SRSF3 and HNRNPH1 regulate alternative splicing of PRMT5 induced by ionizing radiation in hepatocellular carcinoma

doi:10.21203/rs.3.rs-1533802/v1

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SRSF3 and HNRNPH1 regulate alternative splicing of PRMT5 induced by ionizing radiation in hepatocellular carcinoma

https://doi.org/10.21203/rs.3.rs-1533802/v1

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Background

Aberrant splice variants play different roles in the formation of tumors. We observed the splice isoform of Protein arginine methyltransferase 5 (PRMT5-ISO5) increases in HCC patients undergoing stereotactic body radiotherapy, which is associated with improvement of poor prognosis. However, the mechanism of alternative splicing of PRMT5-ISO5 induced by ionizing radiation (IR) is still unclear.

Methods

The transcriptional changes of PRMT5-ISO5 induced by IR were validated by reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay. Bioinformatic analyses were performed to identify potential splicing factors involved in regulating PRMT5 splicing. Small interferring RNA and overexpressing plasmids for SRSF3 and HNRNPH1 were introduced into HCC cell lines, followed by in vitro functional experiments in regulating PRMT5 splicing by RT-qPCR, western blot and RNA-immunoprecipitation assay in vitro. The roles of IR-induced PRMT5-ISO5 and hepatocyte-specific Prmt5 knockout on HCC progression were evaluated in vivo.

Results

we indicated IR could induce PRMT5-ISO5 transcript in HCC cells by virtue of splicing factors SRSF3 and HNRNPH1. Mechanistically, HNRNPH1 silencing resulted in the decrease of PRMT5-ISO5 while SRSF3 silencing led to the increase of PRMT5-ISO5. In addition, both SRSF3 and HNRNPH1 bound to PRMT5 precursor mRNA on the region around 3' splicing site of intron 2 and alternative 3’ splicing site on exon 4, leading to their opposite functions on regulating PRMT5 splicing. In vivo, the increase of PRMT5-ISO5 induced by IR led to tumor regression, and liver-specific Prmt5 depletion decelerated the progression of Akt/N-Ras-derived spontaneous HCC.

Conclusion

Our study not only provides mechanistic views that IR-induced SRSF3 downregulation leads to the imbalance of SRSF3 and HNRNPH1 in regulating PRMT5-ISO5 transcript, but also indicates a potential radiotherapeutic of PRMT5-ISO5 in HCC formation since liver-specific Prmt5 knockout inhibits spontaneous HCC tumorigenesis.

ionizing radiation

PRMT5-ISO5

alternative splicing

SRSF3

HNRNPH1

spontaneous HCC

Protein arginine methyltransferase 5 (PRMT5), a member of the type II protein arginine methyltransferases, has been confirmed to overexpress in solid tumors and hematological tumors. The abnormal expression of PRMT5 is associated with poor prognosis ^[1–4]. PRMT5 catalyzes both monomethylated arginine and symmetric dimethylarginine, either regulating transcription of target genes or post-translationally modulating their functions ^[5]. For now, no inhibitor targeted to PRMT5 is approved for marketing, though some of them (GSK3326595, JNJ64619178, and PF-06939999) have entered clinical trials for multiple cancers. More effective treatment strategies have been evaluated for targeted therapy combined with PRMT5, such as anti-PD-1 immune checkpoint therapies and chemo-radiotherapy ^[6–7].

Considering that alternative splicing (AS) concerns up to 95% of human genes, aberrant AS is suggested to be added to the growing list of cancer hallmarks ^[8–9]. AS can be regulated by cis-elements and the subsequent recruitment of splicing factors. The mutations of cis-elements, as well as the alteration of expression level and activity of splicing factors may lead to AS dysregulation in cancers ^[10]. The disturbed expression of SR and hnRNP proteins in cancers impairs the function of target apoptotic genes through extrinsic and intrinsic pathways. For instance, SRSF1 and hnRNP A1 promote anti-apoptotic splice isoforms of Bcl-x and Mcl-1, leading to programmed cell death fading in chronic myeloid leukemia and bread cancer ^[11]. Aberrant expression of SRSF1 and hnRNP K can contribute to the insensitivity of pancreatic and liver cancers response to chemotherapy.

