基于CRISPR/Cas9技术的法尼醇X受体缺失株的构建及其对HeLa细胞增殖的影响

doi:10.16431/j.cnki.1671-7236.2021.02.006

摘要/Abstract

摘要： 试验旨在利用CRISPR/Cas9基因编辑技术构建法尼醇X受体（FXR）基因稳定敲除细胞株，并考察其对HeLa细胞增殖的影响。根据CRISPR/Cas9靶点设计规则，设计3对特异性识别FXR基因第1外显子相关序列的上下游小向导RNA（small guide RNA，sgRNA），以PX459质粒为载体，构建真核重组表达质粒。酶切和测序鉴定后将重组质粒转染至HeLa细胞中，用嘌呤霉素进行阳性细胞筛选，再用实时荧光定量PCR检测HeLa细胞FXR基因敲除稳定株中FXR的表达量，并用Western blotting方法鉴定HeLa细胞FXR基因敲除效果。最后用结晶紫染色试验检测FXR基因敲除对HeLa细胞增殖的影响。结果显示，经测序后，3对sgRNA位置与方向均正确插入到PX459质粒载体内，成功构建出重组表达质粒PX459-sgRNA；实时荧光定量PCR和Western blotting检测结果显示，转染后筛选出的细胞中FXR蛋白不表达，表明构建出稳定敲除FXR基因的HeLa细胞株；结晶紫试验结果发现，FXR基因敲除的HeLa细胞染色深度明显低于正常HeLa细胞，FXR基因敲除HeLa细胞孔D_{595 nm}值极显著低于野生型HeLa细胞孔（P<0.01），说明HeLa细胞FXR基因敲除株的增殖能力较正常HeLa细胞显著降低。本试验利用CRISPR/Cas9技术成功获得了内源FXR基因敲除细胞株，且FXR基因敲除对癌细胞增殖具有显著抑制作用，为研究FXR的功能和作用机制提供了细胞模型，并为研究FXR在相关癌症发生发展中的作用奠定了基础。

关键词: CRISPR/Cas9; 法尼醇X受体(FXR); HeLa细胞; 细胞增殖

Abstract: This study was aimed to construct a stable FXR gene knockout cell line and its effect on HeLa cell proliferation by using CRISPR/Cas9 gene editing technique.According to the design rules of CRISPR/Cas9 target,three pairs of upstream and downstream small guide RNA (sgRNA) specifically identifying the first exon related sequence of FXR gene were designed,and the recombinant eukaryotic expression plasmid was constructed using PX459 plasmid as the vector.After enzymatic digestion and sequencing identification,the recombinant plasmid was transfected into HeLa cells,puromycin was used for positive cell screening,then the expression level of FXR in the stable FXR knockout strain of HeLa cells was detected by Real-time PCR,and the FXR knockout effect of cells was identified by Western blotting.Finally,the effect of FXR gene knockout on HeLa cell proliferation was detected by crystal violet staining.The results showed that after sequencing,three pairs of sgRNA were inserted in the correct position and direction PX459 plasmid vector,and successfully constructed the recombinant expression vector PX459-sgRNA.The results of Real-time PCR and Western blotting showed that FXR protein was not expressed in the selected cells after transfection,indicating that a stable FXR knockout cell line was constructed.Crystal violet results found FXR knockout HeLa dyeing depth was significantly lower than the normal HeLa.The D_{595 nm} value of FXR knockout HeLa cell hole was extremely significantly lower than that of wild-type HeLa cell hole (P<0.01),indicating that the proliferation capacity of FXR knockout HeLa cell strain was extremely significantly lower than that of normal HeLa cells.Endogenous FXR gene knockout cell lines were successfully obtained by CRISPR/Cas9 technology,and FXR gene had inhibitory effect on the proliferation of cancer cells.This experiment provided an effective tool for studying the function and mechanism of FXR and laid a foundation for studying the occurrence and development of FXR in related cancers.

Key words: CRISPR/Cas9; farnesiod receptor (FXR); HeLa cell; cell proliferation

中图分类号:

马方雪, 安雅男, 王超, 李媛, 栾文静, 王雪飞, 倪丽慧, 周宏, 梁俊超, 于录. 基于CRISPR/Cas9技术的法尼醇X受体缺失株的构建及其对HeLa细胞增殖的影响[J]. 中国畜牧兽医, 2021, 48(2): 450-456.

MA Fangxue, AN Yanan, WANG Chao, LI Yuan, LUAN Wenjing, WANG Xuefei, NI Lihui, ZHOU Hong, LIANG Junchao, YU Lu. Construction of FXR Deletion Strain Based on CRISPR/Cas9 Technology and Its Effect on HeLa Cell Proliferation[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(2): 450-456.

