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.%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.
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