«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:

29卷

期数:

2023年05期

页码:

500-506

栏目:

基础医学

出版日期:

2023-09-20

文章信息/Info

Title:

Construction of FOXQ1 knockout MCF7 breast cancer cell line and itspreliminary functional exploration

文章编号:

1006-8147（2023）05-0500-07

作者:

杨欢; 冯玉梅

（天津医科大学肿瘤医院肿瘤研究所生物化学与分子生物学研究室；国家肿瘤临床医学研究中心；天津市“肿瘤防治”重点实验室；天津市恶性肿瘤临床医学研究中心；乳腺癌防治教育部重点实验室，天津300060）

Author(s):

YANG Huan;  FENG Yu-mei

（Department of Biochemistry and Molecular Biology，Cancer Institute and Hospital，Tianjin Medical University；National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy; Tianjin， Tianjin′s Clinical Research Center for Cancer; Key Laboratory of Breast Cancer Prevention and Therapy，Tianjin Medical University， Ministry of Education， Tianjin 300060，China）

关键词:

CRISPR/Cas9; FOXQ1; 基因敲除; 乳腺癌; 缺氧

Keywords:

CRISPR/Cas9; FOXQ1; gene knockout; breast cancer; hypoxia

分类号:

R737.9

DOI:

-

文献标志码:

A

摘要:

目的：通过CRISPR/Cas9基因编辑技术在乳腺癌细胞MCF7中稳定敲除FOXQ1，并初探其在缺氧环境中的功能。方法：Cas9慢病毒感染MCF7构建工具细胞，在工具细胞中分别转染靶向3个不同靶位点的FOXQ1-crRNA+tracrRNA，用T7E1酶筛选出编辑效率高的细胞挑取单克隆细胞株；利用T7E1酶、Sanger测序和Western印迹检测FOXQ1基因的编辑和表达情况；Western印迹检测常氧/缺氧条件下细胞FOXQ1表达情况；在常氧/缺氧条件下通过划痕实验和transwell实验检测细胞迁移能力；通过METABRIC数据库分析乳腺癌中FOXQ1与HIF1A及其编码蛋白靶基因LOX、LDHA和GLUT3表达的相关性。结果：筛选出转染靶向CCCGTGCGCATCCAGGACAT序列的FOXQ1-crRNA的细胞具有最高编辑效率；T7E1酶切、Sanger测序和Western印迹结果显示稳定敲除FOXQ1的乳腺癌MCF7细胞构建成功；缺氧促进MCF7细胞FOXQ1的表达；缺氧促进MCF7的迁移能力（t=3.78，P<0.01；t=11.94，P<0.001），敲除FOXQ1后缺氧对MCF7迁移能力的促进作用被抑制（t=0.63，P=0.55；t=2.54，P=0.064）、（t=2.46，P<0.05；t=4.95，P<0.05），乳腺癌中FOXQ1与HIF1A、LOX、LDHA和GLUT3的表达均显著正相关（P<0.000 1）。结论：通过CRISPR/Cas9系统成功构建了FOXQ1基因稳定敲除的乳腺癌MCF7细胞株，并发现FOXQ1介导了缺氧对MCF7细胞迁移能力的促进作用，为进一步探究乳腺癌的缺氧微环境中FOXQ1的功能及其机制奠定了基础。

Abstract:

Objective：To stably knock out FOXQ1 using CRISPR/Cas9 gene editing technology in MCF7 breast cancer cells and to explore its function in hypoxic conditions. Methods：Cas9 lentivirus was infected with MCF7 cells to create tool cells， and FOXQ1-crRNA+tracrRNA targeting three different target sites were transfected into the tool cells. T7E1 enzyme was used to screen for cells with high editing efficiency， and single clone cell lines were selected. T7E1 enzyme， Sanger sequencing， and Westernblotting were used to detect the editing and expression of the FOXQ1 gene. Western blotting was used to detect FOXQ1 expression in cells under normoxic/hypoxic conditions. The wound healing and transwell assay were used to detect cell migration ability under normoxic/hypoxic conditions. The METABRIC database was used to analyze the correlation between FOXQ1 and HIF1A， as well as its encoded protein target genes LOX， LDHA and GLUT3 in breast cancer. Results：Cells transfected with FOXQ1-crRNA targeting the CCCGTGCGCATCCAGGACAT sequence showed the highest editing efficiency. T7E1 enzyme cleavage， Sanger sequencing， and Western blotting results confirmed the successful construction of stable FOXQ1-knockout MCF7 breast cancer cells. Hypoxia promoted the expression of FOXQ1 in MCF7 cells and also promoted their migration ability（t=3.78，P<0.01；t=11.94，P<0.001）. Knocking out FOXQ1 inhibited the promoting effect of hypoxia on MCF7 migration（t=0.63，P=0.55；t=2.54，P=0.064）， （t=2.46，P<0.05；t=4.95，P<0.05）.FOXQ1 expression in breast cancer was significantly positively correlated with HIF1A，LOX，LDHA，and GLUT3（P<0.000 1）. Conclusion：Astable FOXQ1-knockout MCF7 breast cancercell line has beensuccessfully constructed using the CRISPR/Cas9 system. It is found that FOXQ1 mediatesthe promoting effect of hypoxia on MCF7 cells migrationability， laying the foundation for further exploration of the function and mechanism of FOXQ1 in the hypoxic microenvironment of breast cancer.

