留言板

尊敬的读者、作者、审稿人, 关于本刊的投稿、审稿、编辑和出版的任何问题, 您可以本页添加留言。我们将尽快给您答复。谢谢您的支持!

姓名
邮箱
手机号码
标题
留言内容
验证码

通过CRISPR/Cas9技术抑制TFDP3基因对前列腺癌PC3细胞生物学功能的影响

李蕊 杨柳 李金洁 刁艳君 苏明权 郝晓柯 刘家云

李蕊, 杨柳, 李金洁, 刁艳君, 苏明权, 郝晓柯, 刘家云. 通过CRISPR/Cas9技术抑制TFDP3基因对前列腺癌PC3细胞生物学功能的影响[J]. 中国肿瘤生物治疗杂志, 2021, 28(5): 443-450. doi: 10.3872/j.issn.1007-385x.2021.05.004
引用本文: 李蕊, 杨柳, 李金洁, 刁艳君, 苏明权, 郝晓柯, 刘家云. 通过CRISPR/Cas9技术抑制TFDP3基因对前列腺癌PC3细胞生物学功能的影响[J]. 中国肿瘤生物治疗杂志, 2021, 28(5): 443-450. doi: 10.3872/j.issn.1007-385x.2021.05.004
LI Rui, YANG Liu, LI Jinjie, DIAO Yanjun, SU Mingquan, HAO Xiaoke, LIU Jiayun. Effects of TFDP3 knock-out by CRISPR/Cas9 on biological function of prostate cancer PC3 cells[J]. Chin J Cancer Biother, 2021, 28(5): 443-450. doi: 10.3872/j.issn.1007-385x.2021.05.004
Citation: LI Rui, YANG Liu, LI Jinjie, DIAO Yanjun, SU Mingquan, HAO Xiaoke, LIU Jiayun. Effects of TFDP3 knock-out by CRISPR/Cas9 on biological function of prostate cancer PC3 cells[J]. Chin J Cancer Biother, 2021, 28(5): 443-450. doi: 10.3872/j.issn.1007-385x.2021.05.004

通过CRISPR/Cas9技术抑制TFDP3基因对前列腺癌PC3细胞生物学功能的影响

doi: 10.3872/j.issn.1007-385x.2021.05.004
基金项目: 国家自然科学基金资助项目(No. 81372747)
详细信息
    作者简介:

    李蕊(1988–),女,硕士,主管技师,主要从事前列腺癌分子机制的研究,E-mail:lirui10305566@126.com

    通讯作者:

    刘家云(LIU Jiayun,corresponding author),博士,副主任医师,副教授,硕士生导师,主要从事临床微生物病原学和肿瘤分子机制的研究,E-mail:1187871836@qq.com

  • 中图分类号: R737.25; R730.54

Effects of TFDP3 knock-out by CRISPR/Cas9 on biological function of prostate cancer PC3 cells

Funds: Project supported by the National Natural Science Foundation of China (No. 81372747)
  • 摘要:   目的:  通过CRISPR/Cas9技术构建前列腺癌PC3细胞TFDP3基因敲除的稳转株,探讨抑制TFDP3表达对PC3细胞周期、凋亡、迁移和侵袭能力的影响。  方法:  通过生物信息学筛选sgRNA,通过CRISPR/Cas9技术、构建抑制TFDP3基因表达的sgRNA-Cas9共转染慢病毒,感染PC3细胞后筛选获取稳转细胞株。通过流式细胞术对TFDP3基因敲除(knock-out,KO)实验组与空白对照组进行细胞周期和凋亡检测,并进一步通过划痕实验和Transwell实验进行细胞迁移和侵袭能力检测。  结果:  通过生物信息学筛选获得3条sgRNA,其中sgRNA2有明显的抑制前列腺癌细胞基因表达的功能;通过CRISPR/Cas9技术成功构建了基于CRISPR/Cas9介导的TFDP3低表达的PC3细胞稳转株。抑制TFDP3基因表达后,相比于对照组,KO组中G0/G1期细胞百分比增加、G2/M期细胞百分比下降(P<0.05或P<0.01),细胞凋亡率显著升高(P<0.05),细胞迁移率明显下降[24 h迁移率:(44.00±1.60)% vs (65.00±4.40)%,P<0.01],穿过聚碳酸酯膜的侵袭细胞数明显下降[(185.89±11.71)vs (248.33±11.95)个,P<0.01]。  结论:  通过CRISPR/Cas9技术抑制TFDP3基因表达后,PC3细胞发生周期阻滞、凋亡率也有所增加、迁移和侵袭能力显著减弱,提示TFDP3是一个前列腺癌促癌基因。
  • 图  1  Luciferase-SSA报告载体活性检测结构示意图

    Figure  1.  The structural representation of the detection of Luciferase-SSA activity