PRMT5, as a prognosis biomarker of various cancers including hepatocellular carcinoma (HCC) ^[7], has been identified to generate different transcripts which may act unequal biological activities. The canonical transcript PRMT5 isoform a (NM_006109.5, PRMT5-ISO1) translated into full-length protein, another transcript PRMT5 isoform e (NM_001282955.2, PRMT5-ISO5) is produced by the skipping of exon 3 and part of exon 4. The latter transcript can exhibit distinct localization and preferential control of critical genes for cell cycle arrest compared to the full-length PRMT5 ^[12]. In our previous results, PRMT5-ISO5 has demonstrated its response to radiation in blood samples from irradiated HCC patients, suggesting the possibility of PRMT5 splicing triggered by IR.

Herein, we identify that serine/arginine-rich splicing factor 3 (SRSF3) and heterogeneous nuclear ribonucleoprotein H 1 (HNRNPH1) have antagonistic interactions in regulating exon 3 and partial exon 4 skipping to generate PRMT5-ISO5 induced by IR. The increase of PRMT5-ISO5 contributes to the response of HCC cells to radiation and against xenograft tumor growth, which could be a potential therapeutic target in HCC progression.

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^[13] was used for expression correlation and survival analysis.

Cell culture

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.

IR treatment

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.

Cell transfection

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

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 A_{450 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).

RNA-immunoprecipitation

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 DynaMag^TM-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 Prmt5^flox/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 Prmt5^flox/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)^[19], 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

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.

The increase of PRMT5-ISO5 improves the poor prognosis of HCC

The splice variant PRMT5-ISO5 lacks exon 3 and part of exon 4 (Fig. 1a), which encodes a shorter isoform and translates the short protein. Using GEPIA2 web server ^[13], we found that the level of PRMT5 was significantly higher in tumors than normal tissues in liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) among 23 types of cancers (Supplementary Fig. 1a). Interestingly, the level of PRMT5-ISO1 (named PRMT-003 in GEPIA2) presented similar distribution among these three cancer types, while the level of PRMT5-ISO5 (named PRMT5-010 in GEPIA2) showed different distribution (the shape of violin-plots) in LIHC compared to the others (Supplementary Fig. 1b). Further investigation revealed that HCC patients with higher level of PRMT5-ISO1 survived for significantly shorter times than lower level patients, while high level of PRMT5-ISO5 improved the poor prognosis of HCC patients, though both transcripts showed increased expression in tumor (Fig. 1b, c). Subsequently, we also observed that the decrease of PRMT5-ISO1 and the increase of PRMT5-ISO5 in blood samples from HCC patients receiving SBRT (Supplementary Fig. 1c). These results indicated that the increase of PRMT5-ISO5 might play a positive role in radiotherapy of HCC patients.

To further confirm that IR could raise the level of PRMT5-ISO5 transcript, three HCC cell lines (Huh7, HepG2, and MHCC-97H cells) were treated with IR (10Gy, 15Gy, and 20Gy) as shown in Supplementary Fig. 1d. By performing the RT-qPCR assay, we found that PRMT5-ISO5 steadily and continuously increased in Huh7 cells after IR (Fig. 1d). Unexpectedly, PRMT5-ISO5 rapidly descended in MHCC-97H cells while mild decreased then recovered in HepG2 cells (Fig. 1e and Supplementary Fig. 1e), showing that the changes of PRMT5-ISO5 level were different among HCC cells. Besides, IR also inhibited the cell proliferation of Huh7 cells (Fig. 1f), supporting that the increase of PRMT5-ISO5 induced by IR might improve the poor prognosis of HCC patients, at least in part, depending on cell proliferation-inhibition in Huh7 cells.

