DDR1对奶牛乳腺上皮细胞增殖与凋亡的调控作用

doi:10.11843/j.issn.0366-6964.2020.09.008

Abstract

Abstract: The aim of this study was to investigate the effect of discoidin domain receptor 1 (DDR1) gene on the proliferation, apoptosis and cell cycle of dairy cow mammary epithelial cells through interfering and overexpressing DDR1. The small RNA interference fragment of DDR1 gene and overexpression vector pcDNA3.1-DDR1 were transfected into dairy cow mammary epithelial cells, qRT-PCR and Western blot were used to detect the interference and overexpression efficiency of DDR1; CCK-8 was employed to detect cell proliferation; Flow cytometry was performed to detect the changes of cell cycle and apoptosis (early apoptosis and late apoptosis). qRT-PCR was used to detect the expression of genes related to proliferation and apoptosis. After interfering DDR1 gene in dairy cow mammary epithelial cells, mRNA and protein expression levels of DDR1 were down-regulated by 94% and 30%, respectively; While after overexpressing DDR1 gene, its mRNA and protein expression were up-regulated 68.24 and 1.38 times, respectively. Interfering DDR1 extremely significantly inhibited the proliferation of cells (P<0.01), the ratio of early and late apoptotic cells was extremely significantly increased (P<0.01); the ratio of G1 phase cells in the interference group was extremely significantly increased (P<0.01), and the proportions of S and G2 phase cells were significantly decreased (P<0.01 and P<0.05), which suggested that the cells were blocked in G0/G1 phase. Conversely, overexpression of DDR1 could significantly promote cell proliferation (P<0.05), significantly inhibit the late apoptosis of cells (P<0.05), the proportion of G1 phase cells was extremely significantly decreased (P<0.01), and the proportion of S phase cells was extremely significantly increased (P<0.01), suggesting the enhancement of transition from G1 to S phase. The results of qRT-PCR detection showed that after interfering DDR1, the expressions of BAX and Caspase9 genes were significantly up-regulated (P<0.05), and the expressions of P53 and FAS genes were extremely significantly up-regulated (P<0.01), while the expressions of PCNA and CyclinB1 genes were significantly down-regulated (P<0.05). After overexpression of DDR1, the expressions of Cytc and BAX genes were significantly (P<0.05) and extremely significantly down-regulated(P<0.01), respectively, and the expression of CyclinB1 gene was extremely significantly up-regulated (P<0.01). In summary, DDR1 can regulate the proliferation, apoptosis and cycle of dairy cow mammary epithelial cells, this result will provide a reference for elucidating the molecular mechanism of DDR1 involved in the growth and development of dairy cow mammary epithelial cells.

Key words: discoidin domain receptor 1, dairy cow mammary epithelial cells, cell proliferation, cell cycle, apoptosis

CLC Number:

S823.91

HUA Liping, LIU Shuanghang, ZHAO Xinzhe, YE Tingzhu, XIONG Jiajun, YANG Liguo, LIANG Aixin. Effects of Interference and Overexpression of DDR1 on Proliferation and Apoptosis of Mammary Epithelial Cells in Dairy Cows[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(9): 2109-2119.

