TY - JOUR AU - Sun Kai AU - Guo Chen AU - Deng Hai-jun AU - Dong Jing-qing AU - Lei Shang-tong AU - Li Guo-xin PY - 2016 TI - Construction of lentivirus-based inhibitor of hsa-microRNA-338-3p with specific secondary structure JF - Acta Pharmacologica Sinica; Vol 34, No 1 (January 2013): Acta Pharmacologica Sinica (Special Feature:Stroke) Y2 - 2016 KW - N2 - Kai SUN1, *, Chen GUO2, Hai-jun DENG1, Jing-qing DONG1, Shang-tong LEI1, Guo-xin LI1 1Department of General Surgery, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China; 2School of Biotechnology, Southern Medical University, Guangzhou 510515, China Aim: To construct a lentivirus-based inhibitor with specific secondary structure that could exert long-term suppression on microRNA-338-3p (miR-338-3p), thus elucidating its molecular function in colorectal carcinoma cells. Methods: The miR-338-3p inhibitor sequence was synthesized and inserted into pLV-THM plasmid. HEK-293T cells were co-transfected with the lentiviral vectors pLV-THM-miR-338-3p-inhibitor, psPAX2, and pMD2.G. The supernatant containing the lentivirus particles was harvested to determine the viral titer, and then used to infect colorectal carcinoma-derived SW-620 cells. eGFP(+) cells were sorted using flow cytometry. The expression of miR-338-3p in SW-620 cells was determined with real-time RT-PCR, and the expression of the smoothened (SMO) protein was detected using Western blot analysis. The migration ability of the transfected SW-620 cells was assessed with transwell assay. Results: Restriction endonuclease analysis and DNA sequencing demonstrated that the lentiviral vector pLV-THM-miR-338-3p-inhibitor was successfully constructed. The expression of miR-338-3p in SW-620 cells was significantly decreased by infection with the lentivirus pLV-THM-miR-338-3p-inhibitor. Moreover, the down-regulated expression of miR-338-3p caused up-regulated expression of the SMO protein in SW-620 cells, which showed significantly enhanced migration in transwell assay. Conclusion: The construction of the lentiviral vector pLV-THM-miR-338-3p-inhibitor with specific secondary structure provides a basis for further studies the molecular function of miR-338-3p in colorectal carcinoma. miR-338-3p may suppress SMO gene expression and thereby inhibit colorectal carcinoma migration. Keywords: microRNAs (miRNAs); colorectal carcinoma; hsa-miR-338-3p; lentivirus This research is supported by a grant from the National Natural Science Foundation of China (No 81101896). * To whom correspondence should be addressed. E-mail sunkai9602@sina.com Received 2012-05-26 Accepted 2012-09-03 UR - http://www.chinaphar.com/article/view/6174