|
|
|
| Construction of miR-362 Knockdown 95-D Cell Line by CRISPR/Cas9 Technology |
| LUO Dan1, WANG Li-juan2, SUN Xiu-xuan2, ZHANG Zheng2, CHEN Zhi-nan1 |
1. College of Life Science and Bioengineering, School of Science, Beijing Jiaotong University, Beijing 100044, China;
2. Cell Engineering Research Center, The Fourth Military Medical University, Xi'an 710032, China |
|
|
|
Abstract Objective:to construct miR-362 knockdown 95-D cells by using CRISPR/Cas9 genome engineering technology, and study the function of miR-362 in cancer.Methods:gRNA sequences targeting the miR-362 gene were selected. Px330-gRNA recombination plasmids were constructed and the validity were evaluated by T7E1 assay. Left and right arms of miR-362 were amplified from genomic DNA by PCR, and sequentially cloned into the donor vector. The CRISPR/Cas9 system and donor vector were co-transfection into 95-D cells, and the selection system that allows for marker genes were integrated into the genome through homologous recombination (HR). The expression of miR-362 of 95-D-KnockDown cells sorted through FACS was detected by qPCR, and the migration and invasion were determined by Transwell assay. Results:Compared with 95-D cells, the expression of miR-362 in 95-D-KD cells was significantly down-regulated, and down-regulation of miR-362 expression can suppress the cell migration and invasion capacity of 95-D-KD cells. Conclusion:The miR-362 knockdown lung cancer cell line(95-D-KD cells)had been successfully constructed by using CRISPR/Cas9 system, which lays the foundation for further study of the mechanism and function of miR-362 in cancers.
|
|
Received: 14 June 2017
Published: 15 November 2017
|
|
|
|
|
| [1] |
Liu Y, Hu X, Xia D, et al. MicroRNA-181b is downregulated in non-small cell lung cancer and inhibits cell motility by directly targeting HMGB1. Oncol Lett, 2016,12(5):4181-4186.
|
|
|
| [2] |
Wang H, Sun T, Hu J, et al. miR-33a promotes glioma-initiating cell self-renewal via PKA and NOTCH pathways. J Clin Invest, 2014,124(10):4489-4502.
|
|
|
| [3] |
Ni F, Zhao H, Cui H, et al. MicroRNA-362-5p promotes tumor growth and metastasis by targeting CYLD in hepatocellular carcinoma. Cancer Lett, 2015,356(2):809-818.
|
|
|
| [4] |
Jung C J, Iyengar S, Blahnik K R, et al. Epigenetic modulation of miR-122 facilitates human embryonic stem cell self-renewal and hepatocellular carcinoma proliferation. PLoS One, 2011,6(11):e27740.
|
|
|
| [5] |
Nicoloso M S, Spizzo R, Shimizu M, et al. MicroRNAs——the micro steering wheel of tumour metastases. Nat Rev Cancer, 2009,9(4):293-302.
|
|
|
| [6] |
Xia J T, Chen L Z, Jian W H, et al. MicroRNA-362 induces cell proliferation and apoptosis resistance in gastric cancer by activation of NF-kappaB signaling. J Transl Med, 2014,12(1):33.
|
|
|
| [7] |
Wu K, Yang L, Chen J, et al. miR-362-5p inhibits proliferation and migration of neuroblastoma cells by targeting phosphatidylinositol 3-kinase-C2beta. FEBS Lett, 2015,589(15):1911-1919.
|
|
|
| [8] |
Pelletier S, Gingras S, Green D R. Mouse genome engineering via CRISPR-Cas9 for study of immune function. Immunity, 2015,42(1):18-27.
|
|
|
| [9] |
Grissa I, Vergnaud G, Pourcel C. The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinformatics, 2007,8(1):172.
|
|
|
| [10] |
Feng Y, Liu J, Kang Y, et al. miR-19a acts as an oncogenic microRNA and is up-regulated in bladder cancer. J Exp Clin Cancer Res, 2014,33(1):67.
|
|
|
| [11] |
Ran F A, Hsu P D, Wright J, et al. Genome engineering using the CRISPR-Cas9 system. Nat Protoc, 2013,8(11):2281-2308.
|
|
|
| [12] |
Xiao A, Cheng Z, Kong L, et al. CasOT:a genome-wide Cas9/gRNA off-target searching tool. Bioinformatics, 2014,30(8):1180-1182.
|
|
|
| [13] |
Cong L, Ran F A, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems. Science, 2013,339(6121):819-823.
|
|
|
| [14] |
Lin Y, Cradick T J, Brown M T, et al. CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Res, 2014,42(11):7473-7485.
|
|
|
| [15] |
Shen B, Zhang W, Zhang J, et al. Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat Methods, 2014,11(4):399-402.
|
|
|
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
