«上一篇/Previous Article|本期目录/Table of Contents|下一篇/Next Article»

《天津医科大学学报》[ISSN:1006-8147/CN:12-1259/R]

卷:

31卷

期数:

2025年03期

页码:

210-217

栏目:

肿瘤疾病专题

出版日期:

2025-05-20

文章信息/Info

Title:

Effect of Cdk12/Cdk13 knockout on migration and proliferation of mouse melanoma cell

文章编号:

1006-8147(2025)03-0210-08

作者:

张榆¹; 邓婷¹; 胡德庆¹; 2; 巴一¹; 3

（1.天津医科大学肿瘤医院消化肿瘤内科，国家恶性肿瘤临床医学研究中心，天津市恶性肿瘤临床医学研究中心，天津市消化系统肿瘤重点实验室；天津市肿瘤防治重点实验室，天津 300060；2.天津医科大学基础医学院细胞生物学系，天津 300060；3.中国医学科学院北京协和医院肿瘤医学中心，北京 100032）

Author(s):

ZHANG Yu¹; DENG Ting¹; HU Deqing¹; 2; BA Yi¹; 3

关键词:

CDK12; CDK13; CRISPR/Cas9; 基因敲除; 迁移; 增殖

Keywords:

-

分类号:

R739.5

DOI:

10.20135/j.issn.1006-8147.2025.03.0210

文献标志码:

A

摘要:

目的：通过CRISPR/Cas9技术构建细胞周期蛋白依赖性激酶（CDK）12和CDK13基因敲除的小鼠黑色素瘤细胞系（B16），观察其迁移和增殖等表型变化。方法：分别设计一对靶向小鼠Cdk12/Cdk13基因第1个内含子和第2个外显子的单向导RNA（sgRNA）。将构建成功的重组质粒共转染至B16细胞中，利用其嘌呤霉素抗性筛选单克隆细胞，进行基因型鉴定及Cdk12/Cdk13 mRNA表达水平检测，并对构建成功的基因敲除细胞进行迁移能力和细胞增殖等表型检测。结果：通过细胞基因组鉴定获得Cdk12/Cdk13正确的单克隆细胞系各 1 株，Cdk12敲除细胞系其Cdk12基因mRNA水平显著降低（t=16.09，P<0.001），Cdk13敲除细胞系其Cdk13基因mRNA水平显著降低（t=14.10，P<0.001）。Cdk12敲除细胞系迁移（t=21.25，P<0.001）和增殖（t=3.106，P<0.05）能力降低，Cdk13敲除细胞系迁移（t=18.99，P<0.001）和增殖（t=10.13，P<0.001）能力增强。结论：成功构建了Cdk12/Cdk13基因敲除的小鼠黑色素瘤B16细胞系，敲除Cdk12基因后细胞迁移和增殖能力减弱，敲除Cdk13基因后细胞迁移和增殖能力增强。

Abstract:

Objective： Cyclin-dependent kinase （CDK）12 and CDK13 knockout mouse melanoma B16 cell lines were constructed with CRISPR/Cas9 system，and to observe the changes of migration and proliferation. Methods：A pair of single guide RNA （sgRNA） was designed targeting the first intron and second exon of the mouse Cdk12/Cdk13 gene. Then the pair of plasmids was co-transfected into B16 cells，screening the monoclonal cells with puromycin resistance. Finally，genotyping was performed with PCR and the levels of Cdk12/Cdk13 mRNA were measured，and the migration ability and cell proliferation of the successfully knocked-out cells were detected. Results：Genotyping was performed and the correct monoclonal cell line of Cdk12/Cdk13 each were obtained. The level of Cdk12 mRNA was significantly decreased in Cdk12 knockout cell line （t=16.09，P<0.001） and the level of Cdk13 mRNA in Cdk12 knockout cell line （t=14.10，P<0.001）. The migration （t=21.25，P<0.001） and proliferation （t=3.106，P<0.05） of Cdk12 knockout cell line were reduced，while the migration （t=18.99，P<0.001） and proliferation （t=10.13，P<0.001） of Cdk13 knockout cell line were enhanced. Conclusion: The Cdk12/Cdk13 knockout mouse melanoma B16 cell lines are constructed successful

