Construction of A549 cell lines silence expressing miR-26a

Objective To construct a lentiviral expression vector stably down-regulating has-miR-26a-5p in human lung adenocarcinoma cell line(A549). Methods Specific complementary sequence of mature hsa-miR-26a-5p was synthesized in vitro. Restriction enzyme cutting sites of Age I and EcoRI was added to this sequence, which was then cloned into the plasmid of GV280 by double digestion to obtain the lentiviral vector named GV280-miR-26a-down. The recombinant plasmid was confirmed by DNA sequencing, subsequently cotransfected into 293T cell with viral pac-kaging plasmid pGC-LV vector, pHelper 1.0 vector, and pHelper 2.0 vector. Virus titer was measured according to the expression level of green fluorescent protein. The lentivirus down-regulating miR-26a was transfected into A549 cells which was screened by puromycin subsequently to obtain stable cell line. One of miR-26a's target gene SMAD1 was detected by Western blot to ensure the function of this A549 cell line. Results The lentivirus vector of GV280-miR-26a-down was successfully constructed confirmed by DNA sequencing, and then was packaged in 293T cells to produce lentiviral particles and the virus titer reached to 2×1012 TU/L. The efficiency of recombinant lentivirus transfecting A549 cells was up to 95%. Nearly 100% A549 cells expressed GFP after being screened by 2.5 mg/L puromycin for about 2 weeks. Western blot results showed that the expression of miR-26a's target gene SMAD1 significantly upregulated in miR-26a-down group compared with viruses in the control group and the blank control group. Conclusion The lentivirus vector of GV280-miR-26a-down and the A549 cell line stably down-regulating has-miR-26a-5p can be successfully constructed, which provides the basis for further study of miR-26a. Key words: miR-26a; A549 cell line; Lentivirus vector