参考文献/References:

[1] VOSS M J，M?魻LLER M F，POWE D G，et al.Luminal and basal-like breast cancer cells show increased migration induced by hypoxia，mediated by an autocrine mechanism[J]. BMC Cancer，2011，11：158.
[2] VAUPEL P，MAYER A，HOCKEL M. Tumour hypoxia and malignant progression[J]. Methods Enzymol，2004，381：335-354.
[3] VAUPEL P. Prognostic potential of the pre-therapeutic tumour oxygenation status[J]. Adv Exp Med Biol，2009，645：241-246.
[4] RANKIN E B，GIACCIA A J. Hypoxic control of metastasis[J]. Science，2016，352（6282）：175-180.
[5] DENGLER V L，GALBRAITH M D，ESPINOSA J M. Transcriptional regulation by hypoxia inducible factors[J]. Crit Rev Biochem Mol Biol，2014，49：1-15.
[6] LIU Z J，SEMENZA G L，ZHANG H F. Hypoxia-inducible factor 1 and breast cancer metastasis[J]. J Zhejiang Univ Sci B，2015，16（1）：32-43.
[7] WANG J，LI W，ZHAO Y，et al.Members of FOX family could be drug targets of cancers[J]. Pharmacol Ther ，2018，181：183-196.
[8] RAEIS V，PHILIP-COUDERC P，ROATTIA，et al. Central venous hypoxemia is a determinant of human atrial ATP-sensitive potassium channel expression：evidence for a novel hypoxia-inducible factor 1alpha-Forkhead box class O signaling pathway[J]. Hypertension，2010，55（5）：1186-1192.
[9] ABDELHAFIZ A S，FOUAD M A，SAYED-AHMED M M，et al.Upregulation of FOXP3 is associated with severity of hypoxia and poor outcomes in COVID-19 patients[J]. Virology，2021，563：74-81.
[10] WANG X H，JIANG Z H，YANG H M，et al. Hypoxia-induced FOXO4/LDHA axis modulates gastric cancer cell glycolysis and progression[J]. Clin Transl Med，2021，11（1）：e279.
[11] TAO Y，LIU Q，WU R，et al. Long noncoding RNA LUCAT1 enhances the survival and therapeutic effects of mesenchymal stromal cells post-myocardial infarction[J]. Mol Ther Nucleic Acids，2021，27：412-426.
[12] FENG J，ZHANG X，ZHU H，et al. FoxQ1 overexpression influences poor prognosis in non-small cell lung cancer，associates with the phenomenon of EMT[J]. PLoS ONE，2012，7：e39937.
[13] QIAO Y，JIANG X，LEE S T，et al. FOXQ1 regulates epithelial-mesenchymal transition in human cancers[J]. Cancer Res，2011，71（8）：3076-3086.
[14] GAO M，SHIH IEM，WANG T L. The role of forkhead box Q1 transcription factor in ovarian epithelial carcinomas[J]. Int J Mol Sci，2012，13：13881-13893.
[15] ZHANG H，MENG F，LIU G，et al. Forkhead transcription factor foxq1 promotes epithelial-mesenchymal transition and breast cancer metastasis[J]. Cancer Res，2011，71（4）：1292-301.
[16] MALI P，YANG L，ESVELT K M，et al. RNA-guided human genome engineering via Cas9 [J]. Science，2013，339（6121）：823-826.
[17] 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.
[18] WYMAN C，KANAAR R. DNA double-strand break repair：all's well that ends well [J]. Annu Rev Genet，2006，40：363-383.
[19] SAATCI O，KAYMAK A，RAZA U，et al. Targeting lysyl oxidase（LOX） overcomes chemotherapy resistance in triple negative breast cancer [J]. Nat Commun，2020，11（1）：2416.
[20] HU D，LINDERS A，YAMAK A，et al. Metabolic maturation of human pluripotent stem cell-derived cardiomyocytes by inhibition of HIF1α and LDHA [J]. Circ Res，2018，123（9）：1066-1079.
[21] MIMURA I，NANGAKU M，KANKI Y，et al.Dynamic change of chromatin conformation in response to hypoxia enhances the expression of GLUT3（SLC2A3） by cooperative interaction of hypoxia-inducible factor 1 and KDM3A[J]. Mol Cell Biol，2012，32（15）：3018-3032.
[22] CONG L，RAN F A，COX D，et al. Multiplex genome engineering using CRISPR/Cas systems [J]. Science，2013，339（6121）：819-823.
[23] 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.
[24] ZHU H，WANG D，ZHANG L，et al. Upregulation of autophagy by hypoxia-inducible factor-1α promotes EMT and metastatic ability of CD133+ pancreatic cancer stem-like cells during intermittent hypoxia[J]. Oncol Rep，2014，32（3）：935-942.
[25] YANG M H，WU K J. TWIST activation by hypoxia inducible factor-1（HIF-1）：implications in metastasis and development[J]. Cell Cycle，2008，7：2090-2096.
[26] ABBA M，PATIL N，RASHEED K，et al. Unraveling the role of FOXQ1 in colorectal cancer metastasis[J]. Mol Cancer Res，2013，11（9）：1017-1028.
[27] ZHANG J，LIU Y，ZHANG J，et al.FOXQ1 promotes gastric cancer metastasis through upregulation of Snail[J]. Oncol Rep，2016，35（6）：3607-3613.
[28] XIA L，HUANG W，TIAN D，et al. Forkhead box Q1 promotes hepatocellular carcinoma metastasis by transactivating ZEB2 and VersicanV1 expression[J]. Hepatology，2014，59（3）：958-973.

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(05):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(05):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(05):53.
[4]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(05):372.
[5]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(05):128.[doi:10.20135/j.issn.1006-8147.2024.02.0128]

备注/Memo

备注/Memo:

基金项目 国家自然科学基金资助项目（82173349）
作者简介 杨欢（1996-），女，硕士在读，研究方向：肿瘤与分子生物学；通信作者：冯玉梅，E-mail：ymfeng@tmu.edu.cn。

常用功能

更新日期/Last Update: 2023-09-25