敲低Raf激酶抑制蛋白促进LX-2人肝星状细胞增殖
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摘要
目的研究Raf激酶抑制蛋白(RKIP)在LX-2人肝星状细胞增殖中作用。方法将RKIP小干扰RNA(siRNA-RKIP)重组质粒转染至LX-2细胞,培养5 d,并筛选出稳定转染的细胞。MTT法检测沉默RKIP后LX-2细胞的增殖情况;流式细胞术检测细胞凋亡、细胞周期;实时定量PCR检测α平滑肌肌动蛋白(α-SMA)和1型胶原蛋白(Col1)mRNA的表达。Western blot法检测RKIP、α-SMA、Col1及胞外信号调节激酶/丝裂原活化蛋白激酶(ERK/MAPK)信号通路相关蛋白的表达水平。结果与空白对照组相比,RKIP低表达明显诱导LX-2细胞增殖,抑制细胞凋亡,增加G2期细胞比例,提高Col1、α-SMA mRNA和蛋白表达。此外,RKIP的沉默明显提高ERK/MAPK磷酸化水平。结论敲低RKIP的水平促进LX-2细胞的增殖,其机制与激活ERK/MAPK信号通路有关。
Objective To investigate the role of Raf kinase inhibitor protein( RKIP) in the proliferation of LX-2 human hepatic stellate cells. Methods The recombinant plasmid siRNA-RKIP was transfected into LX-2 cells. Five days later,the stably transfected cells were screened and cultured. MTT assay was used to detect cell proliferation after RKIP was silenced.Cell apoptosis and cell cycle distribution were evaluated by flow cytometry. The expressions of α-smooth muscle actin( α-SMA) and collagen type 1( Col1) mRNA were detected by quantitative real-time PCR. The expressions of RKIP,α-SMA,Col1 and extracellular signal-regulated kinases/mitogen-activated protein kinase( ERK/MAPK) signaling pathway related proteins were assessed by Western blot analysis. Results Compared with the control group,knockdown of RKIP significantly induced LX-2 cell proliferation,reduced cell apoptosis,raise cell number in G2,and increased the proteins and mRNA expressions of Col1 and α-SMA. Moreover,low-expression of RKIP significantly enhanced the phosphorylation of ERK/MAPK. Conclusion Knockdown of RKIP promotes LX-2 cell proliferation; its mechanism is related to the activation of ERK/MAPK signaling pathway.
Objective To investigate the role of Raf kinase inhibitor protein( RKIP) in the proliferation of LX-2 human hepatic stellate cells. Methods The recombinant plasmid siRNA-RKIP was transfected into LX-2 cells. Five days later,the stably transfected cells were screened and cultured. MTT assay was used to detect cell proliferation after RKIP was silenced.Cell apoptosis and cell cycle distribution were evaluated by flow cytometry. The expressions of α-smooth muscle actin( α-SMA) and collagen type 1( Col1) mRNA were detected by quantitative real-time PCR. The expressions of RKIP,α-SMA,Col1 and extracellular signal-regulated kinases/mitogen-activated protein kinase( ERK/MAPK) signaling pathway related proteins were assessed by Western blot analysis. Results Compared with the control group,knockdown of RKIP significantly induced LX-2 cell proliferation,reduced cell apoptosis,raise cell number in G2,and increased the proteins and mRNA expressions of Col1 and α-SMA. Moreover,low-expression of RKIP significantly enhanced the phosphorylation of ERK/MAPK. Conclusion Knockdown of RKIP promotes LX-2 cell proliferation; its mechanism is related to the activation of ERK/MAPK signaling pathway.
引文
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[23]Wu Y L,Zhang Y J,Yao Y L,et al.Cucurbitacin E ameliorates hepatic fibrosis in vivo and in vitro through activation of AMPK and blocking m TOR-dependent signaling pathway[J].Toxicol Lett,2016,258:147-158.
[24]Lee H C,Tian B,Sedivy J M,et al.Loss of Raf kinase inhibitor protein promotes cell proliferation and migration of human hepatoma cells[J].Gastroenterology,2006,131(4):1208-1217.
[25]Li B,Sun B,Zhu J,et al.Expression of RKIP in chronic myelogenous leukemia K562 cell and inhibits cell proliferation by regulating the ERK/MAPK pathway[J].Tumour Biol,2014,35(10):10057-10066.
[2]Popov Y,Schuppan D.Targeting liver fibrosis:Strategies for development and validation of antifibrotic therapies[J].Hepatology,2009,50(4):1294-1306.
[3]Peng Y,Yang H,Wang N,et al.Fluorofenidone attenuates hepatic fibrosis by suppressing the proliferation and activation of hepatic stellate cells[J].Am J Physiol Gastrointest Liver Physiol,2014,306(3):G253-G263.
