«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04期

页码:

308-314，348

栏目:

肿瘤疾病专题

出版日期:

2025-07-10

文章信息/Info

Title:

Effects of knockdown of Ldha or Ldhb on the growth in Mll4-deleted mouse melanoma

文章编号:

1006-8147(2025)04-0308-08

作者:

余锐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 300070；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):

YU Rui¹;  DENG Ting¹;  HU Deqing¹; 2;  BA Yi¹; 3

关键词:

CRISPR/Cas9; 乳酸脱氢酶; 肿瘤微环境

Keywords:

CRISPR/Cas9;  lactate dehydrogenase;  tumor microenvironment

分类号:

R3

DOI:

10.20135/j.issn.1006-8147.2025.04.0308

文献标志码:

A

摘要:

目的：利用CRISPR/Cas9系统构建Ldha、Ldhb、Ldha-LdhbDKO的B16 Mll4KO细胞系并验证乳酸脱氢酶（LDH）A和B基因敲除对肿瘤生长的影响。方法：设计靶向Ldha和Ldhb的sgRNA，利用CRISPR-Cas9技术，敲除Ldha、ldhb以及Ldha-ldhbDKO，获得LdhaKO、LdhbKO、Ldha-LdhbDKO的细胞系，进而探索LDHA与LDHB两种乳酸脱氢酶基因敲除对肿瘤生长的影响。基因型鉴定验证基因的缺失情况，qPCR和蛋白印迹实验验证基因的敲除效果，记录小鼠皮下植瘤的生长情况。结果：基因型鉴定和蛋白印迹实验：与亲代B16或M25细胞系相比，Ldha/LdhbKO或Ldha-LdhbDKO的M25细胞系中Ldha（t=11.51，P<0.001）以及Ldhb（t=36.26，P<0.001）明显减少，成功构建了LdhaKO、LdhbKO、Ldha-LdhbDKO的细胞系。qPCR实验：Ldhb基因的mRNA水平明显下降（t=51.36，P<0.001）。皮下植瘤实验结果表明LdhaKO对野生型黑色素瘤（t=0.012 13，P=0.990 5）以及Mll4缺失的肿瘤（t=0.040 22，P=0.968 5）生长没有明显的影响；缺失Ldhb后，Mll4缺失的肿瘤生长明显减慢（t=3.225，P<0.01）。结论：成功构建了Ldha、Ldhb基因敲除的细胞系，LdhaKO对小鼠皮下植瘤的生长没有影响，LdhbKO抑制B16Mll4KO细胞系肿瘤生长。

Abstract:

Objective： To construct B16 Mll4KO cell lines with Ldha， Ldhb and Ldha-LdhbDKO using CRISPR/Cas9 system， and verify the effects of lactate dehydrogenase（LDH） A and B gene knockout on tumor growth. Methods： sgRNA targeting Ldha and Ldhb were designed， and CRISPR-Cas9 technology was used to knock out Ldha， Ldhband Ldha-LdhbDKO to obtain LdhaKO， LdhbKO and Ldha-LdhbDKO cell lines， and then to explore the effects of LDHA and LDHB lactate dehydrogenase gene knockout on tumor growth. Genotype identification was used to verify the deletion of genes， qPCR and Western blotting experiments were conducted to verify the knockout effect of genes， and finally the growth of tumors was recorded by subcutaneous tumor implantation. Results： Genotype identification and Western blotting experiments： compared with the parental B16 or M25 cell lines， the Ldha（t=11.51， P<0.001） and Ldhb（t=36.26， P<0.001） were significantly reduced in the M25 cell lines of Ldha/LdhbKO or Ldha-LdhbDKO， demonstrating the cell lines of LdhaKO， LdhbKO and Ldha-LdhbDKO were successfully constructed. qPCR experiments： the mRNA level of Ldhb gene was significantly decreased（t=51.36， P<0.001）. The results of subcutaneous tumor implantation showed that LdhaKO had no significant effect on the growth of wild-type melanoma（t=0.012 13， P=0.990 5） and Mll4-deficient tumors（t=0.040 22，P=0.968 5）. However， the tumor growth of Mll4 deletion was significantly slowed after Ldhb deletion（t=3.225，P<0.01）. Conclusion： Ldha and Ldhb knockout cell lines are successfully constructed， subcutaneous tumor implantation in mice showes that LdhaKO has no effect on tumor growth， and LdhbKO reduce the tumor growth of B16Mll4KO cell line.

