eIF5A2, eukaryotic translation initiation factor 5A2; Dox, doxorubicin; si, small interfering RNA; NC, unfavorable control; LDH, lactate dehydrogenase. Discussion HCC is the third leading cause of cancer-related death world-wide (2). a tissue microarray, which was consistent with the results of reverse transcription-quantitative PCR analysis in paired HCC and adjacent healthy tissues. HCC patient-derived tumor xenograft mouse model was used for the study, and knockdown of eIF5A2 effectively enhanced the efficacy of doxorubicin chemotherapy compared with that in the control group. Notably, eIF5A2 served as a repressor in regulating autophagy under chemotherapy. Silencing of eIF5A2 induced doxorubicin sensitivity in HCC cells by triggering lethal autophagy. In addition, 5-ethynyl-2-deoxyuridine, lactate dehydrogenase release assay and calcein-AM/PI staining were used to determine the enhanced autophagic cell death induced by the silencing of eIF5A2 under doxorubicin treatment. Suppression of autophagy attenuated the sensitivity of HCC cells to doxorubicin induced by eIF5A2 silencing. The results also exhibited that knockdown of the Beclin 1 gene, which is an autophagy regulator, reversed the enhanced autophagic cell death and doxorubicin sensitivity induced by eIF5A2 silencing. Taken together, these results suggested eIF5A2 may mediate the chemoresistance of HCC cells by suppressing autophagic cell death under chemotherapy through a Beclin 1-dependent pathway, and that eIF5A2 may be a novel potential therapeutic target for HCC treatment. (25) have reported that dendrogenin A, a cholesterol metabolite, directly controls a nuclear receptor to trigger lethal autophagy in melanoma. Autophagy has been identified as a cytoprotective mechanism in gastric carcinoma, leukemia and CTEP squamous cell carcinoma (26-28). In addition, autophagy serves a cytocidal role in breast and colorectal cancer (29,30). However, the role of autophagy in the chemoresistance or chemosensitivity in HCC remains controversial. The present study aimed to determine the potential roles of eIF5A2 in doxorubicin sensitivity and to investigate the effects of autophagy during this process. Materials and methods Ethics statement The present study was approved by the Research Ethics Committee of CTEP the Second Affiliated Hospital of Zhejiang University School of Medicine (approval no. 2018-238; Hangzhou, China). All samples were anonymously coded in accordance with local ethical guidelines (based on the Declaration of Helsinki), and written informed consent was obtained from all patients. All animals used received appropriate care according to the Institutional Animal Care and Use Committee at the Second Affiliated Hospital of Zhejiang University School of Medicine (approval no. 2018-311). All efforts were made to minimize animal suffering. Cell lines and culture The human hepatocellular carcinoma cell lines SNU449, SNU387 and Huh7 were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai Institute of Cell Biology, Chinese Academy of Sciences. SNU449 and SNU387 cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.). Huh7 cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.). All culture media were supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 mg/ml streptomycin, and all cells were maintained at 37C in a humidified incubator with 5% CO2. Antibodies and reagents Anti-LC3B (1:1,000; cat. no. 3868S), SQSTM1/p62 (1:1,000; cat. no. 8025S), Beclin 1 (1:1,000, cat. no. 3495P), HRP-conjugated anti-mouse IgG (1:2,000; cat. no. 7076S) and HRP-conjugated anti-rabbit IgG (1:2,000; cat. no. 7074S) antibodies were purchased from Cell Signaling Technology, Inc. The anti-eIF5A2 (1:1,000; cat. no. ab126735) and anti-KI67 (1:200; cat. no. ab16667) antibodies were obtained from Abcam, and the anti–actin (1:1,000; cat. no. 66009-1-ig) antibody was from ProteinTech Group, Inc.. The CTEP eIF5A2 small interfering RNA (siRNA) and unfavorable control siRNA were synthesized by Shanghai GenePharma Co., Ltd. The PT3-EF1a-eIF5A2-flag and PT3-EF1a plasmids were purchased from Wuhan Yuling Biological Technology Rabbit Polyclonal to KR2_VZVD Co., Ltd. Cell Counting Kit-8 (CCK-8; cat. no. AD10) was obtained from Dojindo Molecular Technologies, Inc. The 5-ethynyl-2-deoxyuridine (EdU) kit (cat. no. A10044) and Lipofectamine? 2000 Transfection Reagent (cat. no. 11668019) were purchased from Invitrogen; Thermo Fisher Scientific, Inc. The autophagy inhibitor chloroquine (CQ; cat. no. C6628) and the mTOR inhibitor rapamycin (Rapa; cat. no. V900930) were obtained from Sigma-Aldrich; Merck KGaA. Doxorubicin (cat. no. S1208) was purchased from Selleck Chemicals. Monomeric red fluorescent protein (mRFP)-green fluorescent protein (GFP)-LC3 adeno-associated virus (AAV) was obtained from Hanbio Biotechnology Co., Ltd. 2-O-methoxyethyl (2-Ome)- and 5-cholesterol (5-chol)-modified eIF5A2 siRNA was chemically synthesized by Guangzhou RiboBio Co., Ltd. Survival analysis The tissue microarray made up of 90 paired HCC and adjacent tissues was obtained from Shanghai Xinchao Biological Technology Co. Ltd. (cat. no. HLiv-HCC180Sur-04). The clinicopathological information about patient age, sex, tumor stage and survival was provided by Shanghai Xinchao Biological Technology Co. Ltd. The follow-up period ranged between 1 and 6 years. Immunohistochemistry (IHC) staining of eIF5A2 was performed as follows: The tissue microarray was incubated with 3% H2O2 for 10 min at room temperature, followed by antigen retrieval in Tris-EDTA.