    图  2  Cas9/sgRNA载体的测序结果

    Figure  2.  Sequencing validation of the construction of Cas9/sgRNA vector

    图  4  lenti CRISPR-TFDP3-T2慢病毒滴度检测荧光

    Figure  4.  Fluorescence detection of Lenti CRISPR-TFDP3-T2 concentration

    图  3  LuciferaseSSA报告载体法检测基因敲除效率

    Figure  3.  Knock-out efficiency detected by Luciferase-SSA activity assay

    ** P<0.01

    图  5  细胞单克隆基因型鉴定

    Figure  5.  Identification of cell genotype

    A: Cell monoclone after LENTI CRISPR-TFDP3-T2 infection; B: WB screening of TFDP3 knock-out positive cell clones;C: TFDP3 KO cell clone, allele A deletion mutation (−7 bp); D: TFDP3 KO cell clone, allele B deletion mutation (−7 bp)

    图  6  TFDP3基因敲除后PC3细胞周期和凋亡的改变

    Figure  6.  Changes of PC3 cell cycle and apoptosis after TFDP3 gene KO

    A:The results of flow cytometry showed that the apoptotic rate of PC3 cells in the KO group was higher than that in the NC group; B: Compared with the NC group, the percentage of the cells in the G1 phase was significantly higher in the KO group,while the percentage of cells in the S phase and G2/M phase decreased, indicating that cells were blocked at G1 phase after TFDP3 KO (NC: Normal control group; KO: TFDP3 gene KO group)

    图  7  TFDP3对前列腺癌细胞PC3迁移能力的影响

    Figure  7.  The effect of TFDP3 on the metastatic ability of prostate cancer PC3 cells

    ** P <0.01A: Migration of the PC3 cells in the NC and KO groups at 0, 8 and 24 h detected by scratching test (magnification: 100 times); B: Invasion of PC3 cells in the NC and KO groups detected by Transwell assay (magnification: 200 times); NC: normal control group; KO: TFDP3 gene KO group