参考文献/References:

[1] KHAN F， LIN Y， ALI H， et al. Lactate dehydrogenase a regulates tumor-macrophage symbiosis to promote glioblastoma progression[J]. Nat Commun， 2024， 15（1）： 1987.
[2] XIE H， HANAI J I， REN J G， et al. Targeting lactate dehydrogenase-a inhibits tumorigenesis and tumor progression in mouse models of lung cancer and impacts tumor-initiating cells[J]. Cell Metab， 2014， 19（5）： 795-809.
[3] ZHANG P， WAN Y， MA J， et al. Epigenetic silencing of LDHB promotes hepatocellular carcinoma by remodeling the tumor microenvironment[J]. Cancer Immunol Immun， 2024， 73（7）： 127.
[4] NING H， HUANG S， LEI Y， et al. Enhancer decommissioning by MLL4 ablation elicits dsRNA-interferon signaling and GSDMD-mediated pyroptosis to potentiate anti-tumor immunity[J]. Nat Commun， 2022， 13（1）： 6578.
[5] SHENG W， LIU Y， CHAKRABORTY D， et al. Simultaneous inhibition of LSD1 and TGFβ enables eradication of poorly immunogenic tumors with anti-PD-1 treatment[J]. Cancer Discov， 2021， 11（8）： 1970-1981.
[6] PICKAR-OLIVER A， GERSBACH C A. The next generation of CRISPR-Cas technologies and applications[J]. Nat Rev Mol Cell Biol， 2019， 20（8）： 490-507.
[7] HUANG J， ZHOU Y， LI J， et al. CRISPR/Cas systems： delivery and application in gene therapy[J]. Front Bioeng Biotechnol， 2022， 10：942325.
[8] MOHANRAJU P， MAKAROVA K S， ZETSCHE B， et al. Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems[J]. Science， 2016， 353（6299）：556.
[9] CUI Y， XU J， CHENG M， et al. Review of CRISPR/Cas9 sgRNA design tools[J]. Interdiscip Sci， 2018， 10（2）： 455-465.
[10] MAKAROVA K S， HAFT D H， BARRANGOU R， et al. Evolution and classification of the CRISPR-Cas systems[J]. Nat Rev Microbiol， 2011， 9（6）： 467-477.
[11] WRIGHT A V， NUNEZ J K， DOUDNA J A. Biology and applicati-ons of CRISPR systems： harnessing nature′s toolbox for genome engineering[J]. Cell， 2016， 164（1-2）： 29-44.
[12] DELTCHEVA E， CHYLINSKI K， SHARMA C M， et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III[J]. Nature， 2011， 471（7340）： 602-607.
[13] SáNCHEZ-RIVERA F J， JACKS T. Applications of the CRISPR-Cas9 system in cancer biology[J]. Nat Rev Cancer， 2015， 15（7）： 387-393.
[14] CHUNG S H， SIN T N， DANG B， et al. CRISPR-based VEGF suppression using paired guide RNAs for treatment of choroidal neovascularization[J]. Mol Ther Nucl Acids， 2022， 28： 613-622.
[15] WANG S W， GAO C， ZHENG Y M， et al. Current applications and future perspective of CRISPR/Cas9 gene editing in cancer[J]. Mol Cancer， 2022， 21（1）：57.
[16] CLAPS G， FAOUZI S， QUIDVILLE V， et al. The multiple roles of LDH in cancer[J]. Nat Rev Clin Oncol， 2022， 19（12）： 749-762.
[17] MACCHI C， MOREGOLA A， GRECO M F， et al. Monocarboxylate transporter 1 deficiency impacts CD8+ T lymphocytes proliferation and recruitment to adipose tissue during obesity[J]. iScience， 2022， 25（6）：104435.
[18] BRAND A， SINGER K， KOEHL G E， et al. LDHA-associated lactic acid production blunts tumor immunosurveillance by T and NK cells[J]. Cell Metab， 2016， 24（5）： 657-671.
[19] SCOTT KRISTEN E N， CLEVELAND JOHN L. Lactate wreaks havoc on tumor-infiltrating T and NK cells[J]. Cell Metab， 2016， 24（5）： 649-650.
[20] BRONTE V. Tumor cells hijack macrophages via lactic acid[J]. Immunol Cell Biol， 2014， 92（8）： 647-649.

