Construction and Expression of Runx1-shRNA Expressiong Plasmid_Journal of Biomedical Engineering

Authors：

CHENYifan ¹ , TANGXiaoju ² ,  LUOFengming ³

1. Department of Respiratory Medicine, West China Hospital, Sichuan University, Chengdu 610041, China;
2. Laboratory of Cardiovascular Diseases, Research Center of Regeneration Medicine, West China Hospital, Sichuan University, Chengdu 610041, China;
3. General Practice Branch, West China Hospital, Sichuan University, Chengdu 610041, China;

Corresponding author：

LUOFengming, Email: Luofengming@hotmail.com

Keywords：

Runx1; pSuper; construction of plasmid

DOI：

10.7507/1001-5515.20140157

Video：

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This study aims to construct the plasmid of human Runx1 and observe its possible effects on Runx1 gene expression in human pulmonary adenocarcinoma cells (A549). The shRNA sequence targeting human Runx1 was designed and synthesized, then inserted into pSuper plasmid by DNA recombination technology. The recombinant plasmid was confirmed by bacterial colonies PCR, enzyme digestion analysis and DNA sequencing. A549 cells were transfected by Runx1 shRNA plasmid. The inhibition efficiency of pSuper-Runx1-shRNA plasmid on Runx1 at mRNA level and protein level were measured with real-time PCR and Western blot. The results of real-time PCR and Western blot indicated that the mRNA and protein levels of Runx1 in A549 cells were inhibited by the pSuper-Runx1-shRNA expression plasmid, and the inhibition rate were 33% and 50%, respectively.

Citation： CHENYifan, TANGXiaoju, LUOFengming. Construction and Expression of Runx1-shRNA Expressiong Plasmid. Journal of Biomedical Engineering, 2014, 31(4): 837-841. doi: 10.7507/1001-5515.20140157 Copy

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13.	吴卫兵,陈亮,王俊,等.Runx1基因mRNA表达与非小细胞肺癌发生及淋巴转移的相关性研究[J].南京医科大学学报:自然科学版,2009(5):682-685.

1. ITO Y. RUNX genes in development and cancer: regulation of viral gene expression and the discovery of RUNX family genes[J]. Adv Cancer Res, 2008, 99: 33-76.
2. PTASINSKA A, ASSI S A, MANNARI D, et al. Depletion of RUNX1/ETO in t(8;21) AML cells leads to genome-wide changes in chromatin structure and transcription factor binding[J]. Leukemia, 2012, 26(8): 1829-1841.
3. LACAUD G, GORE L, KENNEDY M, et al. Runx1 is essential for hematopoietic commitment at the hemangioblast stage of development in vitro[J]. Blood, 2002, 100(2): 458-466.
4. NORTH T, GU T L, STACY T, et al. Cbfa2 is required for the formation of intra-aortic hematopoietic clusters[J]. Development, 1999, 126(11): 2563-2575.
5. NORTH T E, DE BRUIJN M F, STACY T, et al. Runx1 expression marks long-term repopulating hematopoietic stem cells in the midgestation mouse embryo[J]. Immunity, 2002, 16(5): 661-672.
6. OKUDA T, NISHIMURA M, NAKAO M, et al. RUNX1/AML1: a central player in hematopoiesis[J]. Int J Hematol, 2001, 74(3): 252-257.
7. JANES K A. RUNX1 and its understudied role in breast cancer[J]. Cell Cycle, 2011, 10(20): 3461-3465.
8. FIJNEMAN R J, ANDERSON R A, RICHARDS E A, et al. Runx1 is a tumor suppressor gene in the mouse gastrointestinal tract[J]. Cancer Sci, 2012, 103(3): 593-599.
9. TAHIROV T H, INOUE-BUNGO T, MORII H, et al. Structural analyses of DNA recognition by the AML1/Runx-1 Runt domain and its allosteric control by CBFbeta[J]. Cell, 2001, 104(5): 755-767.
10. XU A N, CHEN X H, TAN Y H, et al. Identification of a novel circularized transcript of the AML1 gene[J]. BMB Rep, 2013, 46(3): 163-168.
11. MIYOSHI H, OHIRA M, SHIMIZU K, et al. Alternative splicing and genomic structure of the AML1 gene involved in acute myeloid leukemia[J]. Nucleic Acids Res, 1995, 23(14): 2762-2769.
12. BERNSTEIN E, CAUDY A A, HAMMOND S M, et al. Role for a bidentate ribonuclease in the initiation step of RNA interference[J]. Nature, 2001, 409(6818): 363-366.
13. 吴卫兵,陈亮,王俊,等.Runx1基因mRNA表达与非小细胞肺癌发生及淋巴转移的相关性研究[J].南京医科大学学报:自然科学版,2009(5):682-685.

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