mTOR up-regulation of SNRPA1 contributes to hepatocellular carcinoma development

Abstract Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide. Recent studies showed that snRNPs were implicated in human cancer development. The role of SNRPA1, which is a member of U2 snRNPs, in HCC, remains undocumented. Here, we found that SNRPA1 was highly expressed in HCC tissue compared with normal adjacent liver tissues. Up-regulation of SNRPA1 was correlated with the clinical stage of HCC and the overall survival of HCC patients. In vitro and in vivo results showed that knockdown of SNPRA1 inhibited the cell proliferation, colony formation and xenografted tumorigenesis of HCC cells. Apoptosis was induced by SNPRA1 down-regulation. Mechanistically, SNPRA1 was stimulated by mTOR activation. In addition, whole-genome microarray analysis identified that 262 genes were up-regulated and 462 genes were down-regulated by SNPRA1 knockdown in HCC cells. qPCR analysis suggested that the fibroblast growth factor-2 (FGF2), Alpha-fetoprotein (AFP), β-catenin, Ki-67 and cyclin B1 were down-regulated and caspase 3, p53 as well as p21 were up-regulated after SNRPA1 knockdown. Taken together, our findings implicate that SNPRA1 functions as an oncogene in HCC.


Introduction
Hepatocellular carcinoma is fifth most common cancer and the second leading cause of cancer death in worldwide [1,2]. The treatment options are limited and the effectiveness is far from satisfactory with poor prognosis for HCC patients. Understanding the pathogenic factors in tumorigenesis may help explore novel and promising targets for HCC.
Splicing of pre-mRNA into mRNA in eukaryotes is catalyzed by the spliceosome, a multimegadalton ribonucleoprotein complex composed of small nuclear RNAs (snRNAs) and small nuclear ribonucleoproteins (snRNPs) [3,4]. Dysregulation of spliceosome has been reported to participate in diseases development, including retinal degeneration and Taybi-Linder syndrome [5]. Recently, the spliceosome is correlated with cancer progression as mutation of some spliceosome components are found in various cancers, such as chronic lymphocytic leukemia [6], breast cancer [7], lung adenocarcinoma [8], clear cell carcinoma [9] and uveal melanoma [10]. Of note, mutations are detected mainly in U2 snRNPs and U2-related proteins [11]. Although SNRPA1 is one of U2 snRNPs, the role of SNRPA1 in HCC is unclear.
Aberrant activation of mTOR signaling is found in approximately half of HCC cases, largely due to alterations of upstream tumor suppressors and oncogenes, including PI3K, AKT, PTEN and TSC1/2 [12]. Ribosomal protein S6 kinase beta-1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) are well-known downstream targets of mTOR signaling pathway that regulate ribosome RNA biogenesis and protein synthesis, therefore functioning as a critical regulator in tumor cell proliferation, motility, invasion and cancer metastasis [13,14]. Even though a large amount of studies have revealed the molecular mechanisms of mTOR-induced HCC development, little is known about the association between mTOR and SNRPA1. Since SNRPA1 was highly expressed in HCC tissues and its up-regulation was correlated with the HCC stage and patients' overall survival, the function and mechanistic basis of SNRPA1 in HCC development should be determined. Herein, we aimed to investigate the in vitro and in vivo function and related molecular mechanism of SNRPA1 in HCC. Knockdown of SNRPA1 induced the apoptosis and inhibited the proliferation, colony formation and xenografting tumorigenesis of HCC cells. SNRPA1 expression was increased by mTOR activation. Furthermore, SNRPA1 knockdown caused alterations of numerous genes, among which HCC biomarker, AFP, was down-regulated.

TCGA gene expression data
SNRPA1 mRNA expression and clinical information of HCC patients were downloaded from The Cancer Genome Atlas at http://cancergenome.nih.gov. The 543 samples, which contain 373 tumor tissues and 169 adjacent normal liver tissues (located >5 cm from the tumor tissues), were available for the present study. In Table 1, grade represents the cell differentiation extent (G1/G2 is well/ moderately differentiated; G3/G4 represents poorly differentiated/ anaplastic). And T stage represents tumor stage. AJCC is an abbreviation for the American Joint Committee on Cancer, which was refined and well described previously [15]. For survival analysis, HCC patients were ranked by SNRPA1 mRNA expression from the highest to the lowest. Then, they were equally divided into two groups, among which each patient in the high SNRPA1 expression group had higher SNRPA1 expression than those in the low SNRPA1 expression group. Several samples were removed due to missing survival information.