相似文献/References:

[1]刘郁莹,崔晓腾,高星杰,等.利用改良的CRISPR/Cas9基因编辑系统构建HeLa细胞SND1基因敲除稳定株[J].天津医科大学学报,2015,21(06):480.
　LIU Yu-ying,CUI Xiao-teng,GAO Xing-jie,et al.Construction of HeLa SND1 knockout gene stable strain by using modified CRISPR/Cas9 gene editing system[J].Journal of Tianjin Medical University,2015,21(04):480.
[2]张 静,高 雅,李新宇,等.SUZ12过表达及敲减恶性外周神经鞘瘤稳定细胞株的建立及其意义[J].天津医科大学学报,2019,25(05):429.
　ZHANG Jing,GAO Ya,LI Xin-yu,et al.Establishment and significance of SUZ12 overexpression and knockdown of stable malignant peripheral nerve sheath tumor cell lines[J].Journal of Tianjin Medical University,2019,25(04):429.
[3]武晓静,陈玉霞,麻献华,等.利用CRISPR/Cas9技术体内标记示踪小鼠内源性NPC1L1蛋白[J].天津医科大学学报,2022,28(01):53.
　WU Xiao-jing,CHEN Yu-xia,MA Xian-hua,et al.Genetic labeling and tracing of mouse endogenous NPC1L1 protein by CRISPR/Cas9 technology in vivo[J].Journal of Tianjin Medical University,2022,28(04):53.
[4]况兆忠,裴彧,李会强,等.肺炎支原体肺炎患儿外周血CD64及心肌酶与血清MP-IgM抗体滴度的相关性[J].天津医科大学学报,2022,28(02):177.
　KUANG Zhao-zhong,PEI Yu,WEI Dian-jun,et al.Correlation between peripheral blood CD64, myocardial enzyme and serum MP-IgM antibody titer in children with Mycoplasma pneumoniae pneumonia[J].Journal of Tianjin Medical University,2022,28(04):177.
[5]张佳慧,阎晗,胡德庆.利用CRISPR/Cas9技术构建Aff4基因敲除B16-F10细胞系及AFF4的多克隆抗体制备[J].天津医科大学学报,2023,29(04):372.
　ZHANG Jia-hui,YAN Han,HU De-qing.Aff4 gene knockout stable B16-F10 cell line generation with CRISPR/Cas9 system and anti-AFF4 polyclonal antibody preparation[J].Journal of Tianjin Medical University,2023,29(04):372.
[6]杨欢,冯玉梅.FOXQ1敲除乳腺癌MCF7细胞株的构建及其功能初探[J].天津医科大学学报,2023,29(05):500.
　YANG Huan,FENG Yu-mei.Construction of FOXQ1 knockout MCF7 breast cancer cell line and itspreliminary functional exploration[J].Journal of Tianjin Medical University,2023,29(04):500.
[7]董玥湘,刘岩,靳小石,等.应用CRISPR/Cas9技术敲除SLC5A8基因对黑色素瘤的影响[J].天津医科大学学报,2024,30(02):128.[doi:10.20135/j.issn.1006-8147.2024.02.0128]
　DONG Yuexiang,LIU Yan,JIN Xiaoshi,et al.Effect of knockdown of SLC5A8 gene on melanoma by applying CRISPR/Cas9 technology[J].Journal of Tianjin Medical University,2024,30(04):128.[doi:10.20135/j.issn.1006-8147.2024.02.0128]
[8]于艺晞,胡德庆.靶向SUPT6小鼠胚胎干细胞dTAG细胞系的建立及功能验证[J].天津医科大学学报,2025,31(03):231.[doi:10.20135/j.issn.1006-8147.2025.03.0231]
　YU Yixi,HU Deqing.Establishment and functional verification of mouse embryonic stem cell dTAG cell line targeting SUPT6[J].Journal of Tianjin Medical University,2025,31(04):231.[doi:10.20135/j.issn.1006-8147.2025.03.0231]
[9]张榆,邓婷,胡德庆,等.敲除Cdk12/Cdk13基因对小鼠黑色素瘤细胞迁移及增殖能力的影响[J].天津医科大学学报,2025,31(03):210.[doi:10.20135/j.issn.1006-8147.2025.03.0210]
　ZHANG Yu,DENG Ting,HU Deqing,et al.Effect of Cdk12/Cdk13 knockout on migration and proliferation of mouse melanoma cell[J].Journal of Tianjin Medical University,2025,31(04):210.[doi:10.20135/j.issn.1006-8147.2025.03.0210]

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:余锐（1999-），男，硕士在读，研究方向：肿瘤学；通信作者：巴一，E-mail：bayi@tjmuch.com。

常用功能

更新日期/Last Update: 2025-07-10