Construction and identification of stably transfected ovarian cancer cell lines with lentiviral RNA interference-mediated down-regulation of LSD1 gene

WAN Xiaolei; LAI Wensheng; SHAO Genbao; LI Yuanxia; LIU Xiuwen; JIN Jie; WU Chaoyang

doi:10.3969/j.issn.1002-266X.2016.16.006

Abstract：Objective To construct the stably transfected ovarian cancer cell lines SKOV 3 with lentiviral RNA inter-ference-mediated down-regulation of lysine-specific demethylase 1 (LSD1) gene and to provide basis for the in-depth study of taking LSD1 gene as a target in the epigenetics anti-tumor treatment.Methods The plasmids was amplified and extrac-ted, and the UV spectrophotometer was used to detect their purity , recording A260/A280 .PLKO-TET-On plasmids were transfected into 293T cells via Lipofectamine 2000, The transfection of 293T cells was observed under an inverted fluores-cence microscope .Collecting virus supernatants which were infected by ovarian cancer cells SKOV 3 ( observation group ) and the vector PLKO was used as the negative control ( control group ) .The stably transfected cell lines were selected .The cell lines were induced by doxycycline (0, 1, 10, 100 and 1 000 ng/mL) in the observation group , and then they were both induced by 100 ng/mL doxycycline, the expression of LSD1, H3K4me1 and H3K4me2 in the stably transfected cell lines was detected by Western blotting .Results Ultraviolet spectrophotometer showed that the target plasmid A 260/A280 was around 2.0 with high purity, THM plasmid-transfected 293T cells appeared green fluorescence , which was eukaryotic expression vector of GFP green fluorescent protein , suggesting that transfection was successful .The protein expression level of LSD1 was decreased and its main substration H3K4me1, H3K4me2 expression was increased with the concentration gra-dient of doxycycline.There were statistically significant differences between multiple concentrations (all P<0.01).The relative expression of LSD 1 protein of the observation group induced by 100 ng/mL doxycycline was significantly decreased but the expression of H3K4me1, H3K4me2 was increased as compared with that in the group without doxycycline induction and the control group after the induction of doxycycline (all P<0.01).Conclusion The stably transfected ovarian cancer cell lines SKOV3 with lentiviral RNA interference-mediated down-regulation of LSD1 gene are constructed successfully and identified with concentration gradient of doxycycline induction ;this cell line can be used in the epigenetics anti-tumor treat-ment of taking LSD1 as a target.