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宫颈癌是最常见的妇科恶性肿瘤之一,其发病率和病死率逐年升高,已成为威胁女性健康的主要问题。目前,对宫颈癌患者的治疗主要包括手术、放疗和化疗[1]。然而,放疗和化疗在某些情况下并不有效,患者的5年生存率只有40%~50%[2]。并且由于一些肿瘤细胞对X射线的敏感性降低,导致对宫颈癌患者的治疗远远达不到预期的效果[3]。因此,探讨宫颈癌细胞的耐辐射机制,探索新的治疗策略以改善患者的预后已成为当务之急。
异紫堇碱(isocorydine,ICD)是一种阿朴啡类生物碱,广泛存在于秃疮花[Dicranostigma leptopodum(Maxim.)Fedde]、紫金龙[Dactylicapnos scandens(D.Don)Hutchins]等多种罂粟科植物中,临床用于治疗痉挛和血管舒张,以及抗疟原虫和抗心律失常等治疗[4]。既往研究结果表明,ICD可以通过诱导宫颈癌SiHa细胞的细胞周期阻滞,抑制细胞增殖、侵袭和转移,调控上皮间充质转化来抑制宫颈癌细胞的生长,发挥抗癌活性的作用[5]。
最近的研究结果显示,微小RNA(microRNA,miRNA)参与调节不同癌细胞的辐射敏感性,并在癌症的发生和发展中起着至关重要的作用[6];miR-129-5p在宫颈癌组织中低表达,其表达的上调可以抑制宫颈癌细胞的侵袭、迁移以及荷瘤裸鼠体内肿瘤的生长和血管生成能力[7];知母皂苷A-Ⅲ抑制肾癌细胞转移的特性,有助于抑制磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)/蛋白激酶B(protein kinase B,Akt)通路,提高miR-129-5p的表达,以及下调组织蛋白酶C的表达[8]。然而ICD在宫颈癌辐射敏感性中的作用及其是否通过上调miR-129-5p和抑制PI3K/Akt信号通路来发挥作用尚未可知。
本研究探讨ICD对宫颈癌SiHa细胞增殖、辐射敏感性的影响及其潜在的分子机制,为宫颈癌治疗药物的研发提供新思路。
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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、200 μmol/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(均P<0.05)(图1)。
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与NC组比较,100 μmol/L ICD处理组能够明显降低2、4、6、8 Gy X射线照射的SiHa细胞的存活分数(均P<0.05,图2A),且提高γ-H2AX蛋白的相对表达量(P<0.05,图2B),细胞的SER为1.12(表1)。
图 2 ICD对宫颈癌SiHa细胞辐射敏感性的影响
Figure 2. Effect of isocorydine on the radiosensitivity of cervical cancer SiHa cells
组别 D0(Gy) Dq(Gy) N SF2 k SER(D0比值) 对照组 2.305 2.488 2.943 0.799 0.434 - ICD处理组 2.058 0.562 0.761 0.304 0.486 1.12 注:D0为平均致死剂量;Dq为准闭剂量;N为外推值;SF2为照射剂量为2 Gy时的细胞存活分数;k为细胞存活曲线的钝化常数;SER为放射增敏比;ICD为异紫堇碱;-表示无此项数据 表 1 ICD联合X射线照射对宫颈癌SiHa细胞作用的单击 多靶模型的参数值
Table 1. Statistical results of single-click multi-target model of the effect of isocorydine combined with X-ray irradiation on cervical cancer SiHa cells
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qRT-PCR检测结果显示,与NC组比较,ICD处理组能够明显提高SiHa细胞中miR-129-5p的相对表达量(3.22±0.32对1.00±0.11,t=19.682,P<0.05)。
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由图3可见,与转染miR-NC组比较,转染miR-129-5p组能够明显提高SiHa细胞中miR-129-5p的相对表达量(P<0.05,图3A),这表明高表达miR-129-5p的细胞已构建成功;与转染miR-NC组比较,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)%,P<0.05,图3B],降低2、4、6、8 Gy X射线照射后细胞的存活分数(均P<0.05,图3C),降低cyclinD1蛋白的相对表达量(P<0.05,图3D),并提高γ-H2AX蛋白的相对表达量(P<0.05,图3E),细胞的SER为1.68(表2)。
图 3 高表达miR-129-5p对宫颈癌SiHa细胞的增殖和辐射敏感性的影响
Figure 3. Effect of high expression of miR-129-5p on the proliferation and radiosensitivity of cervical cancer SiHa cells
组别 D0
(Gy)Dq
(Gy)N SF2 k SER
(D0比值)转染miR-NC组 2.329 2.275 2.655 0.769 0.429 - 转染miR-129-5p组 1.383 0.893 1.907 0.401 0.723 1.68 注:D0为平均致死剂量;Dq为准闭剂量;N为外推值;SF2为照射剂量为2 Gy时的细胞存活分数;k为细胞存活曲线的钝化常数;SER为放射增敏比;NC为对照;-表示无此项数据 表 2 高表达miR-129-5p联合X射线照射对宫颈癌SiHa细 胞作用的单击多靶模型的参数值
Table 2. Statistical results of single-click multi-target model of the effects of miR-129-5p overexpression combined with X-ray irradiation on cervical cancer SiHa cells
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由图4可见,与转染anti-miR-NC+ICD处理组比较,转染anti-miR-129-5p(低表达miR-129-5p)+ICD处理组能够明显降低SiHa细胞中miR-129-5p的相对表达量(P<0.05,图4A)、细胞的增殖抑制率[(21.09±2.11)%对(71.49±7.15)%,P<0.05,图4B],能够提高2、4、6、8 Gy X射线照射后细胞的存活分数(均P<0.05,图4C)、cyclinD1蛋白的相对表达量(P<0.05,图4D),并降低γ-H2AX蛋白的相对表达量(P<0.05,图4E),细胞的SER为0.83(表3)。
