DHX15 Regulates Autophagy And Its Effect On The Proliferation Of Hepatoma Cells

Background DEAD/H-box helicases are highly conserved enzymes which mainly participate in RNA metabolism.DEAH-box 15(DHX15)is a member of this family,mainly involved in RNA splicing and ribosome maturation which is important to maintain the stability of RNA metabolism in cells.Recent studies have found a certain correlation between DHX15 and tumorigenesis.Macroautophagy(here after referred as autophagy)is a highly conserved cellular physiological process,widely existing in eukaryotic cells.It mainly degrades superfluous or harmful components in cells through its double membrane structure to maintain the intracellular homeostasis.The abnormality of this cellular protective mechanism is closely related to the development of autoimmune diseases,diabetes mellitus,and especially tumour.Both autophagy and DHX15 are involved in the maintenance of intracellular homeostasis.Whether there is an interaction between them has not been clarified at present,and whether they are jointly involved in the occurrence and development of tumours is still elusive.This study attempted to explore the relationship between DHX15 and autophagy,to clarify the role and mechanism of DHX15 in autophagy,and to explore the influence of DHX15 and autophagy on the proliferation of HCC cells,so as to understand the possible role of DHX15 and autophagy in the development of HCC.Methods1.DHX15 was knocked down in L02,Huh7 and HepG2 cell lines by RNA interference (RNAi);Autophagy level was detected by western blot(WB)which included the conversion rate of microtubule-associated protein 1 light chain 3 beta(LC3B)from type I to type II and the changes of SQSTM1(p62)protein.2.DHX15 was decreased in HEK293 cells with LC3-GFP stable expression.The formation of LC3 fluorescent puncta which named autophagosomes was observed by confocal microscopy.3.Knocking down DHX15 in L02 cells,the formation of autophagosomes was detected at a more microscopic level by transmission electron microscopy.4.Quantitative real-time polymerase chain reaction(qPCR)and DHX15 with ATPase mutation were used to explore the role of the ATPase activity of DHX15 in regulating autophagy.5.The phosphorylated protein in the m TORC1 pathway was detected by WB to explore the mechanism of DHX15 in regulating autophagy.6.Reduction of DHX15 promoted the proliferation of Hep G2 cells was detected by cell viability assay,colony formation assay and Ki-67 proliferation marker.At the same time,downregulating autophagy related protein 5(ATG5)or using autophagy inhibitors Bafilomycin A1(Baf-A1)or 3 – Methyladenine(3-MA)explored the role of DHX15-mediated autophagy caused the cell proliferation of Hep G2 cells.Results1.Knocking down DHX15 strongly promoted autophagy in vivo which leading to significant increase of LC3B-II and decrease of p62.Downregulation of DHX15 remarkably promoted the formation of autophagosmes.2.The results detected by qPCR showed that DHX15 had no effect on splicing the m RNA of autophagy related genes.The ATPase activity of DHX15 was not required to regulation of autophagy.3.Decreasing DHX15 significantly reduced phosphorylated substrates of m TORC1 pathway.These results indicated that DHX15 inhibited autophagy by activating m TORC1.4.Cell viability assay(CCK-8 assay),colony formation assay and Ki-67 proliferation marker assay demonstrated that the proliferation of Hep G2 cells significantly increased by DHX15 depletion(P***<0.001,P**<0.01).In addition,using autophagy inhibitors or the reduction of ATG5 could significantly reverse the proliferation induced by DHX15 knockdown(P***<0.001).Conclusion DHX15 is a novel suppressor of autophagy through activation of MTORC1.DHX15 inhibits the proliferation of hepatoma cells by inhibiting autophagy.