Cell cultures and treatments
HL7702, MIHA, SMMC7721, Bel7402, HCCLM3, SNU387, and SK-Hep-1 cells were obtained from the CAS Cell Bank. HCCLM3, SNU387 cells were maintained in DMEM medium (Gibco, California, USA), SK-Hep-1 cells were in MEM medium (Gibco, California, USA) and other cells were in RPMI 1640 medium (Gibco, California, USA), having 10% FBS (Gibco, California, USA), at 37oC in an atmosphere containing 5% CO2.
Oncofetal splicing factor screening
The data set was download from the Cancer Genome Atlas (TCGA) (https://portal.gdc.cancer.gov/) and the Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/geo/) databases. Differential gene expression was analyzed with limma and edgeR. The change in gene expression was considered significant with q-value less than 0.05 and fold change ≥ 2. Next, the splicerosome genes were obtained from the KEGG database and were presented in Supplementary Table 2. We selected differential spliceosome genes according to the spliceosome genes list and presented in Supplementary Table 3. Then we sorted out the common differential spliceosome genes.
Real-time qPCR analysis
Total RNA was extracted from cells by Trizol reagent (vazyme biotech, Nanjing, China). Reverse transcription of RNA to cDNA was completed with hiscript QRT supermix (qPCR; vazyme biotech). StepOne Real-Time PCR System (Applied Biosystems) was used for PCR analysis. Primer sequences for PCR amplification are listed in Supplementary Table 4. 18S served as an internal control. Relative expression of mRNAs was calculated from comparative Ct formula.
Western blot analysis
Extraction of total protein was done using RIPA lysis buffer (NCM Biotech, China), followed by denaturation by adding 5× loading buffer. Proteins were separated by SDS-PAGE. Membranes was blocked by 5% BSA for 1 hour at room temperature. The membranes were placed in the primary antibodies: SNRPE (Thermo, PA5-96342, 1:1000), FGFR4 (Abcam, ab178396, 1:1000). Next, the membranes were placed in secondary antibodies after washing with TBST. Finally, membranes were observed in a Gel imaging System (TANON, Beijing, China). The images were quantified by Image J software. GAPDH or β-Actin was used as an internal reference.
Gene expression analysis
The expression plot and survival plot of SNRPE were collected from GEPIA (Gene Expression Profiling Interactive Analysis) 44. The correlation among the expression of SNRPE, FGFR4, and CREB3L4 was generated from cBioProtal (https://www.cbioportal.org/).
Tissue microarray and immunohistochemistry
TMA (HLivH180Su16) containing HCC samples were obtained from Outdo Bioth (Shanghai, China). The TMA specimens were used for IHC analysis. Multiplicative staining index (0–3) and the average staining score (0–4) were employed for the quantification of SNRPE protein expression levels. We marked the tumor with IHC score ≥ 8 as SNRPE-high expression and tumor with IHC score < 8 as SNRPE-low expression.
Immunohistochemistry (IHC) was performed as previously described. In brief, tumor tissues were fixed in paraformaldehyde and blocked with FBS. Sections were incubated with Ki67 antibody (1:50) after permeabilization with Triton X-100. Next, sections were washed with PBS and placed in the secondary antibody. After counterstaining with hematoxyline, sections were visualized under a microscope (BX53, Olympus, Japan).
Vector construction and siRNA transfections
Cell growth to 50–60% confluency was followed by the transfection with siRNAs targeting SNRPE, FGFR4, UPF1, SOX2, or negative control (NC) for 8 h. Lipofectamine 3000 (Invitrogen, USA) was used in all transfection experiments. To generate HCCLM3 cell lines stably knocking down SNRPE, shRNA or negative control-shRNA were constructed using a lentiviral shRNA technique. The shRNA and siRNA sequences are listed in Supplementary Table 4.
We transfected pEGFPN1-SNRPE (NM_003094.4) or pEGFPN1-empty vector using lipofectamine 3000 to detect the effect of overexpression of SNRPE on HCC cells. To stably express SNRPE in HCC cells, we also used lentiviral vectors. After lentivirus infection, hepatoma cells were exposed to 5 µg/mL puromycin for one week. Before further analysis, the stable expression of SNRPE in cell lines was detected by Western blot.
Cell proliferation and colony formation assay
Cell growth curve was detected through CCK8 (Vazyme biotech, Nanjing, China) assays. Briefly, an equal number of the cells were dispersed into 96-well plates at day 0, and after treatment absorbance was measured at 450 nm with the help of a multi-detection micro plate reader (thermo, USA). EdU Kit (RiboBio, Guangzhou, China) was used for the EdU immunofluorescence staining. The results were analyzed with a light microscope (Olympus Corporation, Japan) and ImageJ software.