Figure 1 is about here

IR induces PRMT5-ISO5 transcript by virtue of SRSF3 and HNRNPH1

Subsequently, we analyzed RNA-seq data and found the vast majority of genes downregulated after IR (Fig. 2a, b). With further exploration, genes with significant change were observed to enrich in RNA splicing pathway, including dozens of representative splicing factors (Fig. 2c). To verify the potential splicing regulators induced by IR, differential expression, overall survival and correlation analysis were performed among these splicing factors with significant change. Considering these signatures above, though we found IR induced remarkably decrease of SRSF2, SRSF3, SRSF5, SRSF6 and SRSF11, only SRSF3 and SRSF11 showed significance in differential expression between LIHC patients and normal controls, as well as the association with poor prognosis of LIHC patients (Fig. 2d, f, and Supplementary Fig. S2a-c). Further consideration indicated that SRSF3 showed higher correlation means stronger relationship with PRMT5 than SRSF11 (Fig. 2g and Supplementary Fig. S2d). Besides, by analyzing the HCC scRNA-seq dataset (GSE149614), more than 70,000 single-cell transcriptomes for 10 HCC patients were obtained. After standardization and dimensionality reduction and clustering, 15 clusters of cells were defined using the clusterProfiler package based on the transcriptional levels of SRSF3 and HNRNPH1 (Fig. 2h and Supplementary Fig. S2e, f). Specifically, genes with significant change from 2 cell populations (PCT1 and PCT2) which were identified with high level of SRSF3 were observed to enrich in RNA splicing pathway (Fig. 2i, j). These results suggested that SRSF3 might be in a dominant position in regulating PRMT5 splicing.

With the assumption that SRSF3 and HNRNPH1^[5] might be the effectors in PRMT5 splicing induced by IR, we observed SRSF3 continuously decreased in Huh7 cells while decreased then recovered in MHCC-97H cells and HepG2 cells after IR treatment (Fig. 2k and Supplementary Fig. S2g). Although HNRNPH1 quickly decreased then recovered, the increase of PRMT5-ISO5 occurred within 24 h (Supplementary Fig. S2h), suggesting that the level changes of SRSF3 and HNRNPH1 might have their effects in the early stage. Expectedly, SRSF3 sharply decreased in Huh7 cells within 1h while stably expressed in MHCC-97H cells until 12h, while HNRNPH1 changes were similar between the two HCC cells (Fig. 2l). It suggests that SRSF3 and HNRNPH1 might be the endogenous regulators of PRMT5-ISO5 induced by IR, and SRSF3 probably played a primary role in this process.

Figure 2 is about here

SRSF3 and HNRNPH1 have the opposite effects on PRMT5 AS

To evaluate the effect of SRSF3 and HNRNPH1 on PRMT5-ISO5 transcript, wide type Huh7 and MHCC-97H cells were transfected with si-HNRNPH1 or si-SRSF3 (Fig. 3a and Supplementary Fig. S3a, b). We observed that HNRNPH1 depletion caused a reduction of PRMT5-ISO5, while SRSF3 silencing led to the increase of PRMT5-ISO5, both in Huh7 and MHCC-97H cells (Fig. 3b). Furthermore, we also found that SRSF3 overexpression inhibited the increase of PRMT5-ISO5 induced by IR in Huh7 cells (Fig. 3c).Though HNRNPH1 silencing slightly weakened the level of PRMT5-ISO5 induced by IR, there was no significance compared with control. In MHCC-97H cells, HNRNPH1 depletion diminished PRMT5-ISO5 levels compared with control (siNC) in most of the time, but SRSF3 silencing could not recover the downregulation of PRMT5-ISO5 induced by IR (Fig. 3d). It seems that IR somehow leads to a more efficient change of PRMT5-ISO5 levels compared with the interference of SRSF3 and HNRNPH1 in HCC cells. Curiously, SRSF3 expression seemed to be more affected with IR treatment in Huh7 cells, while HNRNPH1 interfere was more influential in rescuing PRMT5-ISO5 downregulation in MHCC-97H cells treated by IR.

