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| Construction of TGFBR2 miRNA Lentiviral Vector in Human Colon Cancer Cell Line and the Preliminary Study of Its Function |
| LI Yuan-fei1, ZHAO He-ping1, LIU Jing1, ZHU Guo-qiang2, ZHANG Ge-hong1, JIA Jun-mei1, YANG Wen-hui1 |
1. The First Hospital of Shanxi Medical University, Taiyuan 030001, China;
2. 264 Hospital of PLA, Taiyuan 030001, China |
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Abstract Objective: To obtain small interfering RNA sequence that can stably block the expression of TGFBR2 in the human colon cancer SW480 cell lines, and to construct the TGFBR2-miRNA lentiviral vector and establish a stably transfected cell line, which lay the foundation of further study in the colon cancer cells. Methods: Four kinds of miRNA sequences targeting TGFBR2 are designed. Recombinant pcDNATM6.2-GW/EmGFP-TGFBR2-miRNA plasmids are constructed. The plasmid pcDNATM6.2-GW/Em GFP-TGFBR2-miRNA with lentiviral packaging plasmid are mixed and co-transfected 293T cells with recombinant TGFBR2 vector, then the most effective miRNA is assessed and determined by real-time RT-PCR and Western blot. Virus solution is collected to infect SW480 cells and a stably transfected cell line is established. Invasion experiment is recorded respectively in SW480 cells after interfered with TGFBR2 and controls. Results: The fourth miRNA sequence shows the best gene silencing effect beyond 70% for mRNA and 70% for protein. The lentiviral vector is constructed successfully and inhibition ratio is 78.45% for TGFBR2 mRNA. After incubation with 10ng/ml TGF-β1and 20ng/mL TNF-a for 72h, the invasion experimental study reveals that the migrated cells are higher in TGFBR2-interferred group( 89.3±7.9)than TGFBR2-uninterferred group(15.1±8.2 cells, P<0.01).Conclusion: It is successful to obtain TGFBR2 -miRNA Lentiviral vector that can stably block the expression of TGFBR2 in the colon cancer cell line SW480 and establish a stably transfected cell line which provides foundation for further studies on the prevention and intervention of earliy matastasis of colon cancer. Furthermore, it is indicated that colon cancer cells with TGFBR2 mutant may be more invasive when the microenvironment riched with TGF-β1and TNF-a.
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Received: 18 February 2013
Published: 25 May 2013
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