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中华肝脏外科手术学电子杂志 ›› 2023, Vol. 12 ›› Issue (03) : 336 -341. doi: 10.3877/cma.j.issn.2095-3232.2023.03.017

基础研究

U2AF2表达对肝细胞癌增殖和迁移的影响及其与预后的关系
莫钊鸿1, 翟航1, 苏日顺1, 孟泓宇1, 罗豪1, 陈文豪1, 许瑞云1,()   
  1. 1. 510630 广州,中山大学附属第三医院肝胆外科
  • 收稿日期:2023-01-19 出版日期:2023-06-10
  • 通信作者: 许瑞云
  • 基金资助:
    广州市科技计划项目(201804010211)

Effect of U2AF2 expression on proliferation and migration of hepatocellular carcinoma and its relationship with prognosis

Zhaohong Mo1, Hang Zhai1, Rishun Su1, Hongyu Meng1, Hao Luo1, Wenhao Chen1, Ruiyun Xu1,()   

  1. 1. Department of Hepatobiliary Surgery, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China
  • Received:2023-01-19 Published:2023-06-10
  • Corresponding author: Ruiyun Xu
引用本文:

莫钊鸿, 翟航, 苏日顺, 孟泓宇, 罗豪, 陈文豪, 许瑞云. U2AF2表达对肝细胞癌增殖和迁移的影响及其与预后的关系[J]. 中华肝脏外科手术学电子杂志, 2023, 12(03): 336-341.

Zhaohong Mo, Hang Zhai, Rishun Su, Hongyu Meng, Hao Luo, Wenhao Chen, Ruiyun Xu. Effect of U2AF2 expression on proliferation and migration of hepatocellular carcinoma and its relationship with prognosis[J]. Chinese Journal of Hepatic Surgery(Electronic Edition), 2023, 12(03): 336-341.

目的

探讨U2小核RNA辅助因子2(U2AF2)表达对肝细胞癌增殖和迁移的影响及其与预后的关系。

方法

采用实时荧光定量PCR(qRT-PCR)检测U2AF2在HCC细胞系(HepG2、Hep3B、Huh-7和HCCLM9)及正常肝脏细胞LO2中的表达水平。采用慢病毒介导的短发夹RNA(shRNA)敲低U2AF2在HCC细胞中的表达(shU2AF2-1、shU2AF2-2为实验组,sh-NC为对照组)。采用CCK-8和细胞划痕实验分别检测shU2AF2-1组和sh-NC组HCC细胞的增殖和迁移能力。Western bolt检测shU2AF2-1组和sh-NC组HCC细胞的哺乳动物雷帕霉素靶蛋白(mTOR)表达水平及其磷酸化(p-mTOR)水平。生物信息学分析采用UALCAN和Kaplan-Meier Plotter数据库分别分析U2AF2在HCC中的表达及其与HCC预后的关系。下载癌症基因组图谱(TCGA)数据库的HCC表达谱,采用基因集富集分析(GSEA)分析U2AF2在HCC中参与的信号通路。多组比较采用单因素方差分析,两组比较采用LSD-tt检验。

结果

TCGA数据库分析显示,HCC组织U2AF2 mRNA表达水平明显高于癌旁正常肝组织(P<0.05),U2AF2 mRNA表达水平随着HCC肿瘤分级的增高而增高(P<0.05)。Kaplan-Meier Plotter数据库分析显示,U2AF2高表达组患者总体生存率及无复发生存率明显低于低表达组(HR=1.86,1.85;P<0.05)。GSEA结果提示,U2AF2的高表达与mTOR通路密切相关,标准化富集得分(NES)为2.113(P<0.05)。qRT-PCR检测显示,U2AF2 mRNA在HepG2、Hep3B、Huh-7和HCCLM9细胞中表达水平明显高于正常肝脏细胞LO2,其中以HCCLM9细胞的表达最高(F=26.003,P<0.05);shU2AF2-1和shU2AF2-2组HCCLM9细胞U2AF2 mRNA平均相对表达量分别为0.27±0.09、0.41±0.13,明显低于sh-NC组的1.02±0.06(LSD-t=-7.643,-6.220;P<0.05)。CCK-8实验显示,shU2AF2-1组HCCLM9细胞的增殖能力明显低于sh-NC组(t=-5.381,P<0.05)。细胞划痕实验显示,shU2AF2-1组HCCLM9细胞的迁移能力明显低于sh-NC组(t=-7.903,P<0.05)。Western blot检测显示,shU2AF2-1组HCCLM9细胞的p-mTOR水平明显低于sh-NC组,而两组mTOR水平无明显差异。

结论

U2AF2在HCC中高表达,且U2AF2高表达和HCC的不良预后相关,U2AF2对HCC细胞增殖和迁移的促进作用可能是通过激活mTOR通路实现。

Objective

To evaluate the effect of U2 small nuclear RNA auxiliary factor 2 (U2AF2) expression on the proliferation and migration of hepatocellular carcinoma and its relationship with prognosis.

