Research Of Lysine Specific Demethylase 1 Involved In Ovarian Cancer Cells Sensitized To Cisplatin

Objective:The effect of cisplatin on function and intracellular LSD1 expression of ovarian cancer cells SKOV3 was preliminary studied. Stably transfected cell lines of knock down and overexpression of LSD1 were constructed as an in vitro model. To evaluate the potential cisplatin chemo-sensitization effect of modulating LSD1 in ovarian cancer cell line SKOV3 from three aspects of cell proliferation, migration and apoptosis.Methods:(1)Ovarian cancer cell line SKOV3 were treated with concentration gradient of cisplatin to observe the morphological and functional variability of cell proliferation,migration and apoptosis, and to validate whether the expression of LSD1 and its reaction substrates H3K4 methylation is affected by cisplatin. The fixed concentration of cisplatin was chosed for the subsequent research.( 2) The related plasmids of knock down and overexpression of LSD1 were amplified and extracted, and purity and base size of the plasmids were checked by agarose gel electrophoresis. Then the plasmids were transfected into 293 T cells via Lipofectamine 2000, collecting both lentivirus supernatants to infect target cells SKOV3. Puromycin-resistant cells were selected and obtained. For overexpression of LSD1, G418 was then additionally used to screen the target cell model. After induced by doxycycline, the expression of LSD1 in the two kinds of stably transfected cell lines was detected by Western blot.( 3) The combination of doxycycline and cisplatin was evaluated on the genetically modified cells of LSD1 knock down and overexpression. We measured proliferation by MTT and Ed U assay, cell migration by transwell assay, and apoptosis by Annexin V/PI staining for FACS analysis. To further demonstrate the above three effects on ovarian cancer cells, expression of proliferation, apoptosis and metastasis related markers were examined by Western blot to study the potential chemo-sensitization effect of targeting LSD1 in ovarian cancer cell line SKOV3.Results:( 1) Our results indicated that cisplatin can inhibite the proliferation and migration of ovarian cancer cells and induce apoptosis; Besides, stimulation with cisplatin downregulated LSD1 protein levels in a dose-dependent manner, and its specific reaction substrate H3K4me1 and H3K4me2 protein expression gradually increased, existing a significant difference between the concentration of multiple comparisons. There was no statistical difference in level of H3K4me3.( 2) After induced by doxycycline, LSD1 was down-regulated obviously in lentiviral interference mediated SKOV3 cells, while up-regulated in LSD1 overexpression of the genetically modified cells.(3)Knock down of LSD1 mediated by sh RNA in SKOV3 stably transfected cells exposed to cisplatin obviously promoted apoptosis, supressed cell proliferation and migration compared with the control group. In contrast, overexpression of LSD1 had an adverse effect on the above three biological functions.Conclusions:The validation that levels of LSD1 and its substration H3K4me1 and H3K4me2can be affected by cisplatin, which is consistent with previous studies. Cisplatin can inhibite the proliferation and migration of ovarian cancer cells and induce apoptosis.We have found that knockdown of LSD1 enhance sensitivity of ovarian cancer cells to cisplatin. Conversely, overexpression of LSD1 has the adverse effect. Besides, the corresponding maker proteins further proved the above point of view. Taken together,these results suggest that knock down of LSD1 represent a novel therapeutic approach for human ovarian carcinoma, while overexpression of LSD1 may be involved in carcinogenesis and progression.