参考文献/References:

[1] LUI G Y L，GRANDORI C，KEMP C J. CDK12：an emerging therapeutic target for cancer[J]. J Clin Pathol，2018，71（11）：957-962.
[2] CHOU J，QUIGLEY D A，ROBINSON T M，et al. Transcription-associated cyclin-dependent kinases as targets and biomarkers for cancer therapy[J]. Cancer Discov，2020，10（3）：351-370.
[3] KOHOUTEK J，BLAZEK D. Cyclin K goes with Cdk12 and Cdk13[J]. Cell Div，2012，7：12.
[4] TIEN J C，LUO J，CHANG Y，et al. CDK12 loss drives prostate cancer progression，transcription-replication conflicts，and synthetic le-thality with paralog CDK13[J]. Cell Rep Med，2024，5（10）：101758.
[5] ZHANG Y，CHEN X N，ZHANG H，et al. CDK13 promotes lipid deposition and prostate cancer progression by stimulating NSUN5-mediated m5C modification of ACC1 mRNA[J]. Cell Death Differ，2023，30（12）：2462-2476.
[6] RESCIGNO P，GUREL B，PEREIRA R，et al. Characterizing CDK12-mutated prostate cancers[J]. Clin Cancer Res，2021，27（2）： 566-574.
[7] QI J C，YANG Z，LIN T，et al. CDK13 upregulation-induced formation of the positive feedback loop among circCDK13，miR-212-5p/miR-449a and E2F5 contributes to prostate carcinogenesis[J]. J Exp Clin Cancer Res，2021，40（1）：2.
[8] CESARI E，CIUCCI A，PIERACCIOLI M，et al. Dual inhibition of CDK12 and CDK13 uncovers actionable vulnerabilities in patient-derived ovarian cancer organoids[J]. J Exp Clin Cancer Res，2023， 42（1）：126.
[9] LU X L，ZHAN R，ZHAO G M，et al. Expression of CDK13 was associated with prognosis and expression of HIF-1α and beclin1 in breast cancer patients[J]. J Invest Surg，2022，35（2）：442-447.
[10] QUEREDA V，BAYLE S，VENA F，et al. Therapeutic targeting of CDK12/CDK13 in triple-negative breast cancer[J]. Cancer Cell，2019， 36（5）：545-558.e547.
[11] PENG F，YANG C，KONG Y，et al. CDK12 promotes breast cancer progression and maintains stemness by activating c-myc/β -catenin signaling[J]. Curr Cancer Drug Targets，2020，20（2）：156-165.
[12] WU C，XIE T，GUO Y，et al. CDK13 phosphorylates the translation machinery and promotes tumorigenic protein synthesis[J]. Oncogene，2023，42（16）：1321-1330.
[13] LIU H，SHIN S H，CHEN H，et al. CDK12 and PAK2 as novel therapeutic targets for human gastric cancer[J]. Theranostics，2020，10（14）：6201-6215.
[14] BARRANGOU R，FREMAUX C，DEVEAU H，et al. CRISPR provides acquired resistance against viruses in prokaryotes[J]. Science，2007，315（5819）：1709-1712.
[15] HORVATH P，ROMERO D A，CO?觠Té-MONVOISIN A C，et al. Diversity，activity，and evolution of CRISPR loci in Streptococcus thermophilus[J]. J Bacteriol，2008，190（4）：1401-1412.
[16] STERNBERG S H，REDDING S，JINEK M，et al. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9[J]. Nature，2014， 507（7490）：62-67.
[17] RAGURAM A，BANSKOTA S，LIU D R. Therapeutic in vivo delivery of gene editing agents[J]. Cell，2022，185（15）：2806-2827.
[18] VAN DER OOST J，PATINIOS C. The genome editing revolution[J]. Trends Biotechnol，2023，41（3）：396-409.