Regarding other effective factors, such as PRMT5-AS1 (an antisense transcript of PRMT5) ^[20], we generated multiple single PRMT5 KO clones in Huh7 and MHCC-97H cell lines using the CRISPR/Cas9 system and serial dilution method (Supplementary Fig. 3c). Despite screening for ~ 100 single clones of each cell line, though we seldom observed complete depletion of PRMT5 at the protein level, the transcriptional levels of PRMT5-minigene achieved dozens times of residual expression of endogenous PRMT5 mRNA (Supplementary Fig. 3d). Subsequently, co-transfection of PRMT5-minigene construct and si-HNRNPH1 or si-SRSF3 were performed in PRMT5 KO cells to simulate AS of PRMT5 (Fig. 4c). We found that IR still resulted in the increase of PRMT5-ISO5 in PRMT5^minigene Huh7 cells. Besides, IR still promoted PRMT5 splicing and SRSF3 silencing showed the impact on PRMT5 splicing somehow, compared with wild type Huh7 cells (Fig. 3c, e). Meanwhile, IR-induced the decrease of PRMT5-ISO5 was constrained in PRMT5^minigene MHCC-97H cells, though the silencing of splicing factors still had appropriate effects on PRMT5 splicing (Fig. 3d, f). The results above indicated that there might be other interferences like PRMT5-AS1 depended on 3’-terminal sequences of PRMT5 precursor mRNA (pre-mRNA) to contribute to the change of PRMT5-ISO5 induced by IR, which was worth exploring further.

Figure 3 is about here

The antagonistic interaction of SRSF3 and HNRNPH1 dependents on their competitive binding with PRMT5

To verify the interaction of SRSF3 and HNRNPH1 on PRMT5 AS, co-transfection of PRMT5-minigene and regulators (a mixture of SRSF3 construct and HNRNPH1 construct) was performed in PRMT5 KO cells. We observed that, along with gradient decrease of exogenous HNRNPH1, gradient increase of exogenous SRSF3 could inhibit PRMT5-ISO5 by degrees (Fig. 4a-d). In addition, the increase of PRMT5-ISO5 seemed to be more sensitive to SRSF3 upregulation in PRMT5^minigene Huh7 cells, while the effect of HNRNPH1 was prominent in PRMT5^minigene MHCC-97H cells.

Considering the potential binding sites of SRSF3 and HNRNPH1 on the position from exon 2 to exon 4 of PRMT5 gene in the Splice Aid database (http://193.206.120.249/splicing_tissue.html), we designed 4 pairs of primers targeted to the potential binding sites (Fig. 4e, f). By performing RIP-qPCR assay, we found that both SRSF3 and HNRNPH1 were significantly enriched on the region around 3’ splicing site of intron 2 and alternative 3’ splicing site on exon 4 of PRMT5 (Fig. 4g, h). In addition, the fold enrichment of SRSF3 was higher than HNRNPH1 in Huh7 cells (Fig. 4g), while HNRNPH1 was prior to enrich on PRMT5 in MHCC-97H cells (Fig. 4h). For endogenous SRSF3 and HNRNPH1, though we didn’t observe significant differences on the fold enrichment of HNRNPH1 using anti-HNRNPH1 antibody in cells treated with si-SRSF3 (37.5 and 50 µM), the faint amount of PRMT5 pre-mRNA acquired by anti-HNRNPH1 antibody limited our investigation (data didn’t show).

