424(2):198-207.
[37]	MJELLE R, HEGRE S A, AAS P A, et al.Cell cycle regulation of human DNA repair and chromatin remodeling genes[J].DNA Repair (Amst), 2015, 30:53-67.
[38]	XU R F, QIN N, XU X X, et al.Inhibitory effect of SLIT2 on granulosa cell proliferation mediated by the CDC42-PAKs-ERK1/2 MAPK pathway in the prehierarchical follicles of the chicken ovary[J].Sci Rep, 2018, 8(1):9168.
[39]	LIU L X, KIMBALL S, LIU H, et al.Genetic alterations of histone lysine methyltransferases and their significance in breast cancer[J].Oncotarget, 2015, 6:2466-2482.
[40]	LI G, YE Z S, SHI C, et al.The Histone methyltransferase Ash1l is required for epidermal homeostasis in mice[J].Sci Rep, 2017, 7:45401.

Effects of Histone Methyltransferase ASH1L Overexpression on Proliferation and Apoptosis of Bovine Cumulus Cells

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References 40

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	LI Qiuyun, TIAN Xinyuan, LIAO Wensheng, ZHANG Huanrong, REN Yupeng, YANG Falong, ZHU Jiangjiang, XIANG Hua. Effects of SOCS2 on Proliferation, Cycle and Apoptosis of Turbinate Bone Cells in Goats [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2226-2240.
[2]	SHAO Peng, TANG Yinmei, LIN Yaqiu, WANG Yong, XIANG Hua, HUANG Lian, ZHU Jiangjiang. Regulation Effect of PSMD9 on Lipid Deposition in Goat Precursor Adipocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3653-3663.
[3]	LIANG Rui, FAN Xiaorui, ZHANG Jinqiang, PANG Quanhai. Effects of Mouse Melanocyte Silencing and Overexpression of Pigment Epithelium-Derived Factor on Melanin Synthesis [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3916-3930.
[4]	ZHANG Wanfeng, ZHAO Tianzhi, LI Jiao, YOU Ziwei, YANG Yang, CAI Chunbo, GAO Pengfei, CAO Guoqing, GUO Xiaohong, LI Bugao. Study on NR2F2 Gene Regulating Proliferation and Apoptosis of Porcine PK15 Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3242-3251.
[5]	ZHANG Peng, WANG Mingxiu, JING Kemin, PENG Wei, TIAN Yuan, LI Yuqian, FU Changqi, SHU Shi, ZHONG Jincheng, CAI Xin. Abnormal Expression of FGFs/FGFRs and Their Mediated Signaling Pathway Genes Affect the Proliferative Activity of Undifferentiated Spermatogonia in Cattleyak [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 2886-2897.
[6]	XU Tiantian, ZHANG Tongtong, WANG Meng, WANG Xin. Transcription Factor Foxq1 Affects the Proliferation of Hair Follicle Stem Cells in Cashmere Goats via WNT/β-catenin Signaling Pathway [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2653-2661.
[7]	QUBI Wuqie, LI Yanyan, LI Xin, WANG Yong, WANG Youli, LIU Wei, ZHU Jiangjiang, LIN Yaqiu. APOA4 Gene Inhibits Intramuscular Adipocyte Differentiation in Goat [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 1927-1938.
[8]	DAN Yixin, YANG Lu, XIANG Hua, ZHANG Huanrong, REN Yupeng, YANG Falong, HE Honghong, ZHU Jiangjiang. Effects of BIRC5 on the Cycle and Apoptosis of Goat Testis Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1511-1524.
[9]	REN Xiaoli, FAN Yuying, HUANGFU Heping, LIU Yun, SHI Dongmei. Effect of GSK126 on Epithelial-mesenchymal Transition of Canine Mammary Tumor Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1721-1729.
[10]	YANG Guang, XU Jing, LI Xin, HU Debao, GUO Yiwen, DING Xiangbin, GUO Hong, ZHANG Linlin. Effect of Interfering lncbMD on Proliferation and Differentiation of Bovine Skeletal Muscle Satellite Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(3): 1015-1025.
[11]	SUN Jinkui, XU Houqiang, SHI Pengfei, RUAN Yong. Construction of MEF2A Gene Interference Vector and Effect of Its Transfection on Myoblasts in Guanling Cattle [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(2): 584-595.
[12]	MA Tianwen, YU Yue, LÜ Liangyu, JIA Lina, RUAN Hongri, WANG Haoran, WANG Xinyu, ZHANG Yuxin, ZHANG Jiantao, GAO Li. Effects of Bilobalide on Autophagy, Proliferation and Apoptosis of IL-1β-induced ATDC5 Chondrocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(2): 837-846.
[13]	JIN Sihua, JIA Fumin, HE Peili, JIA Yuqing, SHUI Fei, LIU Xuling, LIU Xing, WANG Xin, XU Man, GENG Zhaoyu. Effect of IHH Gene Overexpression on Proliferation and Differentiation of Chicken Primary Chondrocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(11): 4569-4576.
[14]	REN Xiaoli, FAN Yuying, HUANGFU Heping, WANG Jun, JIN Shuangxing, LIU Yun, SHI Dongmei. Mechanism of miR-502 Regulating Proliferation, Migration, Invasion and EMT of Canine Mammary Tumor Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10): 4379-4388.
[15]	WEI Jiayuan, ZHU Qian, YANG Yaxing, SHEN Ming. Inhibition of Porcine Follicular Granulosa Cell Proliferation by Cobalt Chloride Induced DNA Oxidative Damage [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(9): 2982-2992.