References 37

[1]	BOUTINAUD M,GUINARD-FLAMENTA J,JAMMES H.The number and activity of mammary epithelial cells,determining factors for milk production[J].Reprod Nutr Dev,2004,44(5):499-508.
[2]	JOHNSON J D,EDMAN J C,RUTTER W J.A receptor tyrosine kinase found in breast carcinoma cells has an extracellular discoidin I-like domain[J].Proc Natl Acad Sci U S A,1993,90(12):5677-5681.
[3]	TAKAI K,DRAIN A P,LAWSON D A,et al.Discoidin domain receptor 1 (DDR1) ablation promotes tissue fibrosis and hypoxia to induce aggressive basal-like breast cancers[J].Genes Dev,2018,32(3-4):244-257.
[4]	YUGE R,KITADAI Y,TAKIGAWA H,et al.Silencing of Discoidin Domain Receptor-1 (DDR1) concurrently Inhibits multiple Steps of metastasis cascade in gastric cancer[J].Transl Oncol,2018,11(3):575-584.
[5]	VILELLA E,GAS C,GARCIA-RUIZ B,et al.Expression of DDR1 in the CNS and in myelinating oligodendrocytes[J].Biochim Biophys Acta Mol Cell Res,2019,1866(11):118483.
[6]	WEINER H L,HUANG H Y,ZAGZAG D,et al.Consistent and selective expression of the discoidin domain receptor-1 tyrosine kinase in human brain tumors[J].Neurosurgery,2000,47(6):1400-1409.
[7]	VALIATHAN R R,MARCO M,LEITINGER B,et al.Discoidin domain receptor tyrosine kinases:New players in cancer progression[J]. Cancer Metastasis Rev,2012,31(1-2):295-321.
[8]	FRIDMAN R,AGARWAL G.New concepts on the interactions of discoidin domain receptors with collagen[J].Biochim Biophys Acta Mol Cell Res,2019,1866(11):118527.
[9]	ORGEL J P R O,MADHURAPANTULA R S.A structural prospective for collagen receptors such as DDR and their binding of the collagen fibril[J].Biochim Biophys Acta Mol Cell Res,2019,1866(11):118478.
[10]	XU H F,RAYNAL N,STATHOPOULOS S,et al.Collagen binding specificity of the discoidin domain receptors:Binding sites on collagens II and III and molecular determinants for collagen IV recognition by DDR1[J].Matrix Biol,2011,30(1):16-26.
[11]	VELLA V,MALAGUARNERA R,NICOLOSI M L,et al.Insulin/IGF signaling and discoidin domain receptors:An emerging functional connection[J].Biochim Biophys Acta Mol Cell Res,2019,1866(11):118522.
[12]	MATSUYAMA W,FAURE M,YOSHIMURA T.Activation of discoidin domain receptor 1 facilitates the maturation of human monocyte-derived dendritic cells through the TNF receptor associated factor 6/TGF-beta-activated Protein Kinase 1 Binding Protein 1 beta/p38 alpha mitogen-activated protein kinase signaling cascade[J].J Immunol,2003,171(7):3520-3532.
[13]	HOU G P,VOGEL W,BENDECK M P.The discoidin domain receptor tyrosine kinase DDR1 in arterial wound repair[J].J Clin Invest,2001,107(6):727-735.
[14]	VOGEL W F,ASZÓDI A,ALVES F,et al.Discoidin domain receptor 1 tyrosine kinase has an essential role in mammary gland development[J].Mol Cell Biol,2001,21(8):2906-2917.
[15]	FARACI-ORF E,MCFADDEN C,VOGEL W F.DDR1 signaling is essential to sustain Stat5 function during lactogenesis[J].J Cell Biochem,2006,97(1):109-121.
[16]	LI S,HUANG S H,QIAO S Y,et al.Cloning and functional characterization of STAT5a and STAT5b genes in buffalo mammary epithelial cells[J].Anim Biotechnol,2020,31(1):59-66.
[17]	SHIN H Y,HENNIGHAUSEN L,YOO K H.STAT5-Driven enhancers tightly control temporal expression of mammary-specific genes[J].J Mammary Gland Biol Neoplasia,2019,24(1):61-71.
[18]	刘晓飞,高学军,李庆章,等.stat5高效表达对奶牛乳腺上皮细胞泌乳能力的影响[J].畜牧兽医学报,2011,42(4):508-512.LIU X F,GAO X J,LI Q Z,et al.Effects on lactation ability of dairy cow mammary gland epithelial cells by stat5 high expression[J].Acta Veterinaria et Zootechnica Sinica,2011,42(4):508-512.(in Chinese)
