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目的:研究Hh信号通路抑制剂Cyclopamine作用Hela细胞后Gli1、FoxM1、上皮间质转化(EMT)过程相关标记物表达的改变及对其增殖、侵袭迁移能力的影响,探究Gli1、FoxM1、EMT过程的可能作用关系,以期为宫颈癌的分子靶向治疗提供多个靶点。方法:采用实时定量RT-PCR、Western Blot技术检测Hela、Siha细胞系Hh信号通路中Shh、Ptch1、Smo的表达情况,选择Hh信号通路表达较高的Hela细胞系作为后续实验细胞,利用四甲基偶氮唑蓝比色法(MTT法)检测不同浓度Cyclopamine作用Hela细胞后其增殖能力的改变;采用Transwell小室试验检测Hela细胞侵袭、迁移能力的改变,并采用实时定量RT-PCR、Western Blot技术检测Gli1、FoxM1、EMT过程标记物的表达情况。结果:(1)Hela细胞中Hh信号通路成分(Shh、Ptch1、Smo)在mRNA水平显著高于Siha细胞(P<0.01),且相应蛋白的表达强度也高于Siha细胞;(2)Cyclopamine对Hela细胞增殖的抑制作用具有时间、剂量依赖性;(3)Cyclopamine作用Hela细胞48h后,细胞侵袭试验显示,实验组侵袭细胞数较对照组明显降低(P<0.01)。细胞迁移试验显示,实验组细胞迁移细胞数较对照组明显降低(P<0.01);(4)Cyclopamine作用Hela细胞48h后,实验组Gli1、FoxM1、Vimentin在mRNA水平的表达显著低于对照组(P<0.05),且相应蛋白的表达强度也低于对照组;E-cadherin在实验组中mRNA层面的表达显著高于对照组(P<0.05),且相应蛋白的表达强度也高于对照组。结论:抑制Hh信号通路可显著降低Hela细胞系增殖、侵袭迁移能力,FoxM1很可能是Hh信号通路末端转录因子Gli1的下游靶基因,且其对宫颈癌细胞系增殖、侵袭迁移的作用很可能是通过EMT实现的。
Aims: To investigate the effects of Hh signaling pathway inhibitor Cyclopamine on the expression of Gli1, FoxM1 and EMT in Hela cells and its effect on the proliferation, invasion and migration of Hela cells, and to explore the role of Gli1, FoxM1 and EMT May play a role in the relationship with the aim of molecular targeted therapy for cervical cancer to provide multiple targets. Methods: The expression of Shh, Ptch1 and Smo in Hh signaling pathway of Hela and Siha cell lines were detected by real-time quantitative RT-PCR and Western Blot. Hela cell line with higher Hh signaling pathway was selected as the follow-up experimental cells. MTT assay was used to detect the proliferation of Hela cells treated with different concentrations of Cyclopamine. Transwell assay was used to detect the invasion and migration of Hela cells. Real-time quantitative RT-PCR, Western Blot Technology to detect the expression of Gli1, FoxM1 and EMT markers. Results: (1) The expression of Hh signaling pathway (Shh, Ptch1, Smo) in Hela cells was significantly higher than that in Siha cells (P <0.01), and the expression of Hh signaling pathway was also higher than that of Siha cells. (2) (3) Cyclopamine treatment Hela cells 48h, cell invasion assay showed that the number of invasive cells in the experimental group was significantly lower than the control group (P <0.01). (4) Cyclopamine treated Hela cells 48h, the experimental group Gli1, FoxM1, Vimentin mRNA expression was significantly lower than the control group (P <0.01) P <0.05). The expression of E-cadherin in the experimental group was significantly higher than that in the control group (P <0.05), and the corresponding protein expression intensity was also higher than that of the control group . CONCLUSION: Inhibition of Hh signaling pathway can significantly reduce the proliferation, invasion and migration of Hela cell lines. FoxM1 is likely to be the downstream target gene of transcriptional factor Gli1 at the end of Hh signaling pathway, and its effect on proliferation, invasion and migration of cervical cancer cell lines is likely to be Achieved through EMT.