p53/p21信号转导通路在石英诱导人胚肺成纤维细胞恶性转化中的作用
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摘要
目的观察石英诱导恶性转化的人胚肺成纤维细胞(T-HELF)中p53蛋白的表达量,并探讨p53蛋白对p21、细胞周期蛋白D1和细胞周期蛋白依赖激酶4(CDK4)蛋白表达量及蛋白间相互作用的影响。方法利用胞浆胞核分离技术,分别提取人胚肺成纤维细胞(HELF)、T-HELF胞浆和胞核蛋白,运用Western blot检测p53、磷酸化p53及p21蛋白在细胞中的表达量及分布。利用RNA干扰技术,在T-HELF中转染2μg p53 siRNA,同时设立转染CMV-neo空白载体质粒的对照组,观察p53、磷酸化p53、p21、细胞周期蛋白D1和CDK4的蛋白表达量及改变。用免疫沉淀方法检测HELF和T-HELF中p21、细胞周期蛋白D1及CDK4蛋白结合物水平的变化,并在T-HELF中分别加入20μmol/L p53化学抑制剂pifithrin-α(PFT-α)和2μg p53转染质粒,观察其对p21、细胞周期蛋白D1及CDK4蛋白间相互作用的影响。结果石英刺激HELF可引起胞核中p53及磷酸化p53蛋白表达量显著增高(P<0.05),T-HELF胞核中p53的蛋白表达量较HELF显著降低(P<0.05)。转染p53 siRNA后,p53蛋白与对照组相比表达下降,p21及细胞周期蛋白D1蛋白表达量增加,差异均有统计学意义(P<0.05),尚未观察到CDK4蛋白表达量的变化(P>0.05)。免疫沉淀结果显示抑制p53的表达可下调p21与细胞周期蛋白D1蛋白复合物的表达量(P<0.05),但尚未观察到其对p21、细胞周期蛋白D1与CDK4蛋白结合的影响(P>0.05)。结论 p53可通过调控p21、细胞周期蛋白D1的蛋白表达和蛋白间相互作用参与石英诱导的恶性转化过程。
OBJECTIVE To observe the expression of p53 protein and investigate the roles of p53 in the expressions and interactions of p21, cyclin D1 and cyclin dependent kinase 4(CDK4) proteins in malignant transformation of human embryonic lung fibroblasts(T-HELF) induced by quartz. METHODS The cytosolic protein and nuclear protein of both HELF and T-HELF cells were extracted by the separation technique of cytoplasm and nuclei. The distribution and expression of p53, phosphorylated p53 and p21 proteins were detected by Western blot. Based on the RNA interference technique, p53 siRNA was transfected into T-HELF cells to observe the protein expression and change of p53, phosphorylated p53, p21, cyclin D1 and CDK4, while the control group was conducted by transfecting the CMV-neo blank vector into the plasmid. The expression levels of p21, cyclin D1 and CDK4 protein complex in HELF and T-HELF cells were detected by immunoprecipitation. After adding 20 μmol/L of p53 chemical inhibitor pifithrin-α(PFT-α) and 2 μg of p53 siRNA into T-HELF cells respectively, the effect of p53 protein inhibition on p21, cyclin D1 and CDK4 protein complex was also observed. RESULTS Quartz stimulation of HELF caused a significant increase in the expression of p53 and phosphorylated p53 protein in the nucleus(P<0.05). The protein expression of p53 in the nucleus of T-HELF was significantly lower than that of HELF(P<0.05). After transfection of p53 siRNA, the expression of p53 protein was decreased and the expression of p21 and cyclin D1 protein was increased compared with the control group(P<0.05), while the change of expression in CDK4 was not observed(P>0.05). Additionally, the result of immunoprecipitation showed that the inhibition of p53 expression could down-regulate the expression level of the binding complex between p21 and cyclin D1 protein(P<0.05). However, this effect on p21-CDK4 and cyclin D1-CDK4 protein complex was not observed(P>0.05). CONCLUSION By regulating the expression and protein-protein interaction between p21 and cyclin D1, p53 would participate in quartz-induced malignant transformation.