[19]	VOGEL W F,ABDULHUSSEIN R,FORD C E.Sensing extracellular matrix:An update on discoidin domain receptor function[J]. Cell Signal,2006,18(8):1108-1116.
[20]	LEITINGER B.Discoidin domain receptor functions in physiological and pathological conditions[J].Int Rev Cell Mol Biol,2014, 310:39-87.
[21]	ONGUSAHA P P,KIM J I,FANG L,et al.p53 induction and activation of DDR1 kinase counteract p53-mediated apoptosis and influence p53 regulation through a positive feedback loop[J].EMBO J,2003,22(6):1289-1301.
[22]	VALENCIA K,ORMAZÁBAL C,ZANDUETA C,et al.Inhibition of collagen receptor discoidin domain receptor-1 (DDR1) reduces cell survival,homing,and colonization in lung cancer bone metastasis[J].Clin Cancer Res,2012,18(4):969-980.
[23]	CHOU S T,PENG H Y,MO K C,et al.MicroRNA-486-3p functions as a tumor suppressor in oral cancer by targeting DDR1[J].J Exp Clin Cancer Res,2019,38(1):281.
[24]	ZHONG X,ZHANG W W,SUN T H.DDR1 promotes breast tumor growth by suppressing antitumor immunity[J].Oncol Rep,2019,42(6):2844-2854.
[25]	XIE R X,WANG X Y,QI G Q,et al.DDR1 enhances invasion and metastasis of gastric cancer via epithelial-mesenchymal transition[J].Tumor Biol,2016,37(9):12049-12059.
[26]	WANG C Z,HSU Y M,TANG M J.Function of discoidin domain receptor I in HGF-induced branching tubulogenesis of MDCK cells in collagen gel[J].J Cell Physiol,2005,203(1):295-304.
[27]	AZIZI R,SALEMI Z,FALLAHIAN F,et al.Inhibition of didscoidin domain receptor 1 reduces epithelial-mesenchymal transition and induce cell-cycle arrest and apoptosis in prostate cancer cell lines[J].J Cell Physiol,2019,234(11):19539-19552.
[28]	DENG Y,ZHAO F,HUI L,et al.Suppressing miR-199a-3p by promoter methylation contributes to tumor aggressiveness and cisplatin resistance of ovarian cancer through promoting DDR1 expression[J].J Ovarian Res,2017,10(1):50.
[29]	陆杏蓉,段安琴,马小娅,等.DDR1基因过表达对水牛乳腺上皮细胞的影响[J].中国畜牧兽医,2019,46(12):3635-3641.LU X R,DUAN A Q,MA X Y,et al.Effect of DDR1 gene over expression on buffalo mammary epithelial cells[J].Chinese Animal Husbandry & Veterinary Medicine,2019,46(12):3635-3641.(in Chinese)
[30]	CURAT C A,VOGEL W F.Discoidin domain receptor 1 controls growth and adhesion of mesangial cells[J].J Am Soc Nephrol, 2002,13(11):2648-2656.
[31]	SAITO M,KORSMEYER S J,SCHLESINGER P H.BAX-dependent transport of cytochrome C reconstituted in pure liposomes[J]. Nat Cell Biol,2000,2(8):553-555.
[32]	POP C,TIMMER J,SPERANDIO S,et al.The apoptosome activates caspase-9 by dimerization[J].Mol cell,2006,22(2):269-275.
[33]	JIANG X J,WANG X D.Cytochrome c promotes caspase-9 activation by inducing nucleotide binding to Apaf-1[J].J Biol Chem, 2000, 275(40):31199-31203.
[34]	LAPTENKO O,PRIVES C.Transcriptional regulation by p53:one protein,many possibilities[J].Cell Death Differ,2006, 13(6):951-961.
[35]	LIU X M,YANG Z M,LIU X K.Fas/FasL induces myocardial cell apoptosis in myocardial ischemia-reperfusion rat model[J].Eur Rev Med Pharmacol Sci,2017,21(12):2913-2918.
[36]	MORRIS G F,MATHEWS M B.Regulation of proliferating cell nuclear antigen during the cell cycle[J].J Biol Chem,1989, 264(23):13856-13864.
[37]	马淑梅,刘晓冬,鞠桂芝.CyclinB1、p34cdc2在X射线诱导的K562细胞G2期阻滞中的作用[J].中华放射医学与防护杂志,2002,22(2):105-107.MA S M,LIU X D,JU G Z.The role of CyclinB1 and p34cdc2 in the X2-induced G2 arrest of K562 cells[J].Chinese Journal of Radiological Medicine and Protection,2002,22(2):105-107.(in Chinese)

