Construction of lentivirus vector carrying COX2 gene and its effect on proliferation and apoptosis of non-small cell lung cancer A549 cells

Liu Lianbin; ZHANG Xiangmin; ZENG Wen; ZHOU Maohua; YE Guilin; YE Yongqiang; WAN Gang; LI Shaojin

doi:10.3969/j.issn.1009-0460.2018.01.002

Abstract：Objective To construct a recombinant short hairpin RNA (shRNA) lentiviral vector of cyclooxygenase 2(COX2) gene, and investigate its effects on proliferation and apoptosis of non-small cell lung cancer (NSCLC). Methods Three oligonucleotides targeting COX2 gene were synthesized and cloned into lentivirus vector GV248. The lentivirus vector COX2-shRNA targeted to COX2 gene was constructed and screened for the best inhibition efficiency. The human NSCLC A549 cells were selected for lentivirus transfection during the logarithmic growth period. Specially, shRNA vector targeting COX2 gene was transfected into A549 cells as shRNA group, and empty vector plasmid was transfected as negative control (NC) group. A549 cell without transfection plasmid were selected as blank control group (CON). COX2 expressions were assessed by real-time PCR and Western blotting. The effects of interfering COX2 expression on the proliferation and apoptosis of A549 cells were detected by MIT and flow cytometry. Results The recombinant lentivirus vector targeting COX2 was constructed successfully. Especially COX2-shRNAl showed the best transfection efficiency. In shRNA group, the level of COX2 mRNA in shRNA1 transfected A549 cells was 0.17±0.06, lower than 0.81±0.11 of NC group and 1.09±0.07 of CON group (P<0.05). The proliferative activity of shRNA group was lower than those of NC group and CON group. For example, the absorbance value at 5 d in shRNA group was 0.976±0.016, which was lower than 1.557±0.015 of NC group and 1.880±0.034 of CON group (P<0.05). The apoptotic rate of shRNA group was (6.28±0.31)％, which was higher than (3.08±0.05)％ of NC group and (3.01±0.31)％ of CON group, and the difference was statistically significant (P<0.05). Conclusion The recombinant lentivirus shRNA vector target COX2, with best transfection efficiency, is constructed successfully. RNAi-mediated silencing of the COX2 gene can inhibit the proliferation of A549 cells and promote its cell apoptosis.