Figure 4 is about here

IR-induced PRMT5-ISO5 inhibits tumor growth in vivo

To evaluate the tumorigenic effect of IR accompanied with RNA interference which were associated with PRMT5-ISO5 transcript, a xenograft HCC model was constructed via subcutaneous injection of Huh7 cells (Fig. 5a). After 8 days post-treatment, we confirmed the significant downregulation of SRSF3 in all experimental groups (Fig. 5b and Supplementary Fig. 4a). We found that, although HNRNPH1 silencing reversed the increase of PRMT5-ISO5 induced by IR while SRSF3 knockdown reinforced it, there was no remarkable difference in tumor growth among experimental groups (Fig. 5c). Furthermore, compared with the control group, tumor sizes and tumor growth were significantly reduced in experimental groups (Fig. 5d). Besides, accompanied by lymphocytic infiltration, varying degrees of cell degeneration and necrosis were observed in tumor tissues as well (Fig. 5e). These results suggested that IR inhibited HCC tumor growth and caused tumor regression partially due to the increase of PRMT5-ISO5 transcript.

Figure 5 is about here

So far, some researches proved the high level of PRMT5 was closely associated with HCC, the limitation is the usage of HCC cell lines or xenograft models by passing over the efficacy in the remedy of spontaneous HCC ^[21]. To confirm whether liver-specific Prmt5 deficiency could achieve remission of autochthonous HCC, Akt/N-Ras-based HTVi and tamoxifen-inducible deletion of Prmt5 specifically within liver were used in Prmt5^flox/flox-Alb-CreERT2 mouse models (Fig. 6a). Previously, intraperitoneal injection of tamoxifen were performed to acquire liver-specific Prmt5 KO. We confirmed significant KO efficiency of Prmt5 in liver tissues within continuous injection for 5 and 7 days, and negative or trace amount of Prmt5 was detected 30 days after completing the injection (Supplementary Fig. 4b). Furthermore, regularly sacrificing one cohort of C57BL/6J mice after HTVi, we found that exogenetic Akt1 expression, the number of tumor nodules and the size of liver were increased within HCC progression, along with splenomegaly (Supplementary Fig. 4c, d). Besides, H&E and Oil Red O staining of liver sections demonstrated that vacuolar denaturation, inflammation and intracellular lipid accumulation presented on the liver after HTVi treatment (Supplementary Fig. 4e). In this study, when tumors were visible at day 52 post-HTVi treatment, intraperitoneal injection of tamoxifen was performed in Prmt5^flox/flox-Alb-CreERT2 mice. At day 62 post-HTVi treatment, all the mice were sacrificed. In the results, we observed the exogenous expression of Akt1 remarkably reduced, along with Prmt5 deficiency (Fig. 6b). In addition, dramatically decreased in number of tumor nodules was visible in the surface of liver from liver-specific Prmt5 KO mice (Fig. 6c). H&E and Oil Red O staining of liver sections from liver-specific Prmt5 KO mice also showed reduced pathology associated with steatosis, hepatocyte bubble morohology, and inflammation in livers (Fig. 6d), suggesting that Prmt5 deficiency prominently inhibits Akt/N-Ras-derived hepatocarcinogenesis.

Figure 6 is about here

According to the 2020 WHO report (Globocan 2020), HCC, with the fourth rank of new cases and the second rank of deaths among multiple cancers in China, has remained to show the increased mortality and poor 5-year prevalence. Considering that more than 70% of patients harboring high tumor burden or liver dysfunction are still unsuitable for definitive resection ^[22]. SBRT was a new non-invasive treatment for HCC patients who are not suitable for radical surgery ^[23]. Yet, there is still insufficient evidence on the molecular mechanism of SBRT in HCC patients.

Transcriptomic analyses have revealed that splicing aberrations, not just expression dysregulation of certain genes occur in HCC tumor tissues, which are correlated with HCC patient survival ^[24]. Herein, we reported PRMT5-ISO5, a specific splicing variant of PRMT5, increased in blood samples from HCC patients who received SBRT. PRMT5-ISO5 lacks exon 3 and partial exon 4 and translates the shorter protein, which regulates genes involved in apoptosis and differentiation ^[25]. By observing the high level of PRMT5-ISO5 is associated with improvement of poor prognosis of HCC patients, we inferred that PRMT5 splicing responded to radiation probably contributed to the effect of radiotherapy for HCC.

