LGR5对宫颈癌细胞上皮间质转化的影响及潜在机制研究

摘要

Objctive To investigate the effect and potential mechanism of leucine-rich repeat-containing G-protein-coupled recep-tor 5(LGR5)on cervical mesenchymal transition. Methods The cervical cancer cell Hela was used as the research object to construct the over-expressing and silencing LGR5 cells. The expression of LGR5 was detected by RT-PCR and Western blot. Hela cells transfected with pcDNA3. 1-LGR5 were named as LGR5 group,the cells transfected with pcDNA3.1 empty vector was recorded as pcDNA3.1 group;the cells transfected with LGR5 siRNA or negative control siRNA were named as siLGR5 or NC group,respectively. The expression of E-cadherin,N-cadherin,vi-mentin,Wnt/β-cadherin pathway protein β-catenin and downstream proteins c-Myc and Cyclin D1 in each group were detected by Western blot. Transwell assay was used to detect the changes of cell migration and invasion. Results Hela cells overexpressing and knocking down LGR5 were successfully constructed. Compared with pcDNA3.1 group,the expression of epithelial marker E-cadherin in Hela cells transfected with pcDNA3.1-LGR5 was significantly decreased(P <0.05). The expression of vimentin and N-cadherin in Hela cells was significantly in-creased(P <0.05). Compared with NC group,the expression of E-cadherin in HeLa cells transfected with siLGR5 was significantly increased (P <0.05). The expression of vimentin and N-cadherin was significantly decreased(P <0.05). Compared with pcDNA3.1 group,HeLa cells overexpression LGR5 migration and invasion ability,Wnt / β-catenin pathway protein β-catenin and its downstream genes c-Myc,Cyclin D1 expression were significantly increased(P <0.05). The expression of β-catenin,c-Myc and Cyclin D1 in Hela cells was significantly low-er than that in NC group(P <0.05). Conclusion Overexpression of LGR5 can promote the epithelial-mesenchymal transition,invasion and migration of cervical cancer cells,while the interference of LGR5 inhibits these functions of cervical cancer cells. Thus,LGR5 might induce EMT and advocate HCC cell invasion by activating Wnt/β-catenin signaling pathway.%目的 探讨富含亮氨酸重复序列的G蛋白偶联受体5(LGR5)对宫颈癌细胞上皮间质转化的影响及潜在机制.方法 采用前瞻性研究,以宫颈癌细胞Hela为研究对象,构建过表达和敲低LGR5的细胞株,RT-PCR和West-ern blot检测LGR5表达.将转染pcDNA3.1-LGR5的Hela细胞作为LGR5组,以转染pcDNA3.1空载体的细胞记为pcDNA3.1组;将转染LGR5 siRNA细胞作为siLGR5组,以转染阴性对照siRNA细胞记为NC组.Western blot分别检测各组细胞中上皮间质标记物钙黏附蛋白E(E-cadherin)、钙黏附蛋白N(N-cadherin)、波形蛋白(vimentin)及Wnt/β-cadherin通路蛋白β链蛋白(β-catenin)和下游蛋白c-Myc、细胞周期蛋白(Cyclin D1)表达变化.Transwell实验检测各组细胞迁移侵袭能力变化.结果 Hela细胞中成功过表达和敲低了LGR5;与pcDNA3.1组相比,Hela细胞转染pcD-NA3.1-LGR5后细胞中上皮标志物E-cadherin表达显著降低(P <0.05),间质标记物vimentin、N-cadherin表达显著增加(P <0.05).与NC组相比,Hela细胞转染siLGR5后细胞中E-cadherin表达明显增加(P <0.05),vimentin、N-cadherin表达明显下降(P <0.05).与pcDNA3.1组相比,Hela细胞过表达LGR5后细胞迁移侵袭能力、Wnt/β-cate-nin通路蛋白β-catenin及其下游基因c-Myc、Cyclin D1表达均显著增加(P <0.05);而Hela细胞沉默LGR5后较NC组细胞迁移侵袭能力、Hela细胞中β-catenin、c-Myc和Cyclin D1表达均明显降低(P <0.05).结论 过表达LGR5能够促进宫颈癌细胞Hela上皮间质转化和侵袭迁移,而干扰LGR5则抑制了细胞上皮间质转化和迁移侵袭,其中机制可能与LGR5调控Wnt/β-catenin信号转导有关.