18β-甘草次酸通过调控PIM1对宫颈癌细胞及裸鼠移植瘤自噬与凋亡的影响

Effects of 18β-glycyrrhetinic acid on autophagy and apoptosis of cervical cancer cells and transplanted tumor in nude mice by regulating PIM1

关键词:  18β-甘草次酸  宫颈癌  PIM1  分子对接

Key words:  18β-glycyrrhetinic acid  cervical cancer  PIM1  molecular docking

摘要:

[目的] 探究18β-甘草次酸（18β-GA）通过调控莫洛尼小鼠白血病病毒前病毒插入位点1（PIM1）对宫颈癌细胞及裸鼠移植瘤自噬与凋亡的影响。[方法] 以浓度梯度（50、100、150 μmol/L）18β-GA作用于宫颈癌Hela细胞，明场与免疫荧光染色观测细胞形态变化，AutoDock Vina软件对18β-GA与PIM1进行分子对接。用100 μmol/L 18β-GA处理敲低及过表达PIM1的Hela细胞，四甲基偶氮唑盐（MTT）比色法检测增殖抑制率，蛋白印迹检测蛋白表达变化。以Hela细胞构建裸鼠宫颈癌移植瘤模型，成瘤后随机分为18β-GA实验组与对照组，每日分别灌胃100 mg/kg 18β-GA与等量生理盐水溶液。14 d后取移植瘤切片，行苏木精-伊红（HE）染色对比两组移植瘤组织形态，免疫荧光染色与蛋白印迹检测对比移植瘤蛋白表达水平。[结果] 与对照组相比，随着18β-GA作用浓度的增加，PIM1、B细胞淋巴瘤-2（BCL-2）表达降低，自噬蛋白Beclin-1表达升高，细胞增殖抑制率增高（P<0.05）。敲低PIM1与敲低PIM1后加18β-GA处理组比较细胞增殖抑制率、PIM1、Beclin-1与BCL-2表达水平无明显差异（P>0.05），而过表达PIM1与过表达PIM1后加18β-GA处理组比较上述结果有显著差异（P<0.05）。18β-GA可以与PIM1稳定结合于PHE-100，结合能为 -7.3 kcal/mol。18β-GA可抑制裸鼠移植瘤组织中PIM1与白细胞介素-6（IL-6）表达，降低信号转导和转录激活因子3（STAT3）磷酸化，进而抑制增殖细胞核抗原（PCNA）、抗凋亡蛋白BCL-2表达，增加Beclin-1表达（P<0.05）。[结论] 18β-GA通过抑制PIM1进而下调BCL-2促进Beclin-1表达，促进宫颈癌细胞自噬与凋亡，减少裸鼠移植瘤STAT3磷酸化，降低PCNA、IL-6表达，进而发挥对Hela宫颈癌小鼠的抑瘤作用。

Abstract:

[Objective] To investigate the effects of 18β-glycyrrhetinic acid（18β-GA） on autophagy and apoptosis of cervical cancer cells and transplanted tumor in nude mice by regulating provirus integration site for moloney murine leukemia virus-1（PIM1）. [Methods] Hela cells were subjected to 18β-GA with concentration gradients（50 μmol/L，100 μmol/L，150 μmol/L），and the morphological changes of the cells were observed by bright field and immunofluorescence staining. The molecular docking of 18β-GA with PIM1 was performed using AutoDock Vina software. Hela cells with PIM1 knock down and overexpressing were treated with 100 μmol/L 18β-GA，the proliferation inhibition rate was detected by MTT method，and the protein expression of Hela cells was detected by western blot. Cervical cancer transplantation tumor model of nude mice was established with cervical cancer Hela cells，and after the tumor formation the mice were randomly divided into 18β-GA experimental group and control group，and 100 mg/kg 18β-GA and the same amount of normal saline solution were administered daily，respectively. After 14 days，the grafted tumor sections were taken，and the tissue morphology of the grafted tumor was compared by HE staining，and the protein expression level of the grafted tumor was compared by immunofluorescence staining and Western blot detection. [Results] Compared with the control group，with the increase of 18β-GA concentration，the expressions of PIM1 and B-cell lymphoma-2（BCL-2） were decreased，the expression of autophagy protein Beclin-1 was increased，and the cell proliferation inhibition rate was increased（P<0.05）. There were no significant differences in cell proliferation inhibition rate，expression levels of PIM1，Beclin-1 and BCL-2 after knocking down PIM1 followed by treating with 18β-GA（P>0.05），but there were significant differences between overexpression of PIM1 and overexpression of PIM1 were followed by addition of 18β-GA（P<0.05）. 18β-GA can be stably bound with PIM1 to PHE-100 with a binding energy of -7.3 kcal/mol. Transplanted tumor detection showed that 18β-GA inhibited the expression of PIM1 and interleukin-6（IL-6），decreased the phosphorylation of signal transducer and activator of transcription 3（STAT3），inhibited the expression of proliferation-related protein proliferating cell nuclear antigen（PCNA），anti-apoptotic protein BCL-2，and increased the expression of Beclin-1（P<0.05）. [Conclusion] By inhibiting PIM1 and then down-regulating BCL-2，18β-GA promotes Beclin-1 expression，promotes autophagy and apoptosis of cervical cancer cells，reduces STAT3 phosphorylation and decreases PCNA and IL-6 expression in transplanted tumor of nude mice，and thus plays a tumor suppressive effect on Hela cervical cancer mice.