Current evidence has indicated that aberrant expression of splicing regulators often elicits changes in AS and promotes HCC development, and SR/hnRNP proteins significantly affect responses to chemotherapy^[25–29]. HNRNPH1, as a member of the hnRNPs family, is reported to contribute to splicing involved in tumorigenic progress of Burkitt lymphoma and HCC^[30–32]. Specifically, HNRNPH/F can inhibit the combination of U2AF65 and the polypyrimidine tract by its binding to G tracts which is situated upstream from exon 3 of PRMT5 ^[12]. On the other hand, as an unfavorable prognostic predictor in HCC ^{[30, 33]}, the decreased expression of SRSF3 can induce p53β transcript and lead to p53-mediated cellular senescence ^[34]. Regarding that SRSF3 and HNRNPH1 have effects on regulating the production of HER2 splice variants in invasive breast cancer ^[35], we found a potential interaction of SRSF3 and HNRNPH1 in regulating PRMT5-ISO5 in response to radiation treatment. Unexpectedly, we observed that PRMT5-ISO5 increased in Huh7 cells but decreased in MHCC-97H cells induced by IR, suggesting that SRSF3 might be more responsive to IR and more efficient in regulating PRMT5 splicing in Huh7 cells. Furthermore, we identified that HNRNPH1 silencing led to PRMT5-ISO5 decrease while SRSF3 silencing resulted in the increase of PRMT5-ISO5. Although HNRNPH1 depletion showed its limited inhibition on PRMT5-ISO5 in PRMT5^mini Huh7 cells, compared with wildtype Huh7 cells. This might be partly due to the loss of PRMT5 which was reported to be the methylase of HNRNPH1 ^[36]. Besides, IR appeared to be less effective in reducing PRMT5-ISO5 in PRMT5^mini MHCC-97H cells, suggesting that the absence of PRMT5 3’-terminal might eliminate the effect of PRMT5-AS1 on IR-induced PRMT5-minigene AS ^[20]. By observing that SRSF3 and HNRNPH1 shared similar affinities to the binding region around 3’ splicing site of intron 2 and alternative 3’ splicing site on exon 4, we indicated that SRSF3 and HNRNPH1 probably have a competitive interaction on binding to PRMT5 pre-mRNA. However, there were minor differences between Huh7 and MHCC-97H cells on the fold enrichment of SRSF3 and HNRNPH1 binding to the intron 3 and partial Exon 4, which might contribute to varying PRMT5-ISO5 between the two HCC cells. Still, the molecular mechanism of IR-induced PRMT5-ISO5 transcriptional changes between Huh7 and MHCC-97H cells is needed to study in-depth.

In vivo, we revealed that tumor volume reduction and structural damage presented in xenograft tumors after IR treatment. Although intratumoral injection of in vivo siRNA either enhanced or slightly interfered with the anti-tumorigenic effect of IR, PRMT5-ISO5 probably improved the efficiency of radiotherapy.

