CRISPR/Cas9载体优化及CHD1L条件性敲除肝癌细胞系的构建

doi:10.13418/j.issn.1001-165x.2020.01.009

中国临床解剖学杂志 ›› 2020, Vol. 38 ›› Issue (1) : 39-44. DOI: 10.13418/j.issn.1001-165x.2020.01.009

实验研究

CRISPR/Cas9载体优化及CHD1L条件性敲除肝癌细胞系的构建

黄丽¹，　刘珊珊²，　张晓锋¹，　白银山³，　刘铭¹

作者信息 +

The CRISPR/Cas9 system optimization and construction of CHD1L conditional knockout liver cancer cell lines

HUANG Li¹, LIU Shan-shan², ZHANG Xiao-feng¹, BAI Yin-shan³, LIU Ming¹

Author information +

文章历史 +

摘要

目的　利用优化的pLV-Tet3G-CRISPR/Cas9载体系统构建条件性敲除CHD1L的QGY-7703肝癌细胞系，验证敲除效果及其对细胞生物学的影响，为研究CHD1L促肿瘤细胞恶性表型机制提供重要的细胞模型。方法　用点突变方法优化pLV-Tet3G-Cas9载体以降低其脱靶效应，进而将Cas9改造成eSpCas9；其次，通过筛选获得稳定表达eSpCas9的QGY-7703细胞株；将mCherry基因插入载体pLVX-hU6-SgRNA中，获得携带mCherry荧光基因的pLVX-mCherry-hU6-SgRNA载体；设计和筛选特异性靶向CHD1L的SgRNA序列，用重叠PCR方法获得hU6-CHD1L-SgRNA片段，筛选具有CHD1L切割活性的靶点，随后，将其克隆到pLVX-mCherry-hU6-SgRNA载体中；用293FT细胞进行病毒包装，获得慢病毒颗粒；转染7703eSpCas9细胞株，利用Western blot验证Dox诱导下的CHD1L敲除效果，划痕和Transwell实验检测Dox诱导的CHD1L敲除对肝癌细胞生物学功能的影响。结果　pLV-Tet3G-Cas9载体Cas9成功优化为eSpCas9序列；成功构建pLVX-mCherry-hU6-CHD1L-SgRNA载体；通过转染及筛选，获得Dox诱导的CHD1L敲除QGY-7703肝癌细胞系；细胞实验显示Dox可诱导eSpCas9表达，靶向性切割CHD1L，抑制QGY-7703细胞的迁移侵袭。结论　成功构建Dox诱导的CHD1L敲除肝癌细胞株，此载体系统可为靶向肿瘤特异性基因研究提供细胞模型。

Abstract

Objective　To verify the effect of CHD1L QGY-7703 hepatoma cell line knockout and the effect on the cell biological functions and then to provide important cells models for studying the role of CHD1L in the development of mechanism of malignant phenotype in cancer cell by using the optimized pLV-Tet3G-CRISPR/Cas9 technology to construct a QGY-7703 hepatoma cell line.　Methods　pLV-Tet3G-Cas9 vector was optimized by point mutation technique to reduce its off-target effect and increase specificity. Cas9 was transformed into eSpCas9. Then, QGY-7703 cell line stably expressing eSpCas9 was obtained by screening. pLVX-mCherry-hU6-SgRNA vector carrying mCherry fluorescent gene was obtained by inserting mCherry gene into pLVX-hU6-SgRNA vector. The sgRNA sequence targeting the CHD1L gene was designed and screened, and the targeting sequence of Hu6-CHD1L-sgRNA was obtained by overlapping PCR method. Cloning the site with cleavage activity into the pLVX-mCherry-hU6-SgRNA vector. The vector was packaged as lentivirus by lentiviral packaging method and transfected into 7703 eSpCas9 cell line. The CHD1L knockout induced by Dox was verified by Western blot. The effects of Dox-induced CHD1L knockout on the biological function of hepatocellular carcinoma cells were detected by Wound Healing and Transwell assay. Results　In this experiment, vector Cas9 of pLV-Tet3G-Cas9 was successfully optimized as sequence eSpCas9. pLVX-mCherry-hU6-CHD1L-SgRNA vector was constructed. The QGY-7703 liver cancer cell line that conditional induction CHD1L gene knocking out was successfully constructed. Cell function experiments showed that the expression of eSpCas9 can be induced by Dox, the migration and invasion ability of these hepatoma cells were significantly decreased after knockout of CHD1L.　Conclusions　This study successfully constructed a conditional CHD1L knockout QGY-7703 liver cancer cell line. This model can be used as an effective tool to study the function and mechanism of specific genes targeting tumors.