Methods

The expression level of U2AF2 in HCC cell lines (HepG2, Hep3B, Huh-7 and HCCLM9) and normal liver cell LO2 was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The short hairpin RNA (shRNA) mediated by lentivirus was employed to knock down the expression of U2AF2 in HCC cells (shU2AF2-1 and shU2AF2-2 were assigned into the experimental group and sh-NC as the control group). The proliferation and migration capability of HCC cells in the shU2AF2-1 and sh-NC groups were evaluated by CCK-8 assay and wound healing assay, respectively. The expression levels of mammalian target of rapamycin (mTOR) and phosphorylated mTOR (p-mTOR) in HCC cells in the shU2AF2-1 and sh-NC groups were measured by Western blot. Regarding bioinformatics analysis, the expression level of U2AF2 in HCC and its relationship with prognosis were analyzed by using UALCAN and Kaplan-Meier Plotter databases, respectively. The expression profile of HCC was downloaded from The Cancer Genome Atlas (TCGA) database. The signal pathways of U2AF2 involved in HCC were analyzed by Gene Set Enrichment Analysis (GSEA). Multi-group comparison was analyzed by one-way ANOVA. Two-group comparison was conducted by LSD-t or t test.

Results

TCGA database analysis showed that the expression level of U2AF2 mRNA in HCC tissues was significantly higher than that in adjacent normal liver tissues (P<0.05), and the expression level of U2AF2 mRNA was up-regulated with the increase of HCC grade (P<0.05). Kaplan-Meier plotter database analysis revealed that the overall survival and recurrence-free survival rates of patients with high expression of U2AF2 were significantly lower than those of their counterparts with low expression (HR=1.86, 1.85; P<0.05). GSEA analysis indicated that high expression of U2AF2 was closely correlated with the mTOR signaling pathway, and the normalized enrichment score (NES) was 2.113 (P<0.05).qRT-PCR showed that the expression level of U2AF2 mRNA in HepG2, Hep3B, Huh-7 and HCCLM9 cells was significantly higher than that in normal liver cell LO2, with the highest expression level in HCCLM9 cells (F=26.003, P<0.05). In the shU2AF2-1 and shU2AF2-2 groups, the average relative expression levels of U2AF2 mRNA in HCCLM9 cells were 0.27±0.09 and 0.41±0.13, significantly lower than 1.02±0.06 in the sh-NC group (LSD-t=-7.643, -6.220; P<0.05). CCK-8 assay showed that compared with the sh-NC group, the proliferation capability of HCCLM9 cells was significantly lower in the shU2AF2-1 group (t=-5.381, P<0.05). Wound healing assay found that the migration capability of HCCLM9 cells in the shU2AF2-1 group was significantly lower than that in the sh-NC group (t=-7.903, P<0.05). Western blot showed that the expression level of p-mTOR of HCCLM9 cells in the shU2AF2-1 group was significantly lower than that in the sh-NC group, whereas there was no significant difference in the expression level of mTOR between two groups.

Conclusions

U2AF2 is highly expressed in HCC. High expression of U2AF2 is correlated with poor prognosis of HCC patients. U2AF2 can promote the proliferation and migration of HCC cells probably by activating the mTOR signaling pathway.

图1 HCC组织和细胞系中U2AF2的表达注:a为TCGA数据库分析HCC组织中U2AF2的表达水平;b为TCGA数据库分析不同肿瘤分级HCC组织中U2AF2的表达水平(G1~G4为肿瘤分级1~4级);c为qRT-PCR检测正常肝细胞LO2及HCC细胞系(HepG2、Hep3B、Huh-7和HCCLM9)中U2AF2的表达水平;*为P<0.05,**为P<0.01,***为P<0.001;U2AF2为U2小核RNA辅助因子2,HCC为肝细胞癌,TCGA为癌症基因组图谱,qRT-PCR为实时荧光定量PCR,TPM为每百万条reads的转录本
图2 HCC患者U2AF2表达与预后关系的Kaplan-Meier生存曲线注:a、b分别为总体生存曲线和无复发生存曲线;U2AF2为U2小核RNA辅助因子2,HCC为肝细胞癌
图3 沉默U2AF2对HCC细胞增殖和迁移能力的影响注:a为U2AF2在不同敲低位点处理的HCCLM9细胞中的表达水平;b为沉默U2AF2的HCCLM9细胞CCK-8实验;c为HCCLM9细胞划痕实验;*为P<0.01,**为P<0.001;U2AF2为U2小核RNA辅助因子2,HCC为肝细胞癌,shU2AF2-1和shU2AF2-2为沉默U2AF2-1和U2AF2-2,sh-NC为沉默正常对照细胞
图4 GSEA显示U2AF2与mTOR信号通路相关注:U2AF2为U2小核RNA辅助因子2,GSEA为基因集富集分析,HCC为肝细胞癌,NES为标准化富集得分
图5 Western blot检测HCCLM9细胞mTOR和p-mTOR水平注:U2AF2为U2小核RNA辅助因子2,GAPDH为甘油醛-3-磷酸脱氢酶,mTOR为哺乳动物雷帕霉素靶蛋白,p-mTOR为磷酸化哺乳动物雷帕霉素靶蛋白,shU2AF2-1为沉默U2AF2-1,sh-NC为沉默正常对照细胞
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