DHX15 Inhibits Autophagy and the Proliferation of Hepatoma Cells

Abstract

Autophagy is a highly conserved process by which superfluous or harmful components in eukaryotic cells are degraded by autophagosomes. This cytoprotective mechanism is strongly related to various human diseases, such as cancer, autoimmune diseases, and diabetes. DEAH-box helicase 15 (DHX15), a member of the DEAH box family, is mainly involved in RNA splicing and ribosome maturation. Recently, DHX15 was identified as a tumor-related factor. Although both autophagy and DHX15 are involved in cellular metabolism and cancer progression, their exact relationship and mechanism remain elusive. In this study, we discovered a non-classic function of DHX15 and identified DHX15 as a suppressive protein in autophagy for the first time. We further found that mTORC1 is involved in DHX15-mediated regulation of autophagy and that DHX15 inhibits proliferation of hepatocellular carcinoma (HCC) cells by suppressing autophagy. In conclusion, our study demonstrates a non-classical function of DHX15 as a negative regulator of autophagy related to the mTORC1 pathway and reveals that DHX15-related autophagy dysfunction promotes HCC cell proliferation, indicating that DHX15 may be a target for liver cancer treatment.

Keywords: DEAH-box helicase 15; HCC cells; RNA helicase; autophagy; mTORC1.

Figure 1

Knockdown of DHX15 induces autophagy. (A) GFP-LC3 puncta were analyzed in HEK293 cells stably expressing GFP-LC3 (293-GFP-LC3 cells) after transient transfection with DHX15 siRNA (siDHX15) or a scrambled control (siControl). Representative images were obtained at 600× magnification. Scale bars: 20 μm. (B) The number of GFP-LC3 puncta per cell was quantified for each group. The data are presented as the mean ± SEM. Unpaired Student's t-test (n = 5). ***P < 0.001. (C) LC3-II conversion and SQSTM/p62 degradation in L02, Huh7, and HepG2 cells that had been transiently transfected with siDHX15 or siControl were detected by western blotting. LC3-II conversion and SQSTM1/p62 degradation in these three cell lines that had been transiently transfected by DHX15 plasmid or control plasmid were detected by western blotting. *P < 0.05. (D) Autophagosomes in L02 cells were analyzed by transmission electron microscopy. The autophagosomes are indicated by arrows. Scale bars: 1 μm. (E) Numbers of autophagosomes per 40 μm² as determined from the images. The data are presented as the mean ± SEM. Unpaired Student's t-test (n = 10). *P < 0.05.

Figure 2

Depletion of DHX15 stimulates autophagosome generation. (A) GFP-LC3 puncta were analyzed in Huh7 cells transiently cotransfected with DHX15 siRNA (siDHX15) or scrambled control siRNA (siControl) and GFP-LC3 plasmids with or without Baf-A1 (200 nM) treatment for 6 h. Cellular nuclei were counterstained with DAPI. Representative images were obtained at 600× magnification. Scale bars: 20 μm. (B) The number of GFP-LC3 puncta per cell was quantified for each group. The data are presented as the mean ± SEM. Unpaired Student's t-test (n = 5). *P < 0.05, **P < 0.01, ****P < 0.0001. (C) The expression of SQSTM1/p62 and LC3B-II in Huh7 cells transiently transfected with siDHX15 or siControl with or without Baf-A1 (200 nM) treatment for 6 h was measured by western blotting. *P < 0.05. (D) The expression of SQSTM1/p62 and LC3B-II in Huh7 cells transiently transfected with siDHX15 or siControl with or without chloroquine diphosphate(CQ) (20 uM) treatment for 24 h were measured by western blotting.

Figure 3

Depletion of DHX15 induces autophagy associated with suppressing mTORC1. (A) The levels of phosphorylated substrates of the mTORC1 complex in HepG2 cells transiently transfected with DHX15 siRNA (siDHX15) or scrambled control siRNA (siControl) were analyzed by western blotting. Treatment with Torin 1 (250 nM) for 6 h was used as a positive control. Treatment with DMSO was used as a negative control. β-Actin was used as a loading control. *P < 0.05. (B) Data are representative western blots of three independent assays. *P < 0.05, **P < 0.001 (C) HepG2 cells transiently transfected with siDHX15 or siControl were treated with 200 nM rapamycin for 2 h and harvested for western blotting. Treatment with DMSO was used as a negative control. β-Actin was used as a loading control. *P < 0.05. (D) LC3 and SQSTM1/p62 levels in HepG2 cells transiently transfected with siDHX15 or siControl were measured by western blotting. Treatment with Torin 1 (250 nM) for 6 h was used as a positive control. *P < 0.05.

Figure 4

DHX15 suppresses the growth of HCC cells by inhibiting autophagy. (A) The relative viability of DHX15-knockdown cells with or without autophagy inhibition was measured by CCK-8 assay. The data are presented as the mean ± SEM (n = 3). ***P < 0.001. (B) The Ki67-positive puncta in DHX15-knockdown HepG2 cells with or without ATG5 siRNA (si-ATG5) treatment were analyzed by immunofluorescence. Cellular nuclei were counterstained with DAPI. Representative images were obtained at 600× magnification. Scale bars: 20 μm. The graph shows the results of statistical analysis of the numbers of Ki67-positive puncta per cell. The data are presented as the mean ± SEM (n = 10). ***P < 0.001. (C) After HepG2 cells had been cotransfected with or without DHX15 and si-ATG5 and incubated for 12 days, colonies were stained with Coomassie brilliant blue and counted (scale bar: 5 mm). The data are the mean ± SEM (n = 3). **P < 0.01. (D) The relative viability of cells overexpressing DHX15 with or without autophagy inhibition was measured by CCK-8 assay. The data are presented as the mean ± SEM (n = 3). **P < 0.01.

Figure 5

Expression of DHX15 in human liver cancer tissues. (A) Expression of DHX15 was detected with immunohistologic staining in tumor tissues and adjacent non-tumor tissues of the representative liver cancer patients. Scale bars: 50 um. (B) Plots of percentage of DHX15 positive cells to total cells in human liver cancer tissues. The data are presented as the mean ± SEM. Unpaired Student's t-test (n = 20). ****P < 0.0001.