www.nature.com/scientificreports OPEN Identifcation of targets of JS‑K against HBV‑positive human hepatocellular carcinoma HepG2.2.15 cells with iTRAQ proteomics Zhengyun Liu1,4, Yan Xu1,4, Wanling Zhang2, Xinghong Gao1, Guo Luo1, Hong Song2, Jie Liu3 & Huan Wang1,2* JS‑K, a nitric oxide‑releasing diazeniumdiolates, is efective against various tumors. We have discovered that JS‑K was efective against Hepatitis B virus (HBV)‑positive HepG2.2.15 cells. This study used iTRAQ to identify diferentially expressed proteins following JS‑K treatment of HepG2.2.15 cells. Silenced Transgelin (shTAGLN‑2.15) cells were constructed, and the cell viability was analyzed by the CCK8 assay after treatment with JS‑K. There were 182 diferentially expressed proteins in JS‑K treated‑HepG2.2.15 cells; 73 proteins were up‑regulated and 109 proteins were down‑regulated. These proteins were categorized according to GO classifcation. KEGG enrichment analysis showed that Endocytosis, Phagosome and Proteoglycans were the most signifcant pathways. RT‑PCR confrmed that the expression levels of TAGLN, IGFBP1, SMTN, SERPINE1, ANXA3, TMSB10, LGALS1 and KRT19 were signifcantly up‑regulated, and the expression levels of C5, RBP4, CHKA, SIRT5 and TRIM14 were signifcantly down‑regulated in JS‑K treated‑HepG2.2.15 cells. Western blotting confrmed the increased levels of USP13 and TAGLN proteins in JS‑K treated‑HepG2.2.15 cells. Molecular docking revealed the binding of JS‑K to TAGLN and shTAGLN‑2.15 cells were resistant to JS‑K cytotoxicity, suggesting that TAGLN could be an important target in JS‑K anti‑HBV‑positive liver cancer cells. These proteomic fndings could shed new insights into mechanisms underlying the efect of JS‑K against HBV‑related HCC. Hepatitis B virus (HBV) infection is associated with an increased risk of hepatic cirrhosis, hepatic decompensa- tion, and hepatocellular carcinoma (HCC). HCC is one of the leading malignant deaths worldwide. Over 350 million people are chronically infected with HBV who are at greater risk of HCC compared with uninfected people1. Terefore, it is very important to fnd efective drugs to kill HBV-positive HCC. JS-K (O2-(2, 4-dinitrophenyl) 1-[(4-ethoxycarbonyl) piperazin-1-yl] diazen -1-ium-1, 2-diolate) belongs to the diazeniumdiolate class of prodrug designed to be activated for nitric oxide (NO) release by glutathione S-transferase (GST)2. As a novel anticancer drug, JS-K is efective against a variety of cancer cells including human myeloid leukemia cells3, human multiple myeloma cell lines4, renal cancer cells, prostate cancer cells, brain cancer cells, etc.5. In addition, studies from ours and other research groups found that JS-K was also efec- tive against human HCC6,7. Our previous study demonstrated that JS-K decreased the expression of HBsAg and HBeAg in HepG2.2.15 cells8 and was efective against HBV-positive HCC HepG2.2.15 cells and its mechanism is related with DNA damage and apoptosis9. To better understand the mechanisms of JS-K on HepG2.2.15 cells, we utilized recent advances in proteomics technology and bioinformatics to explore diferentially expressed proteins (DEPs), and protein profle associ- ated with HBV-modulated DNA methylation 10. In the current study, we used stable iTRAQ to identify DEPs 1Key Laboratory of Infectious Disease and Biosafety, Provincial Department of Education, Guizhou, Zunyi Medical University, Zunyi 563000, Guizhou, China. 2Department of Microbiology, Zunyi Medical University, Zunyi 563000, Guizhou, China. 3Key Lab for Basic Pharmacology of Ministry of Education and Joint International Research Laboratory of Ethnomedicine, Zunyi Medical University, Zunyi 563000, Guizhou, China. 4These authors contributed equally: Zhengyun Liu and Yan Xu. *email: [email protected] Scientifc Reports | (2021) 11:10381 | https://doi.org/10.1038/s41598-021-90001-3 1 Vol.:(0123456789) www.nature.com/scientificreports/ in HepG2.2.15 cells treated with or without JS-K. Te research will provide novel insights into the molecular mechanisms involved in the anti-HBV and anti-HBV-positive HCC activity of JS-K. Materials and methods Reagents. JS-K was purchased from Santa Cruz Biotechnology (California, USA); Dulbecco’s Modifed Eagle’s Medium/Nutrient Mixture F-12 Ham (DMEM/F12) was obtained from Hyclone (Utah, USA); Fetal bovine serum (FBS) was purchased from Gibco (California, USA); USP13 and TAGLN antibodies were pur- chased from Santa Cruz Biotechnology (California, USA); β-actin antibody was purchased from BIOSS (Beijing, China); ECL reagents were purchased from Solarbi (Beijing, China); PrimeScript RT reagent Kit and TB Green Premix Ex Taq were purchased from Takara (Tokyo, Japan). CCK8 reagent was purchased from Living (Beijing, China). Cell culture and treatment. Te HBV-positive human hepatoma cell line HepG2.2.15 cells, shNC-2.15 cells and shTAGLN1#-2.15 cells (preserved in Key Laboratory of Infectious Disease & Biosafety, Provincial Department of Education, Guizhou, Zunyi Medical University) were maintained with DMEM/F12 medium containing 10% FBS, 0.5 mg/ml G418, 100 μg/mLstreptomycin and 100 IU/mL penicillin at 5% CO2, 37 °C. Te medium was changed two times per week. For iTRAQ-based quantitative proteomics analysis, HepG2.2.15 cells were treated 24 h with 10 µM JS-K. Te experiments were repeated with at least three diferent biological rep- licates. Cell pellets were washed with ice-cold PBS, instantly frozen and stored at − 80 °C until further analysis. Protein preparation. For each sample, 6300 μg of total proteins were diluted in 4% SDS, 100 mM Tris–HCl (pH 8.0), and 100 mM DTT and heated at 100ºC for 5 min. Each sample was then cooled to RT(room tempera- ture) and loaded onto an ultrafltration flter (30 kDa cutof, Sartorius, Germany) containing 200 μL of UA bufer (8 M urea, 150 mM Tris–HCl, pH 8.0) followed by centrifugation at 14,000×g for 30 min and an additional washing step with 200 μL of UA bufer. 100 µL of 50 mM iodoacetamide in UA bufer was subsequently added to the flter to block the reduced cysteine residues, and the samples were then incubated for 30 min at RT in the dark followed by centrifugation at 14,000×g for 30 min. Te flters were washed with 100 μL of UA bufer and centrifuged at 14,000×g for 20 min, and 100 µL dissolution bufer was added to elute protein. Te procedure was repeated twice. Te protein suspensions were then digested with 40 μL of trypsin (Promega, Madison, WI, USA) bufer (2 μg trypsin in 40μL dissolution bufer) at 37 °C for 16–18 h. Finally, the flter unit was transferred to a new tube and centrifuged at 14,000×g for 30 min. Te resulting peptides were collected as a fltrate, and the peptide concentration was determined at absorbance of 280 nm11. iTRAQ labeling and high‑pH reversed‑phase fractionation. iTRAQ labeling was performed accord- ing to the manufacturer’s instructions (Applied Biosystems). Briefy, the peptide mixtures were reconstituted with 30 μL of iTRAQ dissolution bufer. Te label method of every sample (100 μg) used iTRAQ Reagent-8plex Multiplex Kit (AB SCIEX) (JS-K 0-1, 113; JS-K 0-2, 114; JS-K 0-3, 115; JS-K 10-1, 116; JS-K 10-2, 117; JS-K 10-3, 118). Te labeling solution reaction was then incubated at room temperature for 1 h prior to further analysis. Te six labeled samples were pooled into one sample and dried in a vacuum centrifuge at room temperature. Te iTRAQ-labeled peptides were subjected to High-pH Reversed-Phase Fractionation in 1100 Series HPLC Value System (Agilent) equipped with a Gemini-NX (Phenomenex, 00F-4453-E0) column (4.6 × 150 mm, 3 µm, 110 Å)12. Te peptides were eluted at a fow rate of 1 mL/min with Bufer A (10 mM ammonium acetate, pH 10.0). Te following gradients were applied to perform separation: 100% Bufer A for 30 min, 0–5% Bufer B (10 mM ammonium acetate, 90% v/v ACN, pH 3.0) for 5 min, 5–35% Bufer B for 30 min, 35–70% Bufer B for 10 min, 70–100% Bufer B for 5 min, 100% Bufer B for 15 min, and fnally 100% Bufer A for 15 min. Te elu- tion process was monitored by measuring absorbance at 214 nm, and fractions were collected every 1 min. Te collected fractions (approximately 50) were fnally combined into ffeen pools. Each fraction was concentrated via vacuum centrifugation and reconstituted in 40 μL of 0.1% v/v trifuoroacetic acid. All samples were stored at – 80 °C until LC–MS/MS analysis13. LC–MS/MS analysis. Te iTRAQ-labeled samples were analyzed using Easy-nLC nanofow HPLC system connected to Orbitrap Elite mass spectrometer (Termo Fisher Scientifc, San Jose, CA, USA). A total of 1 μg of each sample was loaded onto Termo Scientifc EASY column using an autosampler at a fow rate of 150 nL/ min. Te sequential separation of peptides on Termo Scientifc EASY trap column and analytical column was accomplished using a segmented 1 h gradient from Solvent A (0.1% formic acid in water) to 35% Solvent B (0.1% formic acid in 100% ACN) for 50 min, followed by 35–90% Solvent B for 3 min and then 90% Solvent B for 7 min. Te column was re-equilibrated to its initial highly aqueous solvent composition before each analy- sis. Te mass spectrometer was operated in positive ion mode, and MS spectra were acquired over a range of 300–2000 m/z. Te resolving powers of the MS scan and MS/MS scan at 200 m/z for the Orbitrap Elite were set as 60,000 and 15,000, respectively13. Protein identifcation and quantifcation.