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沉默减数分裂内切酶1(EME1)抑制肝癌细胞增殖的机制

陈椿 王可欣 贺梦雯 李乐 王春艳 刘妍 纪冬

引用本文:
Citation:

沉默减数分裂内切酶1(EME1)抑制肝癌细胞增殖的机制

DOI: 10.12449/JCH240518
基金项目: 

北京市自然科学基金面上项目 (7222173)

医学伦理声明本研究于2020年7月14日通过解放军总医院第五医学中心伦理委员会审批,批号:2020055D。
利益冲突声明:本文不存在任何利益冲突。
作者贡献声明:纪冬负责课题设计及拟定写作思路;陈椿、王可欣、贺梦雯进行实验研究及撰写论文;王春艳、刘妍、纪冬指导撰写文章并最后定稿。
详细信息
    通信作者:

    刘妍, liuyan5360@163.com (ORCID: 0000-0003-1498-4314)

    纪冬, jidg302@126.com (ORCID: 0000-0001-8214-462X)

Silencing essential meiotic endonuclease 1 inhibits the proliferation of liver cancer cells: A study of related mechanisms

Research funding: 

Beijing Municipal National Science Foundation (7222173)

More Information
  • 摘要:   目的  探讨减数分裂内切酶1(EME1)在肝癌组织中的表达及其对肝癌细胞生物学行为的影响。  方法  筛选TCGA数据库肝癌样本中的差异表达基因。采用免疫组化和Western Blot分析EME1在肝癌组织中的表达丰度。通过短发夹RNA(shRNA)构建慢病毒并感染BEL-7404细胞干扰EME1基因表达,分为沉默组(shEME1)和对照组(shCtrl)。通过实时荧光定量PCR法和Western Blot检测两组EME1mRNA和蛋白表达水平,Celigo计数法及MTT活性检测细胞增殖率,流式细胞术检测细胞周期,Caspase3/7活性检测细胞凋亡。两组间比较采用成组t检验。  结果  TCGA结果显示EME1的mRNA表达水平在肝癌组织中是癌旁组织的18.9倍(114.5±153.0 vs 8.0±7.2,t=5.00,P<0.001);EME1的蛋白表达水平在肝癌组织中是癌旁组织的7.0倍(免疫组化检测,8.4±2.6 vs 1.2±0.4,t=7.55,P<0.001)和2.5倍(Western Blot检测,249.0%±35.5% vs 100.0%±77.8%,t=3.02,P<0.05)。慢病毒感染后,相对于对照组,沉默组EME1的mRNA表达水平下降了29.9%(29.9%± 0.9% vs 100.0%±3.6%,t=32.82,P<0.001),蛋白表达水平显著下降了35.7%(35.7%±14.9% vs 100.0%±28.9%,t=3.42,P<0.05);细胞计数下降了45.1%(4 053±167 vs 8 988±477,t=16.91,P<0.001)、细胞活性下降至66.9%(0.518±0.046 vs 0.774±0.022,t=8.74,P<0.001)及细胞克隆形成能力下降至29.0%(75±6 vs 260±9,t=28.92,P<0.001)。与对照组比较,沉默组G1期细胞(49.9% vs 44.0%,t=8.96,P<0.001)比例增多,G2/M期(15.9% vs 17.9%,t=9.13,P<0.001)与S期(34.2% vs 38.1%,t=6.91,P<0.001)的细胞比例减少;Caspase3/7活性增强了1.5倍(145.8%±5.9% vs 100.0%±2.3%,t=12.50,P<0.001)。  结论  EME1在肝癌组织中高表达,沉默EME1基因可抑制肝癌细胞增殖,促进细胞凋亡。

     

  • 图  1  EME1 mRNA在肝癌/癌旁组织中的表达情况

    注: a,TCGA数据库分析EME1 mRNA表达水平;b,BEL-7404/7402、Hep3B细胞的EME1 mRNA表达水平。

    Figure  1.  Expression level of EME1 mRNA in HCC/paracancerous tissues

    图  2  EME1在肝癌/癌旁组织中的表达情况

    注: a,Western Blot结果;b,Western Blot的定量分析;c,免疫组化结果(×200);d,免疫组化的半定量分析。

    Figure  2.  Expression level of EME1 in HCC/paracancerous tissues

    图  3  慢病毒感染BEL-7404细胞48 h后荧光显微镜结果(×100)

    Figure  3.  Fluorescence microscopie observation of BEL-7404 after transfection for 48 hours (×100)

    图  4  BEL-7404细胞的EME1沉默效率

    注: a,RT-qPCR检测不同处理组EME1 mRNA表达水平;b,Western Blot检测不同处理组EME1蛋白表达水平。

    Figure  4.  EME1 silencing efficiency in BEL-7404 cells

    图  5  不同处理组BEL-7404细胞的增殖情况

    注: a,Celigo计数仪在荧光视野下对BEL-7404细胞的单视野成像;b,Celigo计数仪计数单孔BEL-7404细胞的数量;c,MTT法检测BEL-7404细胞的增殖情况;d、e,BEL-7404细胞的克隆形成情况与克隆计数。

    Figure  5.  Proliferation of BEL-7404 cells in different treatment groups

    图  6  不同处理组BEL-7404细胞周期情况

    Figure  6.  Cell cycle of BEL-7404 cells in different treatment groups

    图  7  不同处理组BEL-7404细胞的Caspase3/7活性

    Figure  7.  Caspase3/7 activity of BEL-7404 cells in the negative control group and the experimental group

    表  1  EME1 shRNA引物序列

    Table  1.   Sequences of essential meiotic structure-specific endonuclease 1 shRNA

    名称 序列(5′-3′)
    shRNA 正义链:CCGGCCCTGAGAAGACAGGAAAGAAC TCGAGTTCTTTCCTGTCTTCTCAGGGTTTTTG
    反义链:AATTCAAAAACCCTGAGAAGACAGGAAAG AACTCGAGTTCTTTCCTGTCTTCTCAGGG
    shRNA-NC 正义链:CCGGTTCTCCGAACGTGTCACGTTTCA AGAGAAGTGACACGTTCGGAGAATTTTTG 反义链:AATTCAAAAATTCTCCGAACGTGTCAC GTTCTCTT GAAACGTGACACGTTCGGAGAA
    鉴定引物 正义链:CCTATTTCCCATGATTCCTTCATA
    反义链:GTAATACGGTTATCCACG
    下载: 导出CSV

    表  2  各基因引物序列

    Table  2.   primer sequences

    名称 序列(5′-3′)
    EME1 正义链: TGACTTCAACAGCGACACCCA
    反义链: CACCCTGTTGCTGTAGCCAAA
    GAPDH 正义链: TCTGAGGAGTTGCCAACATTTG
    反义链: GGCTTCACAATCTGAGATGTCAA
    下载: 导出CSV
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  • 收稿日期:  2023-10-11
  • 录用日期:  2023-12-11
  • 出版日期:  2024-05-25
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