表皮生长因子对Hela 细胞紧密连接蛋白3 表达的影响及其可能机制

Effect of epidermal growth factor on expression of Claudin-3 in Hela cells

摘要:

目的 探讨表皮生长因子（EGF）对宫颈癌Hela 细胞紧密连接蛋白3（Claudin-3）表达的影响及 其可能机制。方法 培养Hela 细胞至贴壁，接种至3 组6 孔板，分别进行如下处理：①以0.0、0.5、5.0、10.0 及100.0 ng/ml EGF 处理8 h，测评不同浓度EGF 对Claudin-3 蛋白及其信使RNA（mRNA）的影响；②培养基 中加入10 ng/ml EGF，分别于培养0 min、10 min、30 min、1 h、2 h、4 h、8 h、24 h，测评Claudin-3 蛋白 及mRNA 水平、EGF 相关作用通路各蛋白磷酸化情况；③分别以EGF 受体（EGFR）、磷脂酰肌醇-3- 激 酶（PI3-K）、促分裂素原活化蛋白激酶（MAPK）、p38、c-Jun 氨基末端激酶（JNK）阻断剂干预1 h，随后 加入含EGF 10 ng/ml 的培养基培养，测评Claudin-3 蛋白及mRNA 水平。结果 EGF 对Hela 细胞Claudin-3 蛋白及mRNA 的表达有促进作用，且10 ng/ml EGF 的促进作用最明显，该促进作用随EGF 刺激时间的延长 而增强。EGF 刺激后，EGFR、蛋白激酶B（AKT）、细胞外调节蛋白激酶1/2（ERK1/2）、p38、JNK 均发生 磷酸化，各阻断剂均能特异性阻断EGF 诱导的上述磷酸化现象。阻断EGFR、PI3-K、MAPK、p38 及JNK 信号通路均能有效抑制Claudin-3 蛋白的表达。结论 EGF 能够增加Hela 细胞Claudin-3 的表达，其机制可 能涉及EGFR、PI3-K、MAPK、p38 及JNK 信号通路。

Abstract:

Objective To investigate the effect of epidermal growth factor (EGF) on expression of Claudin-3 in Hela cells. Methods Hela cells were cultured to adherent growth. In the first group, cells were treated with different concentration of EGF for 8 hours (0, 0.5, 5.0, 10.0, 100.0 ng/ml). In the second group, cells were treated with 10 ng/ml EGF for different time of duration (0 min, 10 min, 30 min, 1 h, 2 h, 4 h, 8 h and 24 h). In the third group, cells were treated with antagonist of EGFR, phosphatidylinositol-3-kinase (PI3-K), mitogen-activated protein kinase (MAK), p38, or c-Jun N-terminal kinase (JNK) followed by treatment of 10 ng/ml EGF for 24 h. Expression of Claudin-3 was identified on protein and mRNA level. Phosphorylation of EGF signal pathway related proteins were tested. Results EGF promoted expression of Claudin-3 in time-dependent manner, and optimistic dose was 10 ng/ml. Phosphorylation of EGFR, protein kinase B (AKT), extracellular regulated protein kinase 1/2 (ERK1/2), p38 and JNK were significantly upregulated after EGF stimulation, which was reversed by all molecular inhibitors. Blockage of EGFR, PI3-K, MAPK, p38 and JNK effectively ameliorated EGF induced increase of Claudin-3 protein. Conclusion EGF increases expression of Claudin-3 in Hela cells through EGFR, PI3-K, MAPK, p38 and JNK signaling pathways.