Patients and bioinformatics analysis
The criteria for blood samples from HCC patients who received stereotactic body radiotherapy (SBRT) including 1) hepatocellular carcinoma with relatively preserved liver function (Child-Pugh classification A or B), 2) taking SBRT as the first treatment with biologically effective dose (BED) in correspondence from 80Gy to 100Gy, 3) without other comprehensive therapies affecting indicators in the blood such as chemotherapeutic treatments. All the patients with the written informed consent and study protocols were approved by the Ethics Committee of Wenzhou medical university. RNA sequencing (RNA-seq) and analysis were completed by Novogene Biotech.
The patients’ datasets derived from TSVdb TCGA Splicing Variants Database (http://www.tsvdb.com/plot.html) were analyzed using Kruskal-Wallis test and Kaplan-Meier Curves (Log-Rank Tests) in R software. Single-cell RNA sequencing (scRNA-seq) dataset (GSE149614) derived from the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/) were preprocessed by using the Seurat package in R software. The harmony package and t-distributed stochastic neighbor embedding (t-SNE) algorithm were used to perform normalization, dimensionality reduction and cluster classification. Gene ontology (GO) analysis was conducted with clusterProfiler packages. GEPIA2 was used for expression correlation and survival analysis.
Three human HCC cell lines (Huh7, HepG2, and MHCC-97H cells) were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and Penicillin/streptomycin at 37℃ with 5% CO2. Additional methods are provided in the Supporting Information.
X-Rad 320 Biological Irradiator (Precision X-Ray, USA) was used to irradiate HCC cells and xenograft at a dose rate of 300 cGy/min.
Plasmid construction and siRNAs
The PRMT5-minigene, a segment of the PRMT5, was amplified by polymerase chain reaction (PCR). The human HNRNPH1 and SRSF3 open reading frames (ORFs) were amplified by RT-PCR. The primers are listed in Supplementary Table S1. Next, the PCR products were gel-purified with the Gel Extraction Kit (Takara, China), and then in-fusion cloned into the pcDNA 3.1-FLAG vector which had been digested by BamHI and XhoI restriction endonucleases (NEB, China) using ExonArt Seamless Cloning and Assembly Kit (Exongen, China).
The normal siRNA and in vivo siRNA (2' O-methyl+5' cholesterol-modified) against HNRNPH1 or SRSF3, and nonsense siRNA (RiboBio, China) were dissolved in RNase-free water for cell transfection, or phosphate buffered saline (5nmol siRNA with 50 μL PBS) for intratumoral injection. The sequences of si-HNRNPH1 and si-SRSF3 are shown in Supplementary Table S1.
RNA interference and construct overexpression were carried out in 6-well plates using Lipofectamine 2000 (Invitrogen, USA) with 5μL siRNA (the final concentration of 50 nM), or 2.5 μg plasmid. For co-transfection assay, 2 μg PRMT5-minigene plasmid was co-transfected with 4 μL siRNA (the final concentration of 40 nM) or 2μg plasmid in PRMT5 KO cells cultivated in the 6-well plates. For investigation of HNRNPH1 and SRSF3 interaction, 4 μg PRMT5-minigene plasmid was co-transfected with total 4 μg plasmids mixture (pcDNA 3.1-FLAG-HNRNPH1 plus pcDNA 3.1-FLAG-SRSF3) in PRMT5 KO cells grew in the 60 mm petri dishes. The cells were harvested 48h post-transfection. The levels of PRMT5-ISO5 were determined by RT-qPCR and the expression of HNRNPH1 and SRSF3 were detected by western-blot.
Generation of PRMT5 KO clones
Cas9 lentivirus and PRMT5-sgRNA lentivirus were designed and generated (Cyagen Biosciences, China) in HEK 293T cell lines by co-transfection of psPAX2, pMD2.G, and Cas9 or PRMT5-sgRNA plasmids with a 4:3:1 ratio. A total of 2.5 μg plasmids were mixed with Lipofectamine 2000 reagent (Thermo Fisher Scientific, USA) in a well of 6-well plates when HEK 293T cells had 60-70% confluency. Huh7 and MHCC-97H cell lines were infected with Cas9 lentivirus for 24 h and then treated with hygromycin for 7-10 d. Subsequently, Cas9 positive cells were infected with PRMT5-sgRNA lentivirus for another 24 h and then treated with puromycin for 5-7 d. Furthermore, serial dilution was performed to generate single clones of stable PRMT5 knockout (KO) cell lines. Finally, all the clones were determined PRMT5 KO efficiency by western blot.
Cell proliferation was detected using cell counting Kit-8 kit (China). 1.5 × 103 Huh7 cells were seeded into 96 well plates and treated with IR. 0h, 24h, 48h and 72h post-IR treatment, 10μL CCK-8 solution was added to each well and incubated for 1.5h at 37℃. The absorbance at A450 nm was measured on Spectra Max 190 plate reader (Molecular Devices, USA).
Reverse transcription-quantitative PCR and western blotting
Total RNA was extracted using RNAiso Plus (Takara, China) according to the manufacturer’s protocol. Quantitative PCR analysis of PRMT5-ISO5 was carried out with 10μL TB Green™ Premix Ex Taq™ II (Takara, China) by QuantStudio 3 Real-Time PCR System (Applied Biosystems, USA). The primers for RT-qPCR are shown in Supplementary Table S1.
