LSD1 Negatively Regulates Autophagy Through The MTOR Signaling Pathway In Ovarian Cancer Cells

Objective: This article aims to explore the role of LSD1 in autophagy in ovarian cancer cells and reveals its possible mechanism of action,which might provide data and theory support to explore the new pathogenesis and potential molecular targeted therapy programs of ovarian cancer.Methods: 1.The inducible stable knockdown of LSD1 in ovarian cancer HO8910 cells were established by lentiviral transfection,then the effect of LSD1 knockdown and the specific substrate H3K4me2 expression were examined by Western blotting and Quantitation real-time PCR(qRT-PCR).2.The effect of LSD1 knockdown or inhibition on the expression of autophagy-related proteins Beclin1 and LC3 were detected by Western blotting.3.The effect of LSD1 knockdown or inhibition on the expression of LC3 mRNA levels were detected by qRT-PCR.4.Immunofluorescence was used to observe the expression and aggregation of endogenous LC3 after LSD1 knockdown or inhibition,and then to discuss the relationship between LSD1 and autophagosomes formation.5.We transfected GFP-LC3 into HO8910-sh LSD1/HO8910 cells to observe the aggregation of exogenous autophagosomes represented by GFP-LC3 using GFP-LC3 single-fluorescent system.6.Serum starvation and rapamycin are used to activate autophagy in HO8910 cells and the the expression of LSD1 was detected under autophagy condition,which futher confirmed the role of LSD1 in autophagy in ovarian cancer cells.7.mTOR activity was detected after LSD1 by Western blotting,and then the mTOR activator MHY1485 was used to activate mTOR,therefore to discuss the association with LSD1 and mTOR signaling pathway.Thus,the mechanism of LSD1 acted on autophagy in ovarian cancer cells would be discussed.Results: 1.The induction of stable LSD1 knockdown in ovarian cancer HO8910 cells were successfully mediated by lentivirus.With the induction of Dox,protein and mRNA levels of LSD1 were effectively silenced and the degree of LSD1 knockdown was in a dose-and time-dependent manner of Dox.In contrast,the expression of specific methylated substrate H3K4me2 by LSD1 was significantly increased,which confirms that the model of inducible stable interference of LSD1 has been successfully constructed in ovarian cancer HO8910 cells.2.Western blotting showed that depletion or inhibition of LSD1 caused significant accumulation of LC3-? in a time-and dose-dependent manner of Dox,but did not affect the levels of Beclin1 in protein levels.In addition,qRT-PCR results showed that LC3 mRNA expression levels did not change after LSD1 depletion or inhibition 3.Western blotting and immunofluorescence experiments confirmed that knockdown or inhibition of LSD1 induces autophagy in HO8910 cells.4.LSD1 was inhibitied under the serum withdrawal and rapamycin induced autophagy.The knockdown of LSD1 induced by Dox resulted in further accumulation of autophagic flux which induced by serum starvation and rapamycin,suggesting that the inhibition of autophagy by LSD1 is also involved in starvation-and rapamycin-induced autophagy,and the expression of LSD1 is also regulated by the negative feedback of both starvation and rapamycin.5.Knockdown or inhibition of LSD1 lead to the inactivation of mTOR signaling pathway.The mTOR activator MHY1485 treatment abolished the effect of LSD1 knockdown on the induction of autophagy,suggestting that LSD1 knockdown induces autophagy is dependent on inhibiting mTOR signaling.Conclusion: 1.LSD1 negatively regulates the occurrence of autophagy in ovarian cancer.2.Pharmacological inhibition or genetic knockdown of LSD1 induces autophagy and stimulates the autophagic flux.3.Knockdown of LSD1 promotes the serum starvation-and rapamycin-induced autophagy and the expression of LSD1 is regulated by negative feedback of autophagy stimuli.4.The mechanism of LSD1 involved in regulating autophagy in ovarian cancer cells depends on the regulation of mTOR signaling pathway.