Construction of has-miR-335 lentiviral vector and verification of the target gene of miR-335

• Published in: Nan fang yi ke da xue xue bao = Journal of Southern Medical University Vol. 32; no. 3; p. 306
• Main Authors: Yang, Hui, Zhang, Chao, Lu, Yan-Xia, Wu, Xiao-Jin, Yuan, Li, Zhou, Chang, Zhou, Chun-Ping, Liu, Guo-Bing, Li, Xue-Nong
• Format: Journal Article
• Language: Chinese
• Published: China 01.03.2012
• Subjects: Cell Line, Tumor; Colorectal Neoplasms - genetics; Colorectal Neoplasms - metabolism; Colorectal Neoplasms - pathology; Genetic Vectors - genetics; Green Fluorescent Proteins; Humans; Lentivirus - genetics; Lentivirus - metabolism; MicroRNAs - genetics; MicroRNAs - metabolism; p120 GTPase Activating Protein - genetics; p120 GTPase Activating Protein - metabolism; Recombinant Proteins - genetics; Recombinant Proteins - metabolism
• ISSN: 1673-4254

To construct a lentiviral vector of miR-335 gene and verify the target gene of miR-335. The precursor sequence of miR-335 gene was amplified from the genomic DNA by PCR and cloned into the lentiviral vector PLVTHM labeled with GFP. Real-time quantitative RT-PCR was used to detect miR-335 and RASA1 expression in different colorectal cancer cell lines. A recombinant vector psiCHECK-2-RASA1 containing RASA1 3'UTR was constructed followed by site-directed mutagenesis of RASA1 3'UTR to establish the vector psiCHECK-2-RASA1-Mut. Co-transfection of hsa-mir-335 or a NC with these recombined vectors in HEK293A and SW480 cells was performed, and dual-luciferase reporter assay was utilized to examine the changes in luciferase activities. The recombinant PLVTHM-miR335 plasmid was packaged into mature lentivirus by 293FT cells and used to infect SW620 cells. Flow cytometry was employed for sorting the GFP+ cells. The expression of miR-335 and RASA1 were determined by qRT-PCR, and Western blotting was used to detect the ex