参考文献

[1] SINAL C J,TOHKIN M,MIYATA M,et al.Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis[J].Cell,2000,102(6):731-744.
[2] MASSAFRA V,VAN M S.Farnesoid X receptor:A "homeostat" for hepatic nutrient metabolism[J].2018,1864(1):45-59.
[3] WANG H,CHEN J,HOLLISTER K,et al.Endogenous bile acids are ligands for the nuclear receptor FXR/BAR[J].Cell,1999,3(5):543-553.
[4] HOU Y,FAN W,YANG W,et al.Farnesoid X receptor:An important factor in blood glucose regulation[J].Clinica Chimica Acta,2019,495:29-34.
[5] 徐俐洁,蒋思达,季晖,等.胆汁酸核受体FXR在胆汁淤积及其药物治疗中的研究进展[J].药学研究,2018,37(3):169-173. XU L J,JIANG S D,JI H,et al.Role of bile acid nuclear receptor FXR in cholestasis and related pharmacotherapy[J].Journal of Pharmaceutical Research,2018,37(3):169-173.
[6] ZHANG Y,LEE F Y,BARRERA G,et al.Activation of the nuclear receptor FXR improves hyperglycemia and hyperlipid-emia in diabetic mice[J].Proceedings of the National Academy of Sciences of the United States of America,2006,103(4):1006-1011.
[7] ROMEO S.Notch and nonalcoholic fatty liver and fibrosis[J].The New England Journal of Medicine,2019,380(7):681-683.
[8] YUAN A,MA X,He B,et al.Cardiomyocyte-expressed farnesoid-X-receptor is a novel apoptosis mediator and contributes to myocardial ischaemia/reperfusion injury[J].European Heart Journal:The Journal of the European Society of Cardiology,2013,34(24):1834-1845.
[9] SWALES K E,KORBONITS M,CARPENTER R,et al.The farnesoid X receptor is expressed in breast cancer and regulates apoptosis and aromatase expre-ssion[J].Cancer Research,2006,66(20):10120-10126.
[10] JOSE J M,MARIA J M,ALBA G B,et al.The role of reduced intracellular concentrations of active drugs in the lack of response to anticancer chemotherapy[J].Acta Pharmacologica Sinica,2014,35(1):1-10.
[11] YU H,GUANG Q L,JIN Z D,et al.Comparison and optimization of CRISPR/dCas9/gRNA genome-labeling systems for live cell imaging[J].Genome Biology,2018,19(1):39.
[12] BARRANGOU R,FREMAUX C,DEVEAU H,et al.CRISPR provides acquired resistance against viruses in prokaryotes[J].Science,2007,315(5819):1709-1712.
[13] PLAZA R A,LANNER F.Towards a CRISPR view of early human development:Applications,limitations and ethical concerns of genome editing in human embryos[J].Development,2017,144(1):3-7.
[14] GARNEAU J,DUPUIS M,VILLION M,et al.The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA[J].Nature,2010,468(7320):67-71.
[15] DELTCHEVA E,CHYLINSKI K,SHARMA C,et al.CRISPR RNA maturation by trans-encoded small RNA and host factor RNase Ⅲ[J].Nature,2011,471(7340):602-607.
[16] HORII T,HATADA I.Genome editing using mammalian haploid cells[J].International Journal of Molecular Science,2015,16(10):23604-23614.
[17] MODICA S,BELLAFANTE E,MOSCHETTA A.Master regulation of bile acid and xenobiotic metabolism via the FXR,PXR and CAR trio[J].Frontiers in Bioscience (Landmark Edition),2009,14:4719-4745.
[18] MIYATA M,SAKAIDA Y,MATSUZAWA H,et al.Fibroblast growth factor 19 treatment ameliorates disruption of hepatic lipid metabolism in farnesoid X receptor (Fxr)-null mice[J].Biological & Pharmaceutical Bulletin,2011,34(12):1885-1889.
[19] EVANS M J,MAHANEY P E,BORGES-MARCUCCI L,et al.A synthetic farnesoid X receptor (FXR) agonist promotes cholesterol lowering in models of dyslipidemia[J].American Journal of Physiology-Gastrointest and Liver Physiology,2009,296(3):G543-G552.
[20] GAUTIER T,DE H W,GROBER J,et al.Farnesoid X receptor activation increases cholesteryl ester transfer protein expression in humans and transgenic mice[J].Journal of Lipid Research,2013,54(8):2195-2205.
[21] DENG Y,WANG H,LU Y,et al.Identification of chemerin as a novel FXR target gene down-regulated in the progression of nonalcoholic steatohepatitis[J].Endocrinology,2013,154(5):1794-1801.
[22] LIU X,WANG M,QIN Y,et al.Targeted integration in human cells through single crossover mediated by ZFN or CRISPR/Cas9[J].BMC Biotechnology,2018,18(1):66.
[23] HE Z,PROUDFOOT C,WHITELAW C B A,et al.Comparison of CRISPR/Cas9 and TALENs on editing an integrated EGFP gene in the genome of HEK293T cells[J].Springerplus,2016,5(1):814.
[24] MALI P,YANG L,ESVELT K M,et al.RNA-guided human genome engineering via Cas9[J].Science,2013,339(6121):823-826.
[25] GILBERT L A,LARSON M H,MORSUT L,et al.CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes[J].Cell,2013,154(2):442-451.
[26] LONE B A,KARNA S,AHMAD F,et al.CRISPR/Cas9 system:A bacterial tailor for genomic engineering[J].Genetics and Molecular Research,2018,2018:3797214.
[27] WANG H,YANG H,SHIYALILA C S,et al.One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering[J].Cell,2013,153(4):910-918.
[28] 王姗姗,杨军,王中,等.利用CRISPR/Cas9技术的烟草NtLS基因敲除分析[J].烟草科技,2018,51(2):1-8. WANG S S,YANG J,WANG Z,et al.Targeted mutagenesis of NtLS gene using CRISPR/Cas9 system[J].Tobacco Science & Technology,2018,51(2):1-8.(in Chinese)
[29] PLATT R J.CRISPR-Cas9 knockin mice for genome editing and cancer modeling[J].Cell,2014,159(2):440-455.