南方医科大学学报

南方医科大学学报 ›› 2019, Vol. 39 ›› Issue (12): 1436-1442.doi: 10.12122/j.issn.1673-4254.2019.12.07

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Musashi1正调控肝细胞癌细胞的生长并促进其增殖

李 杰，闫 堃，杨 屹，李 华，王志东，徐 心   

  • 出版日期:2019-12-27 发布日期:2019-12-20
  • 基金资助:

Musashi-1 positively regulates growth and proliferation of hepatoma cells in vitro

  

  • Online:2019-12-27 Published:2019-12-20

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摘要： 目的 Musashi1（MSI1）属于RNA结合蛋白（RBP）家族，可特异性地结合mRNA并对蛋白质的翻译起激活或抑制作用，但它在肝细胞癌（HCC）中的功能还未被深入探究。本课题主要探讨MSI1在HCC细胞生长及增殖中的作用及机制。方法 构建MSI1过表达或敲除细胞系。采用免疫组化和Western blot技术检测MSI1在HCC组织中的表达。采用MTT、流式等方法探讨过表达或沉默MSI1对HCC细胞增殖、细胞活力及细胞周期分布的影响，同时检测细胞内PCNA、Cyclin D1以及Wnt/β-catenin通路关键分子APC和β-catenin的表达。结果 MSI1在HCC组织中的表达比非肿瘤的癌旁组织高。与对照细胞相比，HepG2细胞过表达MSI1不仅显著促进了其生长（7 d时的MTT值从0.52±0.12大幅增加至1.01±0.14，P<0.01），且处于G0/G1期的细胞比率也从（58.42±3.18）%降至（40.67±1.22）%，而S期细胞比率则从（28.51±1.93）%增加至（40.06±1.92）%（P<0.01）。与此同时，细胞PCNA和Cyclin D1蛋白的表达明显上调。反之，敲除MSI1使Huh7细胞的生长显著受抑，更多细胞停滞于G0/G1期（G0/G1期细胞比率从（43.83±1.57）%增加至（62.84±1.22）%（P<0.01），同时细胞PCNA和Cyclin D1的表达则显著下调。MSI1的过表达可以降低Wnt/β-catenin信号通路分子APC但增加β-catenin的表达，反之MSI1的敲低却可以增加细胞内APC而降低β-catenin的表达。结论 MSI1可以通过正调控HCC细胞的生长及细胞周期的进程而加速HCC病程的进展，其作用可能是通过激活Wnt/β-catenin信号通路实现的。

Abstract: Objective To investigate the regulatory role of Musashi-1 (MSI1) in the proliferation and growth of hepatocellular carcinoma (HCC) cells. Methods We examined the expression of MSI1 in HCC and paired adjacent tissues from 24 patients using immunohistochemistry and Western blotting. A MSI1-expressing vector was constructed and stably transfected into HepG2 cells, and short hairpin RNAs (shRNAs) that targeted MSI1 mRNA were ligated into the vector and stably transfected in Huh7 cells. The effects of MSI1 overexpression and silencing on the proliferation, viability and cell cycle of HepG2 cells were investigated using flow cytometry or MTT assay. The expressions of PCNA, cyclin D1, APC and β-catenin in the HCC cells were detected with Western blotting. Results MSI1 expression was significantly up-regulated in HCC tissues as compared with that in the adjacent tissues. Overexpression of MSI1 in HepG2 cells resulted in significantly enhanced cell growth (P<0.01) and significantly reduced G0/G1 phase cells from (58.42 ± 3.18)％ to (40.67 ± 1.22)% and increased S phase cells from (28.51 ± 1.93)％ to (40.06±1.92)％ (P<0.01), causing also increases in the expressions of PCNA and Cyclin D1. Knockdown of MSI1 in Huh7 cells obviously inhibited the cell growth and caused cell cycle arrest at the G1/S phase (P<0.01) with reduced protein expressions of PCNA and cyclin D1. Overexpression of MSI1 in HepG2 cells also down-regulated the expression of APC and up-regulated the expression of β-catenin protein, while MSI1 knockdown caused reverse changes in Huh7 cells. Conclusion MSI1 promotes the progression of HCC through positive modulation of cell growth and cell cycle via the Wnt/β-catenin pathway.