Cheng Jiwen,Zhao Pu,Yang Weili,et al.Let-7b modulates the proliferation of hepatoblastoma cells by targeting SALL4[J].Journal of Clinical Pediatric Surgery,2020,19(05):404-409.[doi:10.3969/j.issn.1671-6353.2020.05.006]
let-7b通过靶向调控SALL4影响肝母细胞瘤细胞增殖能力的机制研究
- Title:
- Let-7b modulates the proliferation of hepatoblastoma cells by targeting SALL4
- Keywords:
- let-7b; Liver Neoplasms; SALL4; Cell Proliferation
- 分类号:
- R726;R735.7
- 摘要:
- 目的 初步研究let-7b和SALL4在肝母细胞瘤（hepatoblastoma，HB）细胞恶性行为中的作用及二者间的相互作用机制。方法 采用qRT-PCR检测let-7b在正常肝细胞（HL-7702）与HB细胞系、HB组织及癌旁组织中的表达水平，采用免疫组化法检测SALL4在HB组织及癌旁组织中的表达水平，并分析HB组织中let-7b与SALL4表达水平的相关性。通过MTT、细胞周期和凋亡等实验方法上调或抑制HB细胞中let-7b的表达水平，观察let-7b对HB细胞生物学功能的影响；通过生物信息学预测软件、双荧光素酶报告系统、qRT-PCR和western blot等实验，从体外水平验证let-7b与SALL4的靶向关系。采用Kaplan-Meier生存曲线分析不同let-7b与SALL4表达水平的肝母细胞瘤患者总体生存率的差异。结果 与癌旁组织相比，let-7b在HB组织中表达量显著降低，而SALL4表达量显著升高；let-7b在肝母细胞瘤组织中的表达水平与PRETEXT分期存在相关性（P=0.002），且HB组织标本中let-7b与SALL4的表达水平存在负相关性（r=-0.716，P<0.001）。let-7b低表达组HB患者的总体生存率显著低于let-7b高表达组（P=0.017）。SALL4阳性组HB患者的总体生存率显著低于SALL4阴性和弱阳性组（P=0.034）。MTT、细胞周期和凋亡实验结果显示，let-7b可抑制肝癌HepG2细胞增殖，促进细胞的凋亡。而抑制内源性的let-7b可促进HepG2细胞增殖，抑制细胞的凋亡。生物信息学软件预测及构建双荧光报告载体荧光检测等实验结果显示，let-7b与SALL4存在一定的靶向关系。结论 let-7b和SALL4可作为肝母细胞瘤不良预后的潜在标记物；let-7b作为抑癌基因可以靶向调控SALL4的表达，抑制HB细胞的增殖。
- Abstract:
- Objective To explore the role and mechanism of let-7b and SALL4 in the malignant behaviors of hepatoblastoma (HB) cells. Methods Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was employed for defecting the expression of let-7b in normal liver cells (HL-7702),HB cell lines,HB tissues and adjacent normal tissues.Immunohistochemistry was applied for measuring the expression levels of SALL4 in HB tissues and paracancerous tissue and analyzing the correlation between the expression levels of SALL4 in HB tissues.Up-regulating or inhibiting the let-7b expression level in HB cells was used for examining the effects of let-7b on the biological function of HB cells by methyl thiazolyl tetrazolium (MTT) assay,cell cycle and apoptosis.The targeting relationship between let-7b and SALL4 was assessed through dual-luciferase reporter system,qRT-PCR,Western blot and in vitro immunohistochemical staining. Results let-7b became markedly down-regulated in HB tissues and HB cells (e.g.HepG2 & HuH6) while SALL4 protein was frequently up-regulated in HB tissues.The expression of let-7b was correlated with PRETEXT stage (P=0.002).A significant negative correlation existed between let-7b and SALL4 expression level in HB tissue samples (r=-0.761,P<0.001).Through the detection of proliferative activity,cell cycle and cell apoptosis,let-7b could regulate the proliferating capability of HepG2.The dual-luciferase reporter assays revealed that SALL4 was a novel target of let-7b.Conclusion Serving as a tumor suppressor gene,let-7b suppresses the HepG2 cells growth by targeting SALL4.
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备注/Memo
收稿日期:2019-09-01。
基金项目:陕西省创新人才推进计划-青年科技新星项目（编号：2018KJXX-050）；西安交通大学中央高校基本科研业务项目（编号：XTT2018126，YT（ZD）201704）
通讯作者:李鹏,Email:sienafiat@hotmail.com