    表  1  TFDP3的sgRNA靶位点的核苷酸序列

    Table  1.   Nucleotide sequences of sgRNA targeting TFDP3

    TargetsgRNA sequence5’- guide sequenceOligo sequence
    sgRNA1 GCCGGGCAGCACAACAGGAA CACC
    AAAC
    GCCGGGCAGCACAACAGGAA
    TTCCTGTTGTGCTGCCCGGC
    sgRNA2 GCCGTCTTTCCATGAAGGTC CACC
    AAAC
    GCCGTCTTTCCATGAAGGTC
    GACCTTCATGGAAAGACGGC
    sgRNA3 GGAGGTGTGTTCACGACGGC CACC
    AAAC
    GGAGGTGTGTTCACGACGGC
    GCCGTCGTGAACACACCTCC
    下载: 导出CSV
  • [1] 胡小丹, 游敏, 罗文新. 基因编辑技术[J]. 中国生物化学与分子生物学报, 2018, 34(3): 267-277. DOI: 10.13865/j.cnki.cjbmb.2018.03.06.
    [2] 陈力权, 李谦. ZFNs、TALENs和CRISPR-Cas基因编辑技术在肿瘤治疗中的应用[J]. 药物生物技术, 2018, 25(6): 537-541. DOI: 10.19526/j.cnki.1005-8915.20180617.
    [3] 李成功, 梅恒, 胡豫. 基因编辑技术在CAR-T治疗中的研究进展[J]. 中国肿瘤生物治疗杂志, 2019, 26(3): 338-345. DOI: 10.3872/j.issn.1007-385X.2019.03.015.
    [4] 李佰伦, 黄书琴, 王奕众. CRISPR/Cas系统的研究进展及前沿应用[J]. 生命的化学, 2020, 40(1): 16-24. DOI: 10.13488/j.smhx.20190108.
    [5] QIAO H, DI STEFANO L, TIAN C, et al. Human TFDP3, a novel DP protein, inhibits DNA binding and transactivation by E2F[J]. J Biol Chem, 2007, 282(1): 454-466. DOI: 10.1074/jbc.M606169200.
    [6] WANG Y, HAN K J, PANG X W, et al. Large scale identification of human hepatocellular carcinoma-associated antigens by autoantibodies[J]. J Immunol, 2002, 169(2): 1102-1109. DOI: 10.4049/jimmunol.169.2.1102.
    [7] CHU M, YIN K L, DONG Y J, et al. TFDP3 confers chemoresistance in minimal residual disease within childhood T-cell acute lymphoblastic leukemia[J/OL]. Oncotarget, 2017, 8(1): 1405-1415[2020-09-09]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5352064/. DOI: 10.18632/oncotarget.13630.
    [8] TOVAR H, GARCÍA-HERRERA R, ESPINAL-ENRÍQUEZ J, et al. Transcriptional master regulator analysis in breast cancer genetic networks[J]. Comput Biol Chem, 2015, 59(Pt B): 67-77. DOI: 10.1016/j.compbiolchem.2015.08.007.
    [9] MA Y Y, XIN Y J, LI R, et al. TFDP3 was expressed in coordination with E2F1 to inhibit E2F1-mediated apoptosis in prostate cancer[J]. Gene, 2014, 537(2): 253-259. DOI: 10.1016/j.gene.2013.12.051.
    [10] BRAHMER J, RECKAMP K L, BAAS P, et al. Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer[J/OL]. N Engl J Med, 2015, 373(2): 123-135[2020-01-04]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4681400/. DOI: 10.1056/NEJMoa1504627.
    [11] LI C, GUAN X M, DU T, et al. Inhibition of HIV-1 infection of primary CD4+ T-cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9[J]. J Gen Virol, 2015, 96(8): 2381-2393. DOI: 10.1099/vir.0.000139.
    [12] CONG L, RAN F A, COX D, et al. Multiplex genome engineering using CRISPR/Cas systems[J/OL]. Science, 2013, 339(6121): 819-823[2020-09-09]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3795411/. DOI: 10.1126/science.1231143.
    [13] RUAN G X, BARRY E, YU D, et al. CRISPR/Cas9-mediated genome editing as a therapeutic approach for leber congenital amaurosis 10[J]. Mol Ther, 2017, 25(2): 331-341. DOI: 10.1016/j.ymthe.2016.12.006.
    [14] XU L, WANG J, LIU Y L, et al. CRISPR-edited stem cells in a patient with HIV and acute lymphocytic leukemia[J]. N Engl J Med, 2019, 381(13): 1240-1247. DOI: 10.1056/NEJMoa1817426.
    [15] CHEN W, ZHENG R, BAADE P D, et al. Cancer statistics in China, 2015[J]. CA Cancer J Clin, 2016, 66(2): 115-132. DOI: 10.3322/caac.21338.
    [16] TIAN C, LV D, QIAO H, et al. TFDP3 inhibits E2F1-induced, p53-mediated apoptosis[J]. Biochem Biophys Res Commun, 2007, 361(1): 20-25. DOI: 10.1016/j.bbrc.2007.06.128.
    [17] YIN K L, LIU Y C, CHU M, et al. TFDP3 regulates epithelial-mesenchymal transition in breast cancer[J/OL]. PLoS One, 2017, 12(1): e0170573[2020-09-09]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5256886/. DOI: 10.1371/journal.pone.0170573.
    [18] DING L Y, CHU M, JIAO Y S, et al. TFDP3 regulates the apoptosis and autophagy in breast cancer cell line MDA-MB-231[J]. PLoS One, 2018, 13(9): e0203833. DOI: 10.1371/journal.pone.0203833.
    [19] CHUN J N, CHO M, PARK S, et al. The conflicting role of E2F1 in prostate cancer: a matter of cell context or interpretational flexibility?[J]. Biochim Biophys Acta Rev Cancer, 2020, 1873(1): 188336. DOI: 10.1016/j.bbcan.2019.188336.
    [20] 李蕊, 马越云, 辛艺娟, 等. E2F1对TFDP3表达的调控及其对前列腺癌PC3细胞凋亡的影响[J]. 中国肿瘤生物治疗杂志, 2014, 21(2): 125-129. DOI: 10.3872/j.issn.1007-385X.2013.02.002.
    [21] FERREIRA DE OLIVEIRA J M P, SANTOS C, FERNANDES E. Therapeutic potential of hesperidin and its aglycone hesperetin: Cell cycle regulation and apoptosis induction in cancer models[J]. Phytomedicine, 2020, 73: 152887. DOI: 10.1016/j.phymed.2019.152887.
    [22] ZHANG W W, LI L J, LI D G, et al. The first approved gene therapy product for cancer ad-p53 (gendicine): 12 years in the clinic[J]. Hum Gene Ther, 2018, 29(2): 160-179. DOI: 10.1089/hum.2017.218.
    [23] HAMMER S, HAGEMANN U B, ZITZMANN-KOLBE S, et al. Preclinical efficacy of a PSMA-targeted thorium-227 conjugate (PSMA-TTC), a targeted alpha therapy for prostate cancer[J]. Clin Cancer Res, 2020, 26(8): 1985-1996. DOI: 10.1158/1078-0432.CCR-19-2268.
  • 加载中
图(7) / 表(1)
计量
  • 文章访问数:  32
  • HTML全文浏览量:  21
  • PDF下载量:  5
  • 被引次数: 0
出版历程
  • 收稿日期:  2020-09-09
  • 修回日期:  2021-04-09
  • 刊出日期:  2021-05-01

目录

    /

    返回文章
    返回