OBJECTIVE To observe the expression of p53 protein and investigate the roles of p53 in the expressions and interactions of p21, cyclin D1 and cyclin dependent kinase 4(CDK4) proteins in malignant transformation of human embryonic lung fibroblasts(T-HELF) induced by quartz. METHODS The cytosolic protein and nuclear protein of both HELF and T-HELF cells were extracted by the separation technique of cytoplasm and nuclei. The distribution and expression of p53, phosphorylated p53 and p21 proteins were detected by Western blot. Based on the RNA interference technique, p53 siRNA was transfected into T-HELF cells to observe the protein expression and change of p53, phosphorylated p53, p21, cyclin D1 and CDK4, while the control group was conducted by transfecting the CMV-neo blank vector into the plasmid. The expression levels of p21, cyclin D1 and CDK4 protein complex in HELF and T-HELF cells were detected by immunoprecipitation. After adding 20 μmol/L of p53 chemical inhibitor pifithrin-α(PFT-α) and 2 μg of p53 siRNA into T-HELF cells respectively, the effect of p53 protein inhibition on p21, cyclin D1 and CDK4 protein complex was also observed. RESULTS Quartz stimulation of HELF caused a significant increase in the expression of p53 and phosphorylated p53 protein in the nucleus(P<0.05). The protein expression of p53 in the nucleus of T-HELF was significantly lower than that of HELF(P<0.05). After transfection of p53 siRNA, the expression of p53 protein was decreased and the expression of p21 and cyclin D1 protein was increased compared with the control group(P<0.05), while the change of expression in CDK4 was not observed(P>0.05). Additionally, the result of immunoprecipitation showed that the inhibition of p53 expression could down-regulate the expression level of the binding complex between p21 and cyclin D1 protein(P<0.05). However, this effect on p21-CDK4 and cyclin D1-CDK4 protein complex was not observed(P>0.05). CONCLUSION By regulating the expression and protein-protein interaction between p21 and cyclin D1, p53 would participate in quartz-induced malignant transformation.
引文
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[12] 张夏男,李跃纲,贾效伟,等.p53在苯并[a]芘诱导所致p21和细胞周期蛋白改变中的作用[J].卫生研究,2014,43(2):193-197.
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[14] 刘秉慈,关然,周培宏,等.石英的人类致癌性在DNA分子水平的证据[J].卫生研究,1999,28(5):257-258.
[15] 邹向阳,李连宏.细胞周期调控与肿瘤[J].国际遗传学杂志,2006,29(1):72-75.
[16] WANG Y G,XU L,WANG T,et al.Givinostat inhibition of hepatic stellate cell proliferation and protein acetylation[J].World J Gastroenterol,2015,21(27):8326-8339.
[17] GONG Y F,ZHANG X M,LIU F,et al.Inhibitory effect of recombinant human endostatin on the proliferation of hypertrophic scar fibroblasts in a rabbit ear model[J].Eur J Pharmacol,2016,791:647-654.
[18] SUGIMOTO M,MARTIN N,WILKS D P,et al.Activation of cyclin D1-kinase in murine fibroblasts lacking both p21Cip1 and p27Kip1[J].Oncogene,2002,21(53):8067-8074.
[19] VOUSDEN K H,LANE D P.p53 in health and disease[J].Nat Rev Mol Cell Biol,2007,8(4):275-283.
[20] ZAMIR-NASTA T,RAZI M,SHAPOUR H,et al.Roles of p21,p53,cyclin D1,CDK-4,estrogen receptor α in aflatoxin B1-induced cytotoxicity in testicular tissue of mice[J].Environ Toxicol,2018,33(4):385-395.
[21] EL-DEIRY W S.p21(WAF1) mediates cell-cycle Inhibition,relevant to cancer suppression and therapy[J].Cancer Res,2016,76(18):5189-5191.
[22] GAUTSCHI O,HUGLI B,ZIEGLER A,et al.Cyclin D1 (CCND1) A870G gene polymorphism modulates smoking-induced lung cancer risk and response to platinum-based chemotherapy in non-small cell lung cancer (NSCLC) patients[J].Lung Cancer,2006,51(3):303-311.
[2] CASTRANOVA V,VALLYATHAN V.Silicosis and coal workers’ pneumoconiosis[J].Environ Health Perspect,2000,108(4):675-684.