Cell lines
The human normal liver LO2 cells and HCC cell lines BEL-7404, SMMC-7721, HepG2 and SMMC-7721 were obtained from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cells were maintained in Dulbecco modified Eagle's medium (DMEM, Invitrogen), containing 10% fetal bovine serum (Gibco), 1% penicillin and streptomycin solution (Corning). The cells were cultured in a 37 • C incubator with 5% CO 2 .

siRNA interference
siRNA targeting the human Raptor, Rictoror and negative control were synthesized by GenePharma (Shanghai, China). When TSC2 silencing BEL-7404 cells were seeded in 60-mm plates to reach 30-50% confluency, they were transfected with 100 nM siRNA using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. Forty-eight hours later, cells were lysed and subjected to Western blot with indicated antibodies.

High-content screening for cell proliferation assay
Cell proliferation was measured using multiparametric high-content screening (HCS) assay. BEL-7404 and SMMC-7721 cells expressing shCtrl or shSNRPA1 were maintained in 96-well plates for 5 days. Stained cells were analyzed by the ArrayScan™ HCS system (Cellomics Inc). This system is a fluorescence-imaging microscope that automatically identifies stained cells and detects the intensity and distribution of fluorescence in single cells [16]. Images were acquired and analyzed using suitable filters by 20× objective.

MTT for cell proliferation assay
BEL-7404 cells and SMMC-7721 cells expressing shCtrl or shSNRPA1 were seeded into 96-well plates (2000 cells per well) and maintained for 1, 2, 3, 4 and 5 days. Each well was washed by PBS for three times and 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/ml) was added to each well. Three hours later, the supernatants were removed and 150 μl dimethyl sulfoxide (DMSO) was added. Ten minutes later, the OD was determined at 490 nm on a microplate reader.

Colony formation assay
shCtrl or shSNRPA1 BEL-7404 cells and SMMC-7721 cells were seeded in six-well dishes at a concentration of 800 cells per well. After cultured at 37 • C for 12 days, cell colonies were formed. The colonies were fixed with methanol for at least 30 min and stained with Giemsa solution for 15 min. The number of colonies (>50 cells/colony) was manually counted by the microscopy (Olympus).

Apoptosis assay
Apoptosis was measured using Annexin V-APC apoptosis detection kit (Ebioscience, U.S.A.) following the manufacturer's instruction. BEL-7404 cells or SMMC-7721 cells infected with shCtrl or shSNRPA1 lentiviruses were washed twice with PBS, and then were re-suspended with 5 μl annexin V-APC and 100 μl cell suspension at room Abbreviations: CI, confidence interval; HR, hazard ratio.
temperature. Fifteen minutes later, the cells were subjected to apoptosis analysis on flow cytometry (FACS Calibur, Becton-Dickinson, U.S.A.).

In vivo xenograft assay
Female athymic BALB/C nude mice (4 weeks old) were purchased from the Vital River Co., LTD (Beijing, China). A total of 4 × 10 6 BEL-7404 cells infected with shCtrl or shSNRPA1 virus were subcutaneously injected into the right armpit of mice (ten mice for each group). Tumor growth was monitored and tumor size was measured until day 27 after implantation. By day 27, mice were killed by carbon dioxide euthanasia, according to the protocols as described previously [17]. The lid was closed immediately after introducing 100% carbon dioxide into the cages. The mice will die within 2.5 min. Then, the tumors were excised, weighed and fixed in formalin.