导出引用

黄丽, 刘珊珊, 张晓锋, 白银山, 刘铭. CRISPR/Cas9载体优化及CHD1L条件性敲除肝癌细胞系的构建[J]. 中国临床解剖学杂志. 2020, 38(1): 39-44 https://doi.org/10.13418/j.issn.1001-165x.2020.01.009

HUANG Li, LIU Shan-shan, ZHANG Xiao-feng, BAI Yin-shan, LIU Ming. The CRISPR/Cas9 system optimization and construction of CHD1L conditional knockout liver cancer cell lines[J]. Chinese Journal of Clinical Anatomy. 2020, 38(1): 39-44 https://doi.org/10.13418/j.issn.1001-165x.2020.01.009

中图分类号： R730.2

参考文献

[1] Ma NF, Hu L, Fung JM, et al. Isolation and characterization of a novel oncogene, amplified in liver cancer 1, within a commonly amplified region at 1q21 in hepatocellular carcinoma[J]. Hepatology, 2008, 47(2): 503-510.
[2] Wu J, Zong Y, Fei X, et al. Presence of CHD1L over-expression is associated with aggressive tumor biology and is a novel prognostic biomarker for patient survival in human breast cancer[J]. PLoS One, 2014, 9(8): e98673.
[3] Su Z, Zhao J, Xian G, et al. CHD1L is a novel independent prognostic factor for gastric cancer[J]. Clin Transl Oncol, 2014, 16(8): 702-707.
[4] Ji X, Li J, Zhu L, et al. CHD1L promotes tumor progression and predicts survival in colorectal carcinoma[J]. J Surg Res, 2013, 185(1): 84-91.
[5] He WP, Zhou J, Cai MY, et al. CHD1L Protein is overexpressed in human ovarian carcinomas and is a novel predictive biomarker for patients survival[J]. BMC Cancer, 2012, 12(1): 1-8.
[6] 郑小梅, 张晓立, 于建东, 等. CRISPR-Cas9介导的基因组编辑技术的研究进展[J]. 生物技术进展, 2015, 5(1): 1-9.
[7] Pelletier S, Gingras S, Green DR. Mouse genome engineeringvia CRISPR-Cas9 for study of immune function[J]. Immunity, 2015, 42(1): 18-27.
[8] Gossen M, Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters[J]. Proc Natl Acad Sci U S A, 1992, 89(12): 5547-5551.
[9] Wang T, Wei JJ, Sabatini DM, et al. Genetic screens in human cells using the CRISPR-Cas9 system[J]. Science, 2014, 343(6166): 80-84.
[10] Forner A, Llovet JM, Bruix J. Hepatocellular carcinoma[J]. Lancet, 2012, 379(9822): 1245-1255.
[11] Tang KH, Ma S, Lee TK, et al. Chan YP CD133(+) liver tumor-initiating cells promote tumor angiogenesis, growth, and self-renewal through neurotensin/interleukin-8/CXCL1 signaling[J]. Hepatology, 2012, 55(3): 807-820.
[12] Yang ZF, Ho DW, Nq MN, et al. Significance of CD90+ cancer stem cells in human liver cancer[J]. Cancell cell, 2008, 13(2): 153-166.
[13] Yang W, Wang C, Lin Y, et al. OV6+ tumor-initiating cells contribute to tumor progression and invasion in human hepatocellular carcinoma[J]. J Hepatol, 2012, 57(3): 613-620.
[14]Lee TK, Cheung VC, Ng IO. Liver tumor-initiating cells as a therapeutic target for hepatocellular carcinoma[J]. Cancer Lett, 2013, 338(1): 101-109.
[15] Snider AC, Leong D, Wang QT, et al. The chromatin remodeling factor Chd1l is required in the preimplantation embryo[J]. Biol Open, 2013, 2(2): 121-131.
[16] Dou D, Zhao H, Li Z, et al. CHD1L promotes neuronal differentiation in human embryonic stem cells by upregulatingPAX6[J]. Stem Cells Dev, 2017, 26(22): 1626-1636.
[17] Liu SS, Bai YS, Feng L, et al. Identification of CHD1L as an important regulator for spermatogonial stem cell survival and self-renewal[J]. Stem Cells Int, 2016, 2016: e4069543.
[18] Liu M, Chen L, Ma NF, et al. CHD1L promotes lineage reversion of hepatocellular carcinoma through opening chromatin for key developmental transcription factors[J]. Hepatology, 2016, 63(5): 1544-1559.
[19] He LR, Ma NF, Chen JW, et al. Overexpression of CHD1L is positively associated with metastasis of lung adenocarcinoma and predicts patients poor survival[J]. Oncotarget, 2015, 6(31): 31181-31190.
[20] Liu SS, Maguire EM, Bai YS, et al. A novel regulatory axis, CHD1L-microRNA 486-matrix metalloproteinase 2, controls spermatogonial stem cell properties[J]. Mol Cell Biol, 2019, 39(4): e00357-18.
[21] Slaymaker IM, Gao L, Zetsche B, et al. Rationally engineered Cas9 nucleases with improved specificity[J]. Science, 2016, 351(6268):84-88.
[22] Gajbhiye V, Escalante L, Chen G, et al. Drug-loaded nanoparticles induce gene expression in human pluripotent stem cell derivatives[J]. Nanoscale, 2014, 6(1): 521-531.