沉默GRP94基因对乳腺癌MCF7细胞增殖和凋亡的影响
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摘要
目的:研究沉默葡萄糖调节蛋白GRP94(Glucose regulated protein,GRP94)对乳腺癌MCF7细胞增殖、凋亡的影响及潜在机制。方法:设计并化学合成靶向沉默GRP94基因的小干扰RNA,通过脂质体转染入MCF7细胞中,采用qRTPCR和Western blot分别检测GRP94、cyclin D1、Bax和Bcl-2 mRNA和蛋白的表达水平;通过流式细胞术检测细胞凋亡比例变化,Hoechst 33258染色检测凋亡细胞核变化、CCK8实验检测细胞增殖能力的变化。结果:GRP94 siRNA组GRP94基因的表达水平被有效抑制;与对照组相比,GRP94-siRNA转染组的细胞凋亡比例明显增加;凋亡细胞核形态发生变化;增殖能力明显下降;mRNA及蛋白水平cyclin D1、Bcl-2表达明显下调,Bax表达增加。结论:沉默GRP94基因可明显抑制乳腺癌MCF7细胞增殖能力,促进细胞凋亡的发生,且其可能通过下调cyclin D1、Bcl-2和上调Bax表达参与其中。
Objective: To determine the effects of silencing glucose regulated protein( GRP94) on the proliferation and apoptosis of breast carcinoma MCF7 cells. Methods: Chemically synthesized siRNA targeting GRP94 gene and transfected into MCF7 cells used by Liopfectamine RNAIMAX. The mRNA and protein expression levels of GRP94,cyclin D1,Bax and Bcl-2 were detected by Realtime PCR and Western blot. CCK8 assay was used to detect the effect of specific GRP94 siRNA on cell proliferation and the effect on cell cycle and apoptosis were analyzed by flow cytometry and Hoechst 33258 staining. Results: Compared with the siRNA-NC cells,the expression of GRP94 was significantly down-regulated in MCF7 cells. Knockdown of GRP94 in MCF7 cells decreased cell proliferation and promoted cell apoptosis. The expression of cyclin D1 and Bcl-2 levels were significantly reduced,and Bax level was increased in siRNA-GRP94 MCF7 cells. Conclusion: The siRNA-mediated GRP94 silence significantly inhibits MCF7 cell proliferation,promoted cell apoptosis by down-regulating cyclin D1,Bcl-2 expression and up-regulating the Bax expression in MCF7 cells.
Objective: To determine the effects of silencing glucose regulated protein( GRP94) on the proliferation and apoptosis of breast carcinoma MCF7 cells. Methods: Chemically synthesized siRNA targeting GRP94 gene and transfected into MCF7 cells used by Liopfectamine RNAIMAX. The mRNA and protein expression levels of GRP94,cyclin D1,Bax and Bcl-2 were detected by Realtime PCR and Western blot. CCK8 assay was used to detect the effect of specific GRP94 siRNA on cell proliferation and the effect on cell cycle and apoptosis were analyzed by flow cytometry and Hoechst 33258 staining. Results: Compared with the siRNA-NC cells,the expression of GRP94 was significantly down-regulated in MCF7 cells. Knockdown of GRP94 in MCF7 cells decreased cell proliferation and promoted cell apoptosis. The expression of cyclin D1 and Bcl-2 levels were significantly reduced,and Bax level was increased in siRNA-GRP94 MCF7 cells. Conclusion: The siRNA-mediated GRP94 silence significantly inhibits MCF7 cell proliferation,promoted cell apoptosis by down-regulating cyclin D1,Bcl-2 expression and up-regulating the Bax expression in MCF7 cells.
引文
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[2]Umekita Y,Ohi Y,Sagara Y,et al.Overexpression of cyclin D1 predicts for poor prognosis in estrogen receptor-negative breast cancer patients.[J].Int J Cancer,2002,98(3):415-418.
[3]Beeghly-Fadiel A,Lu W,Shu XO,et al.MMP9 polymorphisms and breast cancer risk:a report from the Shanghai Breast Cancer Genetics Study[J].Breast Cancer Res Treat,2011,126(2):507-513.
[4]Dersh D,Jones SM,Eletto D,et al.OS-9 facilitates turnover of nonnative GRP94 marked by Hyperglycosylation[J].Mol Biol Cell,2014,25(15):2220-2234.
[5]Takahashi H,Wang JP,Zheng HC,et al.Overexpression of GRP78and GRP94 is involved in colorectal carcinogenesis[J].Histol Histopathol,2011,26(6):663-671.
[6]Hodorova I,Rybarova S,Solar P,et al.Gp96 and its different expression in breast carcinomas[J].Neoplasma,2008,55(1):31-35.
[7]樊建军,武家艳,李海玉,等.RNAI沉默GRP94基因对人食管癌ECA109细胞迁移及侵袭能力的影响[J].第三军医大学学报,2015,12(37):1222-1225.
[8]Liu X,Lazenby AJ,Siegal GP.Signal transduction cross-talk during colorectal tumorigenesis[J].Adv Anat Pathol,2006,13(5):270-274.
[9]甄春英,于海丰,叶磊光,等.GRP94生物学特点与肿瘤的关系的研究进展[J].现代生物医学进展,2012,12(22):4380-4383.
[10]Li X,Liu Z,Yan X,et al.Induction of regulatory T cells by highdose gp96 suppresses murine liver immune hyperactivation[J].PLo S One,2013,8(7):e68997.
[11]张黎川,王佳瑞,孟强,等.GRP94表达在肺癌细胞对VP-16化疗耐药中的作用[J].大连医科大学学报,2011,3(33):211-215.
[12]Hua Y,White-Gilbertson S,Kellner J,et al.Molecular chaperone gp96 is a novel therapeutic target of multiple myeloma[J].Clin Cancer Res,2013,19(22):6242-6251.
[13]Chen WT,Ha D,Kanel G,et al.Targeted deletion of ER chaperone GRP94 in the liver results in injury,repopulation of GRP94-positive hepatocytes,and spontaneous hepatocellular carcinoma development in aged mice[J].Hepatology,2014,59(3):947-957.
[14]Peng H,Zhong XY,Liu KP,et al.Expression and significance of adenomatous polyposis coli,beta-catenin,E-cadherin and cyclin D1 inesophageal squamous cell carcinoma assessed by tissue microarray[J].Ai Zheng,2009,28(1):38-41.
[15]陈惠瑜,施腾飞,成玲,等.SU11248对白血病细胞HL-60增殖及凋亡作用的实验研究[J].中国免疫学杂志,2012,28(6):512-551.