Microarray
Total RNA was extracted from shCtrl (n=3) or shSNRPA1 (n=3) BEL-7404 cells using Trizol reagents. The quantity and purity of isolated RNA were measured by NanoDrop 2000 and Agilent Bioanalyzer 2100. The gene expression profile was determined using Affymetrix human GeneChip primeview, following the manufacturer's instruction.
Significantly dysregulated genes in shSNRPA1 BEL-7404 cells compared with those in shCtrl cells were identified with the following criteria: P<0.05 and the absolute fold change >1.5. The biofunction and pathway enrichment analysis were performed using IPA ® Software (http://www.ingenuity.com).

Statistical analysis
The statistical analysis was performed using GrpahPad Prism 6.0 software. The results were represented as the mean + − standard error of mean (SEM) of at least three independent samples. Students' t-test were used to analyze the difference between two groups. One-way ANOVA was used when more than two groups. Difference was considered significant when the P-value was < 0.05.

SNRPA1 is up-regulated in HCC tissues and correlated with the overall survival of HCC patients
First, we investigated the clinical relevance of SNRPA1 by analyzing the expression of HCC-related SNRPA1 in TCGA database. The results showed that SNRPA1 expression was obviously up-regulated in HCC tissues compared with the normal tissues ( Figure 1A). We then sought to examine the abundance of SNRPA1 in 50 HCC tissues and corresponding normal liver tissues. The results showed that SNRPA1 was up-regulated in HCC tissues ( Figure 1B). Furthermore, we explored the correlation between the elevated SNRPA1 expression and clinicopathological features of HCC patients. As shown in Table 1, SNRPA1 up-regulation was strongly associated with T stage (P<0.05). In addition, Kaplan-Meier survival analyses were performed to evaluate the relationship between SNRPA1 expression and the overall survival of HCC patients. The 369 HCC patients were divided into SNRPA1 high expression group and SNRPA1 low expression group. The results revealed that the overall survival time was poorer in SNRPA1 highly expressed patients than in SNRPA1 lowly expressed patients ( Figure 1C). We also employed Multivariate Cox Regression Analysis of Overall Survival for Patients with HCC. The results showed that SNRPA1 high expression was an independent prognostic factor for HCC patients (Table 2). Additionally, the expression of SNRPA1 was increased in different HCC cells comparing with normal liver cells LO2 ( Figure 1D). Collectively, these findings suggest that SNRPA1 is closely associated with HCC.

SNRPA1 knockdown suppresses HCC cells proliferation and growth
Based on the clinical relevance of SNRPA1 in HCC, we next assessed the biological functions of SNRPA1 in HCC by knocking down SNRPA1 in HCC cells, including SMMC-7721 and BEL-7404. qRT-PCR and Western blot were performed to determine SNRPA1 knockdown efficiency by lentivirus-medicated shRNA strategy. The results showed that SNRPA1 was silenced in both SMMC-7721 and BEL-7404 cells ( Figure 2). Then, we measured cell proliferation using high-content screening assay and found that SNRPA1 knockdown significantly suppressed the growth of BEL-7404 cells ( Figure 3A,B). MTT assays showed that the viability of BEL-7404 cells was retarded by SNRPA1 silencing ( Figure 3E). We further examined the function of SNRPA1 in another HCC cells SMMC-7721. Consistently, SNRPA1 silencing inhibited the proliferation of SMMC-7721 cells ( Figure 3C,D,F). To confirm the effect of SNRPA1 knockdown on HCC cell growth, we subjected shCtrl and shSNRPA1 BEL-7404 cells or SMMC-7721 cells to colony formation assay. We observed that SNRPA1 silencing almost completely blunted the colony formation of BEL-7404 cells ( Figure 3G,H). To a less extent, colony growth was also repressed by SNRPA1 knockdown in SMMC-7721 cells ( Figure 3I,J). Taken together, these results indicate that SNRPA1 is critical for the proliferation and growth in HCC cells.

SNRPA1 is required for tumorigenesis of BEL-7404 cells in nude mice
To explore the in vivo role of SNRPA1, we subcutaneously implanted shCtrl or shSNRPA1 BEL-7404 cells into nude mice and evaluated the tumorigenesis. We found that SNRPA1 knockdown inhibited tumor growth of BEL-7404 cells ( Figure 4A). Tumor volume was reduced by SNRPA1 knockdown comparing with Ctrl group ( Figure 4B). In addition, the average tumor weight of shSNRPA1 cells was obviously lower than that of shCtrl cells ( Figure 4C). These results demonstrate that SNRPA1 knockdown blunts the tumorigenic capacity of BEL-7404 cells in vivo.