图 4 低表达miR-129-5p对ICD处理的SiHa细胞的增殖和辐射敏感性的影响
Figure 4. Effect of low expression of miR-129-5p on the proliferation and radiosensitivity of cervical cancer SiHa cells treated with isocorydine
组别 D0(Gy) Dq(Gy) N SF2 k SER(D0比值) 转染anti-miR-NC+ICD处理组 2.144 0.709 0.718 0.302 0.467 - 转染anti-miR-129-5p+ICD处理组 2.569 1.199 1.595 0.625 0.389 0.83 注:D0为平均致死剂量;Dq为准闭剂量;N为外推值;SF2为照射剂量为2 Gy时的细胞存活分数;k为细胞存活曲线的钝化常数;SER为放射增敏比;NC为对照;ICD为异紫堇碱;-表示无此项数据 表 3 低表达miR-129-5p联合X射线照射对ICD处理的宫颈癌SiHa细胞作用的单击多靶模型的参数值
Table 3. Statistical results of single-click multi-target model of the effect of miR-129-5p underexpression combined with X-ray irradiation on cervical cancer SiHa cells treated with isocorydine
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由图5可见,与NC组比较,ICD处理组能够明显降低SiHa细胞的p-PI3K、p-Akt蛋白的相对表达量(均P<0.05);与转染anti-miR-NC+ICD处理组比较,转染anti-miR-129-5p(低表达miR-129-5p)+ICD处理组能够显著提高SiHa细胞中p-PI3K、p-Akt蛋白的相对表达量(均P<0.05)。
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
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摘要:
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、200 μmol/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、200 μmol/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,100 μmol/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。 Abstract: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 200 μmol/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, and γ-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 200 μmol/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 100 μmol/L ICD treatment group had increased radiosensitivity to different doses of X-rays (t=19.135−44.478, all P<0.05), increased γ-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, all P<0.05), and γ-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. -
表 1 ICD联合X射线照射对宫颈癌SiHa细胞作用的单击 多靶模型的参数值
Table 1. Statistical results of single-click multi-target model of the effect of isocorydine combined with X-ray irradiation on cervical cancer SiHa cells
组别 D0(Gy) Dq(Gy) N SF2 k SER(D0比值) 对照组 2.305 2.488 2.943 0.799 0.434 - ICD处理组 2.058 0.562 0.761 0.304 0.486 1.12 注:D0为平均致死剂量;Dq为准闭剂量;N为外推值;SF2为照射剂量为2 Gy时的细胞存活分数;k为细胞存活曲线的钝化常数;SER为放射增敏比;ICD为异紫堇碱;-表示无此项数据 表 2 高表达miR-129-5p联合X射线照射对宫颈癌SiHa细 胞作用的单击多靶模型的参数值
Table 2. Statistical results of single-click multi-target model of the effects of miR-129-5p overexpression combined with X-ray irradiation on cervical cancer SiHa cells
组别 D0
(Gy)Dq
(Gy)N SF2 k SER
(D0比值)转染miR-NC组 2.329 2.275 2.655 0.769 0.429 - 转染miR-129-5p组 1.383 0.893 1.907 0.401 0.723 1.68 注:D0为平均致死剂量;Dq为准闭剂量;N为外推值;SF2为照射剂量为2 Gy时的细胞存活分数;k为细胞存活曲线的钝化常数;SER为放射增敏比;NC为对照;-表示无此项数据 表 3 低表达miR-129-5p联合X射线照射对ICD处理的宫颈癌SiHa细胞作用的单击多靶模型的参数值
Table 3. Statistical results of single-click multi-target model of the effect of miR-129-5p underexpression combined with X-ray irradiation on cervical cancer SiHa cells treated with isocorydine
组别 D0(Gy) Dq(Gy) N SF2 k SER(D0比值) 转染anti-miR-NC+ICD处理组 2.144 0.709 0.718 0.302 0.467 - 转染anti-miR-129-5p+ICD处理组 2.569 1.199 1.595 0.625 0.389 0.83 注:D0为平均致死剂量;Dq为准闭剂量;N为外推值;SF2为照射剂量为2 Gy时的细胞存活分数;k为细胞存活曲线的钝化常数;SER为放射增敏比;NC为对照;ICD为异紫堇碱;-表示无此项数据 -
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