Accumulating researches indicate that PRMT5 might be a potential therapeutic target in HCC, the disadvantage of experimentally induced spontaneous HCC models limits studies in vivo. Currently, a transposon-based liver-specific delivery through HTVi becomes the perfect technique for the establishment of HCC to study the potential oncogenic genes ^{[15, 37–38]}. Akt/N-Ras-based HTVi technology is reported to rapidly induce HCC formation accompanied by fatty change lesions ^[15]. By recapitulating aberrant lipid metabolism involved in the HCC development, we observed that liver specific Prmt5 KO decelerated tumorigenesis of Akt/N-Ras-transformed hepatocytes, compared to non-CKO group. Besides, recent studies also showed that the inhibition of PRMT5 with immune checkpoint therapy diminished growth of murine melanoma tumors and enhanced therapeutic efficacy ^[39], and amounts of suppressive macrophages infiltrate and exhausted-like phenotypes tumor-associated antigen-specific CD8 + T cells presented during Akt/N-Ras-derived HCC progression ^{[15, 37, 40]}. It suggests that PRMT5 depletion inhibits Akt/N-Ras-derived hepatocarcinogenesis might partly due to the dysfunction of methylating Akt1 for oncogenic activation and improve immune dysregulation. Considering that murine Prmt5 has different splicing isoforms compared to human PRMT5-ISO5, whether PRMT5-ISO5 contributes to the anti-tumorigenic function which is analogous to Prmt5 depletion in primary HCC is worth investigating further. And our study provides a suitable model to further elucidate the function of PRMT5-ISO5 on HCC progression and treatment in vivo.

In summary, we presented a potential mechanism that SRSF3 and HNRNPH1 play antagonistic roles in regulating PRMT5-ISO5 induced by IR, owing to their competitive binding with PRMT5 pre-mRNA (Fig. 7). The increase of PRMT5-ISO5 improves the radiosensitivity of HCC in response to radiation treatment, and Akt/N-Ras-derived spontaneoous HCC with liver-specific Prmt5 KO model provides a rationale to investigate the role of the specific spliced isoform on HCC therapy.

Figure 7 is about here

PRMT5, arginine methyltransferase 5, PRMT5-ISO5, PRMT5 isoform e, HNRNPH1, heterogeneous nuclear ribonucleoprotein H 1, SRSF3, serine/arginine-rich splicing factor 3, pre-mRNA, precursor mRNA, SBRT, stereotactic body radiotherapy, IR, ionizing radiation, 5’ss, 5’ splicing site, 3’ss, 3’ splicing site, RBPs, RNA-binding proteins, AS, alternative splicing, exon 3-4A, exon 3 and partial exon 4, RIP: RNA immunoprecipitation, HTVi, hydrodynamic tail-vein injection, AKT: protein kinase B, N-RAS: neuroblastoma RAS viral oncogene homolog, H&E: hematoxylin and eosin.

Authors’ contributions

C Wen and X Liu designed research and wrote the manuscript, C Wen, L Li, Z Liang, Y Ying and P Li performed the in vitro experiments and generated data, C Wen, S Qu, H Chen and J Dai analyzed the data, C Wen, Z Tian, T Chen, M Li and Y Liu performed in vivo experiments and analyzed the data, S Ma and X Liu provided critical evaluation of experimental data and edited the manuscript. All authors reviewed and approved the manuscript.

Funding

This work was supported by National Natural Science Foundation of China (81972969 and 81773363), and Zhejiang Provincial Medical and Health Science and Technology Project of China (2018KY508). The funding body had no role in the design of the study, collection, analysis, and interpretation of the data, or preparation of the manuscript.

Acknowledgements

We thank to Prof. Haishan Huang and Prof. Hezhi Fang in School of Laboratory Medicine and Life Science, Wenzhou Medical University for help during the advice and process of histochemistry assay and RNA immunoprecipitation.

Availability of data and materials

The RNA-seq datasets supporting the conclusions of this article are included within the article and its supplementary files. The scRNA-seq datasets presented in this study can be found below: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE149614.

Ethics approval and consent to participate

The present study was approved by the Ethics Committee of Wenzhou medical university and Institutional Animal Care and Use Committee of Wenzhou Medical University.

Consent for publication

We have received consents from individual patients who have participated in this study. The consent forms will be provided upon request.

Competing interests

The authors declare no potential conflicts of interest.

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SRSF3 and HNRNPH1 regulate alternative splicing of PRMT5 induced by ionizing radiation in hepatocellular carcinoma

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