Depletion of SNRPA1 increases HCC cells apoptosis in vitro
To explore the role of SNRPA1 in apoptosis, BEL-7404 and SMMC-7721 cells expressing shCtrl or shSNRPA1 lentivirus were harvested and examined by flow cytometry. Depletion of SNRPA1 significantly induced apoptosis in both BEL-7404 and SMMC-7721 cells ( Figure 5A-D). Thus, we speculated that SNRPA1 knockdown may inhibit the viability of HCC cells partly through inducing apoptosis.

SNRPA1 expression is activated by mTORC1 in HCC cells
mTOR signaling is frequently hyper-activated in HCC. We then explored the potential association between SNRPA1 expression and mTOR signaling pathway. We found that silencing of TSC2 could cause mTOR activation, as showed by elevated phosphorylation level of S6 ( Figure 6B). In addition, TSC2 knockdown elevated the expression of SNRPA1 in both mRNA and protein levels, which could be efficiently suppressed by rapamycin treatment (Figure 6A,B). These results indicated that the activation of mTOR promoted SNRPA1 expression in HCC cells. As mTOR forms the catalytic subunit of two separate multi-protein complexes, referred to mTOR Complex 1 (mTORC1) and mTOR Complex 2 (mTORC2), which complex contributed to SNRPA1 stimulation should be addressed. Thus, we silenced Raptor or Rictor in TSC2 knockdown HCC cells and found that depletion of Raptor decreased the upregulated SNRPA1 in TSC2 silencing HCC cells, while Rictor knockdown had no effect ( Figure 6C,D). Our results suggested that mTORC1 activation enhances SNRPA1 expression in HCC cells.

SNRPA1 knockdown cause dysregulation of numerous genes
To investigate the potential downstream targets responsible for the biological activity of SNRPA1 in HCC, we conducted GeneChip analysis in shCtrl and shSNRPA1 BEL-7404 cells. The significantly dysregulated genes were defined following the criteria: fold change>1.5 and P<0.05. Heatmap results showed that a total of 262 genes including several oncogenes were up-regulated and 462 genes including some tumor suppressors, and genes relating to the iNOS pathway were downregulated by SNRPA1 knockdown ( Figure 7A). Furthermore, the ingenuity canonical pathways enrichment analyses showed that genes in the iNOS pathway were significantly suppressed by SNRPA1 knockdown in BEL-7404 cells (−log (P-value) = 1.36, Z-score = −2; Figure 7B). To validate the microarray assay results, we measured the mRNA expression of several genes differential expressed in microarray by qRT-PCR assay. Our results showed that fibroblast growth factor-2 (FGF2), alpha-fetoprotein (AFP), β-catenin, Ki-67 and cyclin B1 were down-regulated after SNRPA1 knockdown in BEL-7404 cells ( Figure 7C). Conversely, caspase 3, p53 and p21 were up-regulated in shSNRPA1 cells compared with shCtrl BEL-7404 cells ( Figure 7C). Collectively, our results suggest that SNRPA1 silencing may suppress HCC development via iNOS signaling pathway, as well as numerous oncogenes and tumor suppressors.