For the analysis of colony formation, Cells (500 cells/well) were dispersed into 6-well plates and contained for 14 days. Then methanol was used to fix, crystal violet to stain, and Image J software for counting the number of colonies.
Cell migration assay
Cells dispersed in 250 µL of serum-free medium were seeded into the upper compartment of the transwell. Migrated cells were immobilized and stained using crystal violet. Cell number was counted by getting average from 5 random fields under a light microscope (Olympus Corporation, Japan).
Cell Apoptosis and Cell Cycle Assays
For the determination of cellular apoptosis, cells were collected after treatment and suspended in binding buffer. Cells were then allowed to interact with PI and Annexin V-FITC (Vazyme Biotech, Nanjing, China) for 15 min and analyzed by flow cytometry (BD, Biosciences, USA).
Cells were treated for cell cycle assay, fixed in ethanol and stained by Cell Cycle staining kit (Keygen Biotech, Nanjing, China). Flow cytometer and FlowJo 10.6.2 were used for analysis.
Xenograft assay
All animal experiments were performed following the instructions of the Animal Care Committee of China Pharmaceutical University (Approval No.2110748). Five-week-old female mice (BABL/c nude) were obtained from the Hangzhou Ziyuan Experimental Animal Technology Co., Ltd. HL7702 (4 ×106 cells in 100 µL PBS) or SMMC772 (2.5 ×106 cells in 100 µL PBS) or HCCLM3 (2×106 cells in 100 µL PBS) were administered subcutaneously into the right flank of mice. Time period for tumor growth was 4 weeks. Tumor growth and mice weight were recorded every other day and were calculated. The formula is as follows: tumor volume= (length × width^2)/2. At the end, tumors were removed and photographed.
For in vivo metastasis assays, HCCLM3 cells (2×106 cells in 200 µL PBS) with SNRPE shRNA (HCCLM3-shSNRPE) or negative control HCCLM3 cells (HCCLM3-shNC) was used. The cells were injected into mice via the tail vein. Then mice were killed after 5 weeks of injections. Paraffin-embedded nude mice lungs were stained with H&E to observe metastatic nodules.
RNA sequencing and data analysis
Extraction of total RNA was done using TRIZOL Reagent (Vazyme). After quantification with Agilent 2100 Bioanalyzer and Nano Drop, samples were used for library preparation. Libraries having divergent indices were multiplied and loaded on an Illumina HiSeq instrument. 2×150bp paired-end configuration was carried out for sequencing; base calling and image analysis were carried out on the HiSeq instrument by the HiSeq Control Software + OLB + GAPipeline-1.6 (Illumina). Further analysis of sequences was completed by GENEWIZ. The RNA-Seq data were saved in GEO Database under accession number GSE197092, available at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE197092.
Alternative splicing events are determined by the human transcriptome. CASH was used to analyze differential splicing of SNRPE knockdown samples (compared to the negative control) 45. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was accomplished with DAVID (https://david.ncifcrf.gov/). Differentially spliced genes (PSI ≥ 0.2, Bayes-factor ≥ 10) were used for KEGG enrichment analysis.
Gene set enrichment analysis (GSEA)
Gene Set Enrichment Analysis (GSEA) was performed according to the instructions of the Broad Institute. The RNA-HiSeq data of HCC were divided into SNRPE high expression group and SNRPE low expression group according to the FPKM value of SNRPE. P value was obtained by comparing the enrichment score with the enrichment result produced by the random arrangement of 1000 genes, and its statistical significance was evaluated.
Splicing assay using RT-PCR
Total RNA isolation and single-stranded cDNA were obtained according to the above-mentioned method. PCR was executed with Rapid Taq Master Mix (Vazyme). The PCR program was set as denaturation at 95°C for 3 min, amplification by 35 cycles at 95°C for 15 s, 60°C for 25 s, 72°C for 15 s, finally 72°C for 5 min. Final PCR products were separated by agarose gel electrophoresis followed by the scanning with gel imaging system (TANON, Beijing, China). The primer sequences are presented in Supplementary Table 4.
Data analysis
Graphpad Prism 6.02 was used for statistical analyses. For comparisons, Student’s t-test (two-sided) or the one-way analysis of variance was applied between the data pairs. P˂0.05 was statistically significant. Kaplan–Meier method was applied for survival curve. We used log-rank test to compare survival time between two groups.