[19] CHEN M，MAO A，XU M，et al. CRISPR-Cas9 for cancer therapy：opportunities and challenges[J]. Cancer Lett，2019，447：48-55.
[20] CHENG X，FAN S，WEN C，et al. CRISPR/Cas9 for cancer treatment：technology，clinical applications and challenges[J]. Brief Funct Genomics，2020，19（3）：209-214.
[21] GHAEMI A，BAGHERI E，ABNOUS K，et al. CRISPR-cas9 genome editing delivery systems for targeted cancer therapy[J]. Life Sci，2021，267：118969.
[22] SHARMA G，SHARMA A R，BHATTACHARYA M，et al. CRISPR-Cas9：a preclinical and clinical perspective for the treatment of human diseases[J]. Mol Ther，2021，29（2）：571-586.
[23] ASGHAR U，WITKIEWICZ A K，TURNER N C，et al. The history and future of targeting cyclin-dependent kinases in cancer therapy[J]. Nat Rev Drug Discov，2015，14（2）：130-146.
[24] PACULOVá H，KOHOUTEK J. The emerging roles of CDK12 in tumorigenesis[J]. Cell Div，2017，12（1）：7.
[25] LIANG S，HU L，WU Z，et al. CDK12：a potent target and biomarker for human cancer therapy[J]. Cells，2020，9（6）：1483.
[26] BARTKOWIAK B，LIU P，PHATNANI H P，et al. CDK12 is a transcription elongation-associated CTD kinase，the metazoan ortholog of yeast Ctk1[J]. Genes Dev，2010，24（20）：2303-2316.
[27] BLAZEK D，KOHOUTEK J，BARTHOLOMEEUSEN K，et al. The Cyclin K/Cdk12 complex maintains genomic stability via regulation of expression of DNA damage response genes[J]. Genes Dev，2011， 25（20）：2158-2172.
[28] CHENG S W，KUZYK M A，MORADIAN A，et al. Interaction of cyclin-dependent kinase 12/CrkRS with cyclin K1 is required for the phosphorylation of the C-terminal domain of RNA polymeraseⅡ[J]. Mol Cell Biol，2012，32（22）：4691-4704.
[29] GREENLEAF A L. Human CDK12 and CDK13，multi-tasking CTD kinases for the new millenium[J]. Transcription，2019，10（2）：91-110.
[30] GREIFENBERG A K，H?魻NIG D，PILAROVA K，et al. Structural and Functional Analysis of the Cdk13/Cyclin K Complex[J]. Cell Rep，2016，14（2）：320-331.
[31] QIU M，YIN Z，WANG H，et al. CDK12 and Integrator-PP2A complex modulates LEO1 phosphorylation for processive transcription elongation[J]. Sci Adv，2023，9（20）：eadf8698.
[32] PANZERI V，PIERACCIOLI M，CESARI E，et al. CDK12/13 promote splicing of proximal introns by enhancing the interaction between RNA polymeraseⅡ and the splicing factor SF3B1[J]. Nucleic Acids Res，2023，51（11）：5512-5526.
[33] FAN Z，DEVLIN J R，HOGG S J，et al. CDK13 cooperates with CDK12 to control global RNA polymeraseⅡprocessivity[J]. Sci Adv，2020，6（18）：eaaz5041.
[34] INSCO M L，ABRAHAM B J，DUBBURY S J，et al. Oncogenic CDK13 mutations impede nuclear RNA surveillance[J]. Science，2023，380（6642）：eabn7625.
[35] ZHANG T，KWIATKOWSKI N，OLSON C M，et al. Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors[J]. Nat Chem Biol，2016，12（10）：876-884.

相似文献/References:

备注/Memo

备注/Memo:

基金项目: 天津市教委社科重大项目（2021JWZD22）
作者简介: 张榆（1999-），男，硕士在读，研究方向：肿瘤学；
通信作者：巴一，E-mail：bayi@pumch.cn。

常用功能

更新日期/Last Update: 2025-06-01