异紫堇碱对宫颈癌SiHa细胞增殖和辐射敏感性的影响

Effects of isocorydine on the proliferation and radiosensitivity of cervical cancer SiHa cells

目的探讨异紫堇碱(ICD)对宫颈癌SiHa细胞增殖和辐射敏感性的影响及其作用机制。方法采用不同浓度(25、50、100、200μmol/L)ICD处理宫颈癌SiHa细胞(简称ICD处理组),同时将正常培养未经ICD处理的SiHa细胞设为对照(NC)组。采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)实验检测细胞的增殖抑制率,细胞克隆形成实验检测ICD对细胞辐射敏感性的影响,Western blot检测磷酸化组蛋白H2AX(γ-H2AX)、磷酸化磷脂酰肌醇3-激酶(p-PI3K)和磷酸化蛋白激酶B(p-Akt)的相对表达量,实时荧光定量PCR(qRT-PCR)检测微小RNA-129-5p(miR-129-5p)的表达。在SiHa细胞中分别转染miR-NC和miR-129-5p,观察转染miR-129-5p对细胞增殖、辐射敏感性、细胞周期蛋白D1(cyclinD1)、γ-H2AX蛋白表达的影响。在SiHa细胞中分别转染anti-miR-NC和anti-miR-129-5p并使用ICD处理,分析其对ICD诱导的细胞增殖、辐射敏感性、PI3K/Akt信号通路的影响。2组间数据的比较采用独立样本t检验。结果MTT实验结果显示,与NC组SiHa细胞的增殖抑制率[(0.05±0.01)%]相比,25、50、100、200μmol/L ICD处理组能够明显提高SiHa细胞的增殖抑制率[(10.26±1.03)%、(22.16±2.21)%、(44.09±4.41)%、(70.88±7.09)%],且差异均有统计学意义(t=29.736~30.013,均P<0.05)。细胞克隆形成实验、Western blot和qRT-PCR结果显示,与NC组相比,100μmol/L ICD处理组能够明显提高SiHa细胞对不同剂量X射线的辐射敏感性(t=19.135~44.478,均P<0.05),提高γ-H2AX蛋白和miR-129-5p的相对表达量(t=15.041、19.682,均P<0.05),降低p-PI3K和p-Akt蛋白的相对表达量(t=14.897、15.429,均P<0.05)。与转染miR-NC组相比,转染(高表达)miR-129-5p组能够明显提高SiHa细胞的增殖抑制率[(75.06±7.51)%对(1.04±0.10)%,t=29.566,P<0.05]、对不同剂量X射线的辐射敏感性(t=13.239~37.015,均P<0.05)和γ-H2AX蛋白的相对表达量(t=17.076,P<0.05),能够降低cyclinD1蛋白的相对表达量(t=17.393,P<0.05)。与转染anti-miR-NC+ICD处理组相比,转染anti-miR-129-5p(低表达miR-129-5p)+ICD处理组可以反转ICD抑制SiHa细胞增殖、抑制PI3K/Akt信号通路的活性和增强细胞辐射敏感性的作用,且差异均有统计学意义(t=13.370~28.252,均P<0.05)。结论ICD能够通过上调miR-129-5p表达及抑制PI3K/Akt信号通路的活性来抑制宫颈癌SiHa细胞的增殖以及增强其辐射敏感性。

Objective To investigate the effects of isocorydine(ICD)on the proliferation and radiosensitivity of cervical cancer SiHa cells and determine the underlying mechanism.Methods Different concentrations(25,50,100,and 200μmol/L)of ICD were used to treat cervical cancer SiHa cells(ICD treatment group).Normal cultured SiHa cells without ICD treatment were set as the normal control(NC)group.The 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay was used to detect cell proliferation.Cell clone formation experiment was conducted to determine the radiation sensitization effect of ICD on cells.Western blot analysis was employed to detect phosphorylated histone H2AX(γ-H2AX),phosphorylated phosphatidylinositol 3-kinase(p-PI3K),and phosphorylated protein kinase B(p-Akt)expression.Real-time quantitative PCR(qRT-PCR)was used to detect the expression of microRNA-129-5p(miR-129-5p).SiHa cells were transfected with miR-NC and miR-129-5p,and the roles of transfection of miR-129-5p on cell proliferation,radiosensitivity,cyclinD1,andγ-H2AX protein expression were observed.Anti-miR-NC and anti-miR-129-5p were transfected into SiHa cells,which were then treated with ICD to evaluate the effects on ICD-induced cell proliferation,radiosensitivity,and PI3K/Akt signaling pathway.Data between two groups were compared by independent sample t test.Results The MTT assay showed that the 25,50,100,and 200μmol/L ICD treatment groups exhibited significant increase in the proliferation inhibition rate((10.26±1.03)%,(22.16±2.21)%,(44.09±4.41)%,(70.88±7.09)%)of SiHa cells compared with the NC group((0.05±0.01)%),and the differences were statistically significant(t=29.736-30.013,all P<0.05).The cell clone formation,Western blot,and qRT-PCR analyses showed that the 100μmol/L ICD treatment group had increased radiosensitivity to different doses of X-rays(t=19.135-44.478,allP<0.05),increasedγ-H2AX protein expression(t=15.041,P<0.05)and miR-129-5p expression(t=19.682,P<0.05),and decreased expression of p-PI3K and p-Akt protein compared with the NC group(t=14.897,15.429;both P<0.05).Transfected(highly expressed)miR-129-5p considerably increased the proliferation inhibition rate((75.06±7.51)%vs.(1.04±0.10)%;t=29.566,P<0.05),radiosensitivity to different doses of X-rays(t=13.239-37.015,allP<0.05),andγ-H2AX protein expression(t=17.076,P<0.05)of SiHa cells and evidently decreased the expression of the cyclinD1 protein(t=17.393,P<0.05)compared with transfected miR-NC.The transfection of anti-miR-129-5p(low expression of miR-129-5p)reversed the inhibitory effect of ICD on SiHa cell proliferation and PI3K/Akt signaling pathway and reversed its ability to enhance cell radiosensitivity compared with the transfection of anti-miR-NC,and the differences were statistically significant(t=13.370-28.252,all P<0.05).Conclusion ICD inhibits the proliferation of cervical cancer SiHa cells and enhances radiosensitivity by up-regulating the expression of miR-129-5p and the PI3K/Akt signaling pathway.