清毒栓提取物通过调节Foxp3影响β-catenin通路从而抑制SiHa细胞活性的研究

Study on the inhibition of SiHa cell activity by regulating Foxp3 and affecting the β-catenin pathway with the extract of Qingdushuan

关键词:  Foxp3过表达质粒  清毒栓  宫颈癌  SiHa细胞  β-catenin通路

Key words:  Foxp3 overexpressed plasmid  Qingdushuan  cervical cancer  SiHa cell  β-catenin pathway

摘要:

[目的] 考察清毒栓提取物对宫颈癌SiHa细胞活性的影响，并基于叉状头/翅膀状螺旋转录因子（Foxp3）探讨其可能的作用机制。[方法] 利用脂质体介导转染方法，建立Foxp3过表达人乳头瘤病毒（HPV）感染阳性SiHa细胞株，同时利用pcDNA3.1空载体转染SiHa细胞株作为阴性对照。用蛋白免疫印迹（Western Blot）法检测经空白血清和15%含药血清分别处理的Foxp3过表达质粒组及空白质粒组SiHa细胞中Foxp3蛋白及其下游信号分子水平，并用CCK8法检测4组SiHa细胞活性。[结果] Foxp3过表达载体转染处理可以显著增加SiHa细胞中Foxp3蛋白、Foxp3下游信号分子β-连环蛋白（β-catenin）、c-Myc、细胞周期蛋白D1（cyclinD1）的蛋白含量并增加SiHa细胞活性（P<0.01），而抑制凋亡基因含半胱氨酸的天冬氨酸蛋白水解酶-3（Caspase-3）剪切（P<0.05）；清毒栓含药血清可以显著抑制SiHa细胞中Foxp3及其下游信号分子蛋白水平（P<0.05）并抑制SiHa细胞活性（P<0.01），促进Caspase-3剪切（P<0.01）。Foxp3过表达质粒可以抑制含药血清诱导的SiHa细胞中Foxp3下调和活性下降，抑制含药血清诱导的β-catenin、c-Myc、cyclinD1含量下调以及Caspase-3剪切增加。[结论] Foxp3可通过激活β-catenin通路来增加SiHa细胞活性，在SiHa细胞中扮演着原癌基因角色，而清毒栓含药血清通过调节Foxp3影响β-catenin通路从而抑制SiHa细胞活性。

Abstract:

[Objective] To investigate the effect of Qingdushuan on the activity of SiHa cell in cervical cancer,and to explore its possible mechanism based on forkhead/winged helix transcription factor (Foxp3).[Methods] The method of Liposome-mediated transfection was used to establish Foxp3 overexpressed human papilloma virus (HPV) -positive SiHa cell lines,and pcDNA3.1 empty vector transfected SiHa cell lines were as the negative control. Western Blot method was used to detect the levels of Foxp3 proteins and their downstream signaling molecules in the Foxp3 overexpression plasmid group and blank plasmid group SiHa cells treated with empty white blood and 15% drug-containing serum respectively. CCK8 method was used to detect SiHa cell activity in the four groups.[Results] Foxp3 overexpression vector transfection treatment can significantly increase the protein content of Foxp3 protein,Foxp3 downstream signaling molecules β-catenin,proto-oncogene c-Myc,and cyclin D1 in SiHa cells. Increased SiHa cell activity (P<0.01),while inhibiting the apoptosis gene Caspase-3 shearing (P<0.05). Qingdu Suppository containing serum can significantly inhibit Foxp3 protein (P<0.01) and Foxp3 downstream signaling molecule protein (P<0.05) and inhibited SiHa cell activity (P<0.01),promoted Caspase-3 splicing (P<0.01);Foxp3 overexpression plasmid can inhibit Foxp3 down-regulation and decreased activity in SiHa cells induced by drug-containing serum. It can inhibit the down-regulation of β-catenin,c-Myc and cyclinD1 induced by medicated serum and increase of Caspase-3 shear.[Conclusion] Foxp3 can increase the activity of SiHa cells by activating the β-catenin pathway,which plays the role of proto-oncogene in SiHa cells. However,serum of Qingdushuan can inhibit the activity of SiHa cells by regulating Foxp3 to affect the β-catenin pathway.