[3] RONG Y,SHEN Y,ZHANG Z,et al.Blocking TGF-β expression inhibits silica particle-induced epithelial-mesenchymal transition in human lung epithelial cells[J].Environ Toxicol Pharmacol,2015,40(3):861-869.
[4] STEENLAND K,WARD E.Silica:a lung carcinogen[J].CA Cancer J Clin,2014,64(1):63-69.
[5] PALABIYIK S S,GIRGIN G,TUTKUN E,et al.Immunomodulation and oxidative stress in denim sandblasting workers:changes caused by silica exposure[J].Arh Hig Rada Toksikol,2013,64(3):431-437.
[6] MEYER K C.Pulmonary fibrosis,part I:epidemiology,pathogenesis,and diagnosis[J].Exp Rev Respir Med,2017,11(5):343-359.
[7] QIE S,DIEHL J A.Cyclin D1,cancer progression,and opportunities in cancer treatment[J].J Mol Med,2016,94(12):1313-1326.
[8] 詹启敏,陈杰.细胞周期与肿瘤转化医学[J].中国肿瘤临床,2014,41(1):1-7.
[9] 张凤梅,刘秉慈,刘海峰,等.p53表达在石英诱导的细胞周期改变及DNA损伤修复中的作用[J].中华劳动卫生职业病杂志,2010,28(4):246-249.
[10] 闫克夏,刘秉慈,史香林,等.细胞周期蛋白依赖激酶K4在石英致人细胞恶性转化中的作用[J].中华劳动卫生职业病杂志,2004,22(5):331-335.
[11] 张夏男,李跃纲,贾效伟,等.铜蓝蛋白在石英诱导的PI3K/PTEN信号通路中的作用[J].中华劳动卫生职业病杂志,2014,32(4):241-245.
[12] 张夏男,李跃纲,贾效伟,等.p53在苯并[a]芘诱导所致p21和细胞周期蛋白改变中的作用[J].卫生研究,2014,43(2):193-197.
[13] 叶萌,刘秉慈,贾效伟,等.苯并(a)芘通过活化蛋白-1非依赖性通路诱导细胞中p53蛋白过表达及高磷酸化[J].卫生研究,2008,37(3):255-258.
[14] 刘秉慈,关然,周培宏,等.石英的人类致癌性在DNA分子水平的证据[J].卫生研究,1999,28(5):257-258.
[15] 邹向阳,李连宏.细胞周期调控与肿瘤[J].国际遗传学杂志,2006,29(1):72-75.
[16] WANG Y G,XU L,WANG T,et al.Givinostat inhibition of hepatic stellate cell proliferation and protein acetylation[J].World J Gastroenterol,2015,21(27):8326-8339.
[17] GONG Y F,ZHANG X M,LIU F,et al.Inhibitory effect of recombinant human endostatin on the proliferation of hypertrophic scar fibroblasts in a rabbit ear model[J].Eur J Pharmacol,2016,791:647-654.
[18] SUGIMOTO M,MARTIN N,WILKS D P,et al.Activation of cyclin D1-kinase in murine fibroblasts lacking both p21Cip1 and p27Kip1[J].Oncogene,2002,21(53):8067-8074.
[19] VOUSDEN K H,LANE D P.p53 in health and disease[J].Nat Rev Mol Cell Biol,2007,8(4):275-283.
[20] ZAMIR-NASTA T,RAZI M,SHAPOUR H,et al.Roles of p21,p53,cyclin D1,CDK-4,estrogen receptor α in aflatoxin B1-induced cytotoxicity in testicular tissue of mice[J].Environ Toxicol,2018,33(4):385-395.
[21] EL-DEIRY W S.p21(WAF1) mediates cell-cycle Inhibition,relevant to cancer suppression and therapy[J].Cancer Res,2016,76(18):5189-5191.
[22] GAUTSCHI O,HUGLI B,ZIEGLER A,et al.Cyclin D1 (CCND1) A870G gene polymorphism modulates smoking-induced lung cancer risk and response to platinum-based chemotherapy in non-small cell lung cancer (NSCLC) patients[J].Lung Cancer,2006,51(3):303-311.
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