五味子活性物质五味子乙素联合KLF6基因调控宫颈癌细胞增殖和凋亡的机制研究

Schisandra chinensis active component Schisandrin B combined with KLF6 gene processing in regulating proliferation and apoptosis of cervical cancer cells: a mechanistic study

目的探讨五味子乙素(SchB)单独和联合Kruppel样因子6(KLF6)重组质粒对宫颈癌细胞增殖和凋亡的影响及机制。方法以人正常宫颈上皮细胞End1/E6E7为对照,通过免疫印迹检测人宫颈癌HeLa、Siha、Caski和C33a细胞KLF6蛋白表达。将KLF6重组质粒(pcDNA3.1-KLF6)转染Caski细胞,免疫印迹检测转染效果。25、50、100和200μmol/L的SchB处理Caski细胞48h,MTT法检测细胞增殖活力。将Caski细胞分为空白组、SchB组、pcDNA3.1-KLF6组和SchB+pcDNA3.1-KLF6组,通过MTT、流式细胞术及免疫印迹分别检测4组细胞增殖活力、凋亡率和PI3K、p-AKT、PCNA和survivin蛋白表达。结果KLF6在HeLa、Siha、Caski和C33a细胞表达均明显低于在End1/E6E7细胞表达(P<0.05)。pcDNA3.1-KLF6组KLF6表达明显高于空白组(P<0.05)。不同浓度SchB均可降低Caski细胞活力,与0μmol/LSchB组比较差异有统计学意义(P<0.05)。SchB及pcDNA3.1-KLF6均可抑制Caski细胞增殖活力,诱导细胞凋亡,抑制PI3K、p-AKT、PCNA和survivin蛋白表达,两者联合对细胞增殖活力、凋亡及PI3K、p-AKT、PCNA和survivin蛋白表达影响更显著(P<0.05)。结论SchB及KLF6基因均可抑制宫颈癌细胞增殖,诱导细胞凋亡,两者联合对细胞增殖和凋亡影响更显著,机制可能与抑制PI3K/AKT信号通路有关.

Objective To investigate the effect and mechanism of Schisandrin B (SchB) alone or in combination with Kruppel-like factor 6 (KLF6) recombinant plasmid on proliferation and apoptosis of cervical cancer cells. Methods The expression of KLF6 protein in several human cervical cancer cell-lines (HeLa, Siha, Caski and C33a cells) was detected by Western blotting using human normal cervical epithelial cells End1/E6E7 as control. KLF6 recombinant plasmid (pcDNA3.1-KLF6) was transfected into Caski cells, and the transfection was validated by Western blotting. After the Caski cells were treated with SchB at levels of 25, 50, 100 and 200 μmol/L for 48 h, the cell proliferation was examined by MTT assay. The Caski cells then were divided into blank group, SchB group, pcDNA3.1-KLF6 group and SchB+pcDNA3.1-KLF6 group. The proliferation, apoptosis and protein levels of PI3K p-AKT, PCNA and survivin in the four groups were detected by MTT, flow cytometry and Western blotting, respectively. Results The expression of KLF6 in HeLa, Siha, Caski and C33a cells was significantly lower than that in End1/E6E7 cells (P<0.05). The expression of KLF6 in pcDNA3.1-KLF6 group was significantly higher than that in the blank group (P<0.05). Different concentrations of SchB were shown to decrease the activity of Caski cells, with statistically significant differences compared with 0μmol/L SchB group (P<0.05). Either SchB or pcDNA3.1-KLF6 alone may inhibited the proliferation, induced apoptosis, and inhibited the expression levels of PI3K, p-AKT, PCNA and survivin proteins, of Caski cells. When used in combination, the both exhibited more significant effects on cell proliferation, apoptosis and expression levels of PI3K, p-AKT, PCNA and survivin proteins (P<0.05). Conclusion Both SchB and KLF6 genes may inhibit the proliferation and induce apoptosis of cervical cancer cells. Such effect is more significant when the both are used in combination. The underlying mechanism may be related to the inhibition of PI3K/AKT signaling pathway.