Qian Yuanyuan, Zhang Lin, Zhu Yuling, Zhang Wencui, Niu Yong, Liu Kai, Ye Meng
National Institute of Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention, Beijing 100050, China.
Wei Sheng Yan Jiu. 2019 Mar;48(2):187-192.
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.
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.
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).
By regulating the expression and protein-protein interaction between p21 and cyclin D1, p53 would participate in quartz-induced malignant transformation.
观察p53蛋白的表达情况，探讨p53在石英诱导人胚肺成纤维细胞（T-HELF）恶性转化过程中对p21、细胞周期蛋白D1（cyclin D1）及细胞周期蛋白依赖性激酶4（CDK4）蛋白表达及相互作用的影响。
采用细胞质与细胞核分离技术提取HELF和T-HELF细胞的胞质蛋白和核蛋白。通过蛋白质免疫印迹法检测p53、磷酸化p53及p21蛋白的分布及表达。基于RNA干扰技术，将p53小干扰RNA（siRNA）转染至T-HELF细胞，观察p53、磷酸化p53、p21、cyclin D1及CDK4蛋白表达及变化，对照组转染CMV-neo空载体质粒。采用免疫沉淀法检测HELF和T-HELF细胞中p21、cyclin D1及CDK4蛋白复合物的表达水平。分别向T-HELF细胞中加入20 μmol/L的p53化学抑制剂pifithrin-α（PFT-α）及2 μg p53 siRNA，观察抑制p53蛋白对p21、cyclin D1及CDK4蛋白复合物的影响。
石英刺激HELF细胞后，细胞核中p53及磷酸化p53蛋白表达显著增加（P<0.05）。T-HELF细胞核中p53蛋白表达显著低于HELF（P<0.05）。转染p53 siRNA后，与对照组相比，p53蛋白表达降低，p21及cyclin D1蛋白表达增加（P<0.05），而CDK4表达未见变化（P>0.05）。此外，免疫沉淀结果显示，抑制p53表达可下调p21与cyclin D1蛋白结合复合物的表达水平（P<0.05）。但对p21-CDK4及cyclin D1-CDK4蛋白复合物未见此作用（P>0.05）。
p53可通过调节p21与cyclin D1的表达及蛋白-蛋白相互作用参与石英诱导的恶性转化。
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