[4]Ha T Y,Hwang S,Moon K M,et al.Sorafenib inhibits migration and invasion of hepatocellular carcinoma cells through suppression of matrix metalloproteinase expression[J].Anticancer Res,2015,35(4):1967-1976.
[5]Keller E T.Metastasis suppressor genes:a role for raf kinase inhibitor protein(RKIP)[J].Anticancer Drugs,2004,15(7):663-669.
[6]Yeung K,Seitz T,Li S,et al.Suppression of Raf-1 kinase activity and MAP kinase signalling by RKIP[J].Nature,1999,401(6749):173-177.
[7]Houben R,Michel B,Vetter-Kauczok C S,et al.Absence of classical MAP kinase pathway signalling in Merkel cell carcinoma[J].J Invest Dermatol,2006,126(5):1135-1142.
[8]Vandamme D,Herrero A,Al-Mulla F,et al.Regulation of the MAPK pathway by raf kinase inhibitory protein[J].Crit Rev Oncog,2014,19(6):405-415.
[9]王园园,贺秋艳,易红梅,等.RKIP在鼻咽癌侵袭转移中的作用及机制研究[J].临床与病理杂志,2015,35(3):375-381.
[10]Datar I,Qiu X,Ma H Z,et al.RKIP regulates CCL5 expression to inhibit breast cancer invasion and metastasis by controlling macrophage infiltration[J].Oncotarget,2015,6(36):39050-39061.
[11]Wu X,Yang Y,Xu Z,et al.Raf kinase inhibitor protein mediated signaling inhibits invasion and metastasis of hepatocellular carcinoma[J].Biochim Biophys Acta,2016,1860(2):384-391.
[12]杨大运,齐战,邢亚威,等.RKIP的表达与肺癌侵袭转移的相关性[J].江苏医药,2013,39(13):1525-1527.
[13]Yuan L,Yi H M,Yi H,et al.Reduced RKIP enhances nasopharyngeal carcinoma radioresistance by increasing ERK and AKT activity[J].Oncotarget,2016,7(10):11463-11477.
[14]He Q Y,Yi H M,Yi H,et al.Reduction of RKIP expression promotes nasopharyngeal carcinoma invasion and metastasis by activating Stat3signaling[J].Oncotarget,2015,6(18):16422-16436.
[15]Chen Z,Cheng Q,Ma Z,et al.Overexpression of RKIP inhibits cell invasion in glioma cell lines through upregulation of miR-98[J/OL].Biomed Res Int,2013,2013:695179.doi:10.1155/2013/695179.Epub 2013 Dec 12.
[16]邹堂斌,贾青,朱姗姗,等.PI3K/Akt信号通路在虾青素调节LX-2细胞活化中作用[J/OL].中国公共卫生,2016,http://www.cnki.net/kcms/detail/21.1234.R.20160621.1438.056.html.
[17]马俊骥,赵慧,姜慧卿.RKIP过表达对肝星状细胞在细胞外基质上黏附的抑制[J].世界华人消化杂志,2013,21(14):1303-1308.
[18]Ma J,Li F,Liu L,et al.Raf kinase inhibitor protein inhibits cell proliferation but promotes cell migration in rat hepatic stellate cells[J].Liver Int,2009,29(4):567-574.
[19]Dooley S,ten Dijke P.TGF-beta in progression of liver disease[J].Cell Tissue Res,2012,347(1):245-256.
[20]Jiang M D,Zheng S M,Xu H,et al.An experimental study of extracellular signal-regulated kinase and its interventional treatments in hepatic fibrosis[J].Hepatobiliary Pancreat Dis Int,2008,7(1):51-57.
[21]Nie F,Cao J,Tong J,et al.Role of Raf-kinase inhibitor protein in colorectal cancer and its regulation by hydroxycamptothecine[J/OL].J Biolmed Sci,2015,22:56.Doi:10.1186/s12929-015-0162-y.
[22]马俊骥,姜慧卿,李芳芳,等.RKIP在胆总管结扎肝纤维化大鼠肝脏组织的表达[J].中国老年学杂志,2008,28(20):1998-2000.
[23]Wu Y L,Zhang Y J,Yao Y L,et al.Cucurbitacin E ameliorates hepatic fibrosis in vivo and in vitro through activation of AMPK and blocking m TOR-dependent signaling pathway[J].Toxicol Lett,2016,258:147-158.
[24]Lee H C,Tian B,Sedivy J M,et al.Loss of Raf kinase inhibitor protein promotes cell proliferation and migration of human hepatoma cells[J].Gastroenterology,2006,131(4):1208-1217.
[25]Li B,Sun B,Zhu J,et al.Expression of RKIP in chronic myelogenous leukemia K562 cell and inhibits cell proliferation by regulating the ERK/MAPK pathway[J].Tumour Biol,2014,35(10):10057-10066.