ObjectiveTo investigate the role of ski in proliferation of astrocytes and the molecular mechanisms in rats. MethodsAstrocytes were obtained from cerebral cortex of a three-day old rat and cultured in vitro. siRNA targeted to ski and negative control sequences were prepared. The astrocytes were divided into ski-siRNA group, siRNA negative control group and untreated control group, while the specific siRNA targeting ski negative control sequences were transfected into astrocytes with Lipofectamine® RNAiMAX Reagent. The protein levels of ski, glial fibrillary acidic protein (GFAP) and Cyclin D1 were determined with Western blotting. The proliferation of astrocytes were measured with CCK8 assay. The cell-cycle of astrocytes were analyzed with flow cytometer. ResultsThe protein level of ski (F=38.611, P<0.01), GFAP (F=7.547, P<0.05) and Cyclin D1 (F=3.901, P<0.05) reduced in ski-siRNA group, the proliferation of astrocyte was significantly inhibited since twelve hours after culture (F>30.507, P<0.01), and less cells were in S phase and more in G1/G0 phase (F>48.425, P<0.01), compared with the control groups. Conclusionski knocking down by siRNA significantly inhibits the proliferation of astrocytes, which may associate with the down-regulation of Cyclin D1 expression.
ZHAO Xin
,
GUO Yong-qiang
,
KOU Jiang-li
,
DING Ning
,
ZHOU Kai-sheng
,
NAN Wei
,
ZHANG Hai-hong
. Effects of Knocking Down ski on Proliferation of Astrocytes and Expression of Cyclin D1 in Rats[J]. Chinese Journal of Rehabilitation Theory and Practice, 2017
, 23(9)
: 1032
-1036
.
DOI: 10.3969/j.issn.1006-9771.2017.09.009
[1] Stavnezer E, Barkas AE, Brennan LA, et al. Transforming Sloan-Kettering viruses generated from the cloned v-ski oncogene by in vitro and in vivo recombinations [J]. J Virol, 1986, 57(3): 1073-1083.
[2] 周开升,朱彦东,张海鸿,等. Ski在神经系统中的作用及机制的研究进展[J]. 中国康复理论与实践, 2016, 22(7): 797-800.
[3] 赵鑫,郭永强,王明,等. 脂多糖对大鼠星形胶质细胞Ski蛋白表达的影响[J]. 中国康复理论与实践, 2017, 23(5): 514-519.
[4] Bonnon C, Atanasoski S. c-Ski in health and disease [J]. Cell Tissue Res, 2012, 347(1): 51-64.
[5] 周开升,朱彦东,赵鑫,等. Ski在大鼠脊髓损伤后不同时间的表达变化[J]. 中国康复理论与实践, 2016, 22(9): 1015-1019.
[6] Zhou K, Nan W, Feng D, et al. Spatiotemporal expression of Ski after rat spinal cord injury [J]. Neuroreport, 2017, 28(3): 149-157.
[7] 赵鑫,寇江力,郭永强. Ski在大鼠活化星型胶质细胞中表达的变化[J]. 中国病理生理杂志, 2017, 33(6): 968-974.
[8] 赵鑫,王兴文,寇江力,等. 沉默ski基因对大鼠星形胶质细胞迁移的影响[J]. 中国康复理论与实践, 2017, 23(8): 905-911.
[9] Atanasoski S, Notterpek L, Lee HY, et al. The protooncogene Ski controls Schwann cell proliferation and myelination [J]. Neuron, 2004, 43(4): 499-511.
[10] Ritter M, Kattmann D, Teichler S, et al. Inhibition of retinoic acid receptor signaling by Ski in acute myeloid leukemia [J]. Leukemia, 2006, 20(3): 437-443.
[11] Li J, Li P, Zhang Y, et al. c-Ski inhibits the proliferation of vascular smooth muscle cells via suppressing Smad3 signaling but stimulating p38 pathway [J]. Cell Signal, 2013, 25(1): 159-167.
[12] van Gijsel-Bonnello M, Baranger K, Benech P, et al. Metabolic changes and inflammation in cultured astrocytes from the 5xFAD mouse model of Alzheimer's disease: Alleviation by pantethine [J]. PLoS One, 2017, 12(4): e0175369.
[13] Liu T, Xue CC, Shi YL, et al. Overexpression of mitofusin 2 inhibits reactive astrogliosis proliferation in vitro [J]. Neurosci Lett, 2014, 579: 24-29.
[14] Liu Z, Xin H, Chopp M. Reactive astrocytes promote axonal remodeling and neurological recovery after stroke [J]. Neural Regen Res, 2014, 9(21): 1874-1875.
[15] Hol EM, Pekny M. Glial fibrillary acidic protein (GFAP) and the astrocyte intermediate filament system in diseases of the central nervous system [J]. Curr Opin Cell Biol, 2015, 32: 121-130.
[16] Pekny M, Wilhelmsson U, Pekna M. The dual role of astrocyte activation and reactive gliosis [J]. Neurosci Lett, 2014, 565: 30-38.
[17] Sofroniew MV. Astrogliosis [J]. Cold Spring Harb Perspect Biol, 2014, 7(2): a020420.
[18] Mohr SE, Smith JA, Shamu CE, et al. RNAi screening comes of age: improved techniques and complementary approaches [J]. Nat Rev Mol Cell Biol, 2014, 15(9): 591-600.
[19] Kim SJ, Kim JM, Shim SH, et al. Shikonin induces cell cycle arrest in human gastric cancer (AGS) by early growth response 1 (Egr1)-mediated p21 gene expression [J]. J Ethnopharmacol, 2014, 151(3): 1064-1071.
[20] Chen H, Xu X, Wang G, et al. CDK4 is degraded by APC/C in mitosis and reaccumulates in early G1 phase to initiate a new cell cycle in HeLa cells [J]. J Biol Chem, 2017, 292: 10131-10141.
[21] Schafer KA. The cell cycle: a review [J]. Vet Pathol, 1998, 35(6): 461-478.
[22] Godínez-Palma SK, Rosas-Bringas FR, Rosas-Bringas OG, et al. Two maize Kip-related proteins differentially interact with, inhibit and are phosphorylated by cyclin D-cyclin-dependent kinase complexes [J]. J Exp Bot, 2017, 68(7): 1585-1597.
[23] Godínez-Palma SK, García E, Sánchez ML, et al. Complexes of D-type cyclins with CDKs during maize germination [J]. J Exp Bot, 2013, 64(18): 5661-5671.
[24] Zhang Q, Sakamoto K, Wagner KU. D-type Cyclins are important downstream effectors of cytokine signaling that regulate the proliferation of normal and neoplastic mammary epithelial cells [J]. Mol Cell Endocrinol, 2014, 382(1): 583-592.
[25] de la Cruz JF, Vergara EJ, Cho Y, et al. Glehnia littoralis root extract induces G0/G1 phase cell cycle arrest in the MCF-7 human breast cancer cell line [J]. Asian Pac J Cancer Prev, 2015, 16(18): 8113-8117.