Discussion
In the present study, we found that of SNRPA1 expression was up-regulated in HCC tissues compared with normal liver tissues. SNRPA1 expression was associated with not only the clinical T stage of HCC, but also the overall survival of HCC patients. Depletion of SNRPA1 promoted apoptosis and suppressed the proliferation, growth and xenografting tumorigenesis in HCC. Additionally, mTORC1 activation potentiated the expression of SNRPA1 in HCC cells. Knockdown of SNRPA1 caused dysregulation of several important oncogenes and tumor suppressors and iNOS pathway in HCC cells.
SNRPA1 is a U2 snRNPs belonging to the spliceosome family that regulates the splicing of pre-mRNA into mRNA. Although some evidences have shown that spliceosome may play a key role in cancer development, little is known about the biological function of SNRPA1 in HCC. TCGA data showed that SNRPA1 was significantly overexpressed in HCC tissues and its expression was inversely associated with the disease prognosis. In addition, the mRNA expression of SNRPA1 was relatively higher in HCC cells than in normal liver cells LO2. Because we intended to explore the function of SNRPA1 in HCC, we silenced SNRPA1 in HCC cells BEL-7404 and SMMC-7721 and subjected them to cell proliferation, apoptosis and tumor growth analysis. We observed that depletion of SNRPA1 in BEL-7404 and SMMC-7721 cells largely reduced HCC cell viability. Nude mice experiments also demonstrated the essential contribution of SNRPA1 in tumor formation. It is well known that suppressed apoptosis leads to a state of uncontrolled proliferation of cancer cells. Here, SNRPA1 knockdown increased the apoptosis of both BEL-7404 and SMMC-7721 cells, suggesting that SNRPA1 knockdown inhibits the viability of HCC cells partly through inducing apoptosis.
Hyper-activation of mTOR signaling is frequently observed in human HCC tissues, mainly caused by dysregulation of upstream tumor suppressors, such as PTEN or TSC1/2 [12]. Evidences have shown that mTOR activation is sufficient to induce HCC development in mice [18,19]. Even though rapamycin, an inhibitor of mTOR, effectively reduces HCC with mTOR activation [18], long-term usage of rapamycin in a HCC mouse model causes liver injury, inflammation and subsequently higher tumor burden [20]. Rapamycin has been widely used in clinic for tumors, whereas the outcomes of rapamycin treatment in HCC remain far from satisfactory. Therefore, exploring the potential downstream targets may help us develop promising therapeutic targets for HCC. Here, we revealed that mTOR activation stimulated SNRPA1. Rapamycin treatment reversed the up-regulated SNRPA1 in HCC cells. Knockdown of Raptor, a member of mTORC1, suppressed the highly expressed SNRPA1 in TSC2 silencing HCC cells, while silencing of Rictor, a component of mTORC2, had no effect. We suggested that mTORC1 activation promoted SNRPA1 expression.
Because SNRPA1 regulates the splicing of pre-mRNA into mRNA, we next attempted to detect dys-regulated genes after SNRPA1 knockdown. Our GeneChip results showed that a total of 262 genes were up-regulated and 462 genes were down-regulated in shSNRPA1 compared with shCtrl BEL-7404 cells. Among these alterations, iNOS pathway was significantly inhibited. Overexpression of iNOS has been found in various cancers, including ovarian cancer [21], lung cancer [22] and osteosarcoma [23]. Thus, iNOS pathway may contribute to SNRPA1-mediated HCC development. It has been demonstrated that FGF2 stimulates HCC proliferation, invasion, and induces angiogenesis [24]. A novel FGF2 monoclonal antibody effectively suppressed the growth of hepatocellular carcinoma xenografts [25]. β-catenin is the second oncogene in HCC [26,27]. AFP is a well-known biomarker for HCC [28,29]. Here, we found that SNRPA1 knockdown in BEL-7404 cells resulted in down-regulation of FGF2, β-catenin, AFP, Ki-67 and cyclin B1. Additionally, apoptosis and cell cycle-associated genes, caspase 3, p53 and p21 were up-regulated by SNRPA1 silencing. These altered genes might explain why SNRPA1 silencing suppresses the proliferation and tumor formation of HCC cells. However, we still need further investigate which gene regulated by SNRPA1 is critical for HCC development.
In conclusion, we provided the first evidence that SNRPA1 functioned as an oncogene in HCC. SNRPA1 was up-regulated in HCC tissues and its expression was significantly correlated with the prognosis of the HCC patients. SNRPA1 silencing inhibited the proliferation and tumorigenesis of HCC cells. Mechanistically, SNRPA1 was stimulated by mTOR activation and it regulated various oncogenes and tumor suppressors. Our study suggests that SNRPA1 may serve as a promising target for HCC. Further study is needed to investigate the underlying mechanism by which SNRPA1 regulates proliferation and apoptosis.