Effects of Interference and Overexpression of DDR1 on Proliferation and Apoptosis of Mammary Epithelial Cells in Dairy Cows

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References 37

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	DONG Shucan, MAO Shuaixiang, WU Cuiying, LI Yaokun, SUN Baoli, GUO Yongqing, DENG Ming, LIU Dewu, LIU Guangbin. The Effect of the Androgen Receptor Inhibitor Enzalutamide on Proliferation and Apoptosis of Goat Ovarian Granulosa Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2022-2031.
[2]	WANG Jiying, YIN Ruiru, XIE Xing, WANG Haiyan, LIU Hudong, HU Hui, XIONG Qiyan, FENG Zhixin, SHAO Guoqing, YU Yanfei. Effects of LDH in Mesomycoplasma (Mycoplasma) hyopneumoniae on Apoptosis of Porcine Bronchial Epithelial Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2195-2205.
[3]	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.
[4]	LAN Xinrui, ZHAO Baobao, ZHANG Bihan, LIN Xiaoyu, MA Huiming, WANG Yongsheng. Effects of β-sitosterol on Porcine Oocyte Maturation and Embryonic Development in Vitro [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1629-1637.
[5]	HU Qiaoyan, ZHAI Xiangqin, LI Yidan, HAN Jiale, LEI Chuzhao, DANG Ruihua. Effects of bta-miR-101 on Proliferation, Apoptosis and Secretion of Bovine Testicular Sertoli Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(3): 1040-1051.
[6]	HUO Yuannan, QIU Meijia, ZHANG Jiaojiao, YANG Weirong, WANG Xianzhong. Arginine and Its Metabolites Attenuate Heat Stress-induced Apoptosis of Immature Boar Sertoli Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 587-597.
[7]	QIU Wenyue, SU Yiman, YE Jiali, ZHANG Xinting, PANG Xiaoyue, WANG Rongmei, XIE Zimao, ZHANG Hui, TANG Zhaoxin, SU Rongsheng. Study on Asiatic Acid Alleviates LPS-induced Acute Kidney Injury by Regulating Apoptosis and Autophagy of Broilers [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 809-821.
[8]	CHEN Songbiao, LIU Feifei, SHANG Ke, YU Zuhua, HE Lei, WEI Ying, CHEN Jian, ZHANG Chunjie, CHENG Xiangchao, DING Ke. Molecular Mechanism of the “Battle” between Virus Infection and Host Antiviral Immunity-Apoptosis, Necroptosis and Pyroptosis [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 59-70.
[9]	DUAN Xiangru, KANG Jia, YANG Ruochen, SHAN Xinyu, LI Taichun, ZHAO Wen, ZHANG Yingjie, LIU Yueqin. Effect of L-cysteine on Proliferation, Apoptosis and the Secretion of Steroid Hormone in Ovine Ovarian Granulosa Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 179-191.
[10]	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.
[11]	SONG Meijun, HAO Kexing, HAI Siyu, CHEN Yan, WANG Jing, HU Guangdong. Effects of SRIF-14 on Proliferation and Apoptosis of Endometrial Epithelial Cells in Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3325-3334.
[12]	XU Xi, YANG Baigao, ZHANG Hang, FENG Xiaoyi, HAO Haisheng, DU Weihua, ZHU Huabin, ZHANG Peipei, ZHAO Xueming. Effects of NMN on Lipid Droplet Content and Cryopreservation Effect of Bovine Oocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3348-3357.
[13]	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.
[14]	SHEN Tong, WANG Mengjie, WU Hua, LI Jinming, WANG Shuo, WU Xiaoqing, CHEN Xinlei, XING Qianwen, LIU Bo. Effect of Lycium ruthenicum Murray Anthocyanin on Hypoxia-induced Apoptosis in H9c2 Rat Cardiomyocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3490-3499.
[15]	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.