Construction of lentivirus-based inhibitor of hsa- microRNA-338-3p with specific secondary structure

• Published in: Acta pharmacologica Sinica Vol. 34; no. 1; pp. 167 - 175
• Main Authors: Sun, Kai, Guo, Chen, Deng, Hai-jun, Dong, Jing-qing, Lei, Shang-tong, Li, Guo-xin
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.01.2013; Nature Publishing Group
• Subjects: Base Sequence; Biomedical and Life Sciences; Biomedicine; blot分析; Cell Line, Tumor; Cell migration; Colon - cytology; Colon - metabolism; Colorectal carcinoma; Colorectal Neoplasms - genetics; DNA sequencing; Down-Regulation; Endonuclease; Expression vectors; Flow cytometry; Gene expression; Gene Expression Regulation, Neoplastic; Genetic Vectors - chemistry; Genetic Vectors - genetics; HEK293 Cells; Humans; Immunology; Infection; Internal Medicine; Lentivirus; Lentivirus - genetics; Medical Microbiology; MicroRNAs - genetics; miRNA; Molecular Sequence Data; Nucleic Acid Conformation; Original; original-article; Pharmacology/Toxicology; Plasmids; Polymerase chain reaction; Protein structure; Rectum - cytology; Rectum - metabolism; Secondary structure; Transduction, Genetic; Vaccine; Western blotting; 二级结构; 人血清白蛋白; 实时RT-PCR; 慢病毒载体; 病毒感染; 病毒抑制剂; 限制性内切酶; hsa-miR-338-3p; colorectal carcinoma; microRNAs (miRNAs); lentivirus
• ISSN: 1671-4083; 1745-7254
• DOI: 10.1038/aps.2012.172

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-trans- fected with the lentiviral vectors pLV-THM-miR-338-3p-inhibitor, psPAX2, and pMD2.G. The supernatant containing the lentivirus par- ticles 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 lenti- virus 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 cotorectal carcinoma, miR-338-3p may suppress SMO gene expression and thereby inhibit colorectal carcinoma migration.