Proteins extracted from cells and tissues were measured and heat-denatured. Equal amounts of proteins were separated using 10% or 12% SDS-PAGE and transferred on the PVDF membrane (Millipore, USA). After saturating, membranes were incubated with anti-PRMT5 (ab109451, Abcam, USA), anti- HNRNPH1 (ab10374, Abcam, USA), anti-SRSF3 (ab198291, Abcam, USA), anti-AKT1 (75692S, CST, USA), anti-GAPDH (60004-1-Ig, Proteintech, China), and subsequently incubated with HRP-conjugated secondary antibodies (Biosharp, China). Finally, membranes were detected using Pierce™ ECL Western Blotting Substrate (Thermo Scientific™, USA) and visualized with ChemiScope 6100 (CliNX Science Instruments, China).
Cells were transfected with FLAG-tagged SRSF3 or HNRNPH1 using Lipofectamine 2000 reagent (Thermo Fisher Scientific, USA). 48 h post-transfection, cells were washed and collected in lysis buffer (10 mM Tris-HCl (pH 7.5), 100mM NaCl, 5mM MgCl2，0.5% NP-40, 1% Triton X-100) containing EDTA-free Protease Inhibitor Cocktail (Roche, Switzerland), 1mM DTT (Thermo Scientific™, USA) and 200 units/mL RNase OUT (Invitrogen, USA). Then 10% of cell lysate was taken as “input” and the remains were divided into equal amounts and incubated overnight at 4℃ with anti-FLAG magnetic beads (Bimake, China) or pre-prepared IgG-protein A/G magnetic beads (Bimake, China). Subsequently, all the mixtures were washed 6 times with 1×NT-2 buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM MgCl2, 0.05% NP-40) using DynaMagTM-2 Magnet (Invitrogen, USA). Next, washed samples were digested at 55℃ for 30 min with Proteinase K digestion buffer (1×NT-2 buffer containing 1 % SDS, 1.2mg/mL Proteinase K). Finally, the immunoprecipitated RNA was extracted using RNAiso Plus (Takara, China). Fold enrichment of target region was determined after normalization to the input and compared with IgG control. The primers for RT-qPCR are shown in Supplementary Table S1.
Hydrodynamic tail-vein injection and Prmt5 conditional knockout experiments
Male Prmt5flox/flox-Alb-CreERT2 mice with C57BL/6J background (4-5 weeks old) were generated and purchased from Cyagen Biosciences. The mice were housed under pathogen-free conditions with a 12 h light/dark cycle and freely accessed to water and food. pCMV(CAT)T7-SB100 and pT3-myr-AKT-HA were bought from Fenghui Biotechnology (Addgene plasmids #34879 and #31789), and pT3-EF1α-NRasV12 (RPT-ZL 2101C2) was purchased from RiboBio. After acclimation for a week, hydrodynamic tail-vein injection (HTVi) of a volume equivalent to 10% body weight of endotoxin-free plasmids (10 μg of pCMV(CAT)T7-SB100, 10 μg of pT3-myr-AKT-HA, and 10 μg of pT3-EF1α-NRasV12 for each mice [14-16]) dissolved in PBS was given to Prmt5flox/flox-Alb-CreERT2 mice within 7s [17-18]. Registering morbidity (white spots presented on the liver) according to pre-specified criteria, the mice were randomly divided into conditional KO (CKO) cohort and non-CKO control around 7.4 weeks post-HTVi. The mice from the CKO group were received continuous intraperitoneal injection of 100 μL tamoxifen (pre-dissolved in corn oil at 10 mg/mL, 40 mg/kg per mice, Sigma-Aldrich, USA) for 7 days. The mice were euthanized, and liver (tumor) tissues were collected for further analyses. All animal care and experimental procedures were carried out in accordance with protocols approved by Wenzhou Medical University Institutional Animal Care and Use Committee.
Xenograft model and treatments
Male BALB/C nude mice (4-5 weeks old, Zhejiang Vital River Laboratory Animal Technology) were kept under pathogen-free conditions with a 12 h light/dark cycle and freely accessed to water and food. After acclimation for a week, 1×107 Huh7 cells were mixed with Matrigel Basement Membrane Matrix (BD, USA) at 4℃ and then injected into the right flank of nude mice. When the tumors were visible (160 mm3 in volume), the mice were divided into 4 groups and respectively treated with 1) 15Gy IR, 2) 15Gy IR, and intratumoral injection of si-HNRNPH1 (5 nmol for each), 3) 15Gy IR, and intratumoral injection of si-SRSF3 (5 nmol for each), and 4) untreated control. All animal care and experimental procedures were carried out in accordance with protocols approved by Wenzhou Medical University Institutional Animal Care and Use Committee.
Tumor growth and histochemistry assay
The xenograft tumor volume was measured every 2 days and calculated as follows: volume = longest tumor diameter × (shortest tumor diameter)2/2. At the end of the experiment, mice were sacrificed by euthanasia, tumor tissues were excised, weighted, images captured, and immunohistochemistry analyzed.
Liver (tumor) tissue sections were paraffin-embedded, deparaffinized, and rehydrated, followed by hematoxylin and eosin (H&E) staining. For detection of lipid droplets, liver tissue sections were incubated with oil red O dye and washed with 60% isopropanol. The images were captured and have been evaluated by an experienced pathologist (SFH).
Statistical analysis for biological assays was performed by Graphpad Prism 7 software, using one way or two-way ANOVA, and two-tailed unpaired t-test, as appropriate. All data are presented as mean ± S.D. differences were considered statistically significant if P<0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.