Prognostic Value of Survival-Associated Splicing Factor SNRPA1 Overexpression and its Potential Mechanism in Liver Cancer

Prognostic Value of Survival-Associated Splicing Factor SNRPA1 Overexpression and its Potential Mechanism in Liver Cancer. Abstract Background: Small Nuclear Ribonucleoprotein Polypeptide A ( SNRPA1 ) is a Splicing Factor (SF) responsible for the processing of pre-mRNA into mRNA. The expression level of SNRPA1 associated with several cancer types. However, the expression level of SNRPA1 and its role as a splicing factor in hepatocellular carcinoma remain unclear. The purpose of this study was to explore the clinicopathological characteristics and prognostic significance of SNRPA1 mRNA expression level and survival status (p=3.022e−04 and p=2.932e−03). Finally, SNRPA1 protein expression level was significantly up-regulated in tumor tissues compared with normal tissues (p=3.197e−47) in CPTAC database. Our clinical samples also support the results of TCGA, with a significantly up-regulated in tumor tissues compared with normal tissues (p=0.029). Conclusions: The expression level of SNRPA1 and the PSI value of SNRPA1 could be the biomarkers of liver cancer. Furthermore, the PSI value of splicing factor SNRPA1 is superior to its mRNA expression level in predicting the prognosis of liver cancer. SNRPA1 plays an important role in tumorigenesis as a splicing factor in hepatocellular carcinoma.


Introduction
Liver cancer is predicted to be the sixth most commonly diagnosed cancer and the fourth most common cause of cancer death worldwide in 2018, with about 841,000 new cases and 7,82,000 deaths each year [1]. Similar to other malignant tumors, the pathogenesis of liver cancer is very complicated. The factors contributing to the occurrence of liver cancer include chronic hepatitis virus infection, alcohol, drugs, and genetic factors [2]. So far, a surgical operation is still the first choice in treatment of liver cancer.
It was reported that 5-year Recurrence Free Survival Rate (RFS) and 5-year Overall Survival Rates (OS) were only 30.8%-42.8% and 42.9%-60, respectively [3,4]. Even in the early stage of liver cancer, its 5-year cumulative recurrence rate was reported to be as high as 57.2%, and the 5-year overall survival rate was only 76.4% [5]. Therefore, it is a clinically important to identify a reliable biomarker that can be used for diagnosis and to predict the prognosis of liver cancer.
Multiple studies have shown that specific Alternative Splicing (AS) events such as cell proliferation, angiogenesis, tumor metastasis, and immune escape, are associated with the development and progression of cancer [6,7]. There are seven common patterns of AS events: Alternate Acceptor Site (AA), Alternate Donor site (AD), Alternate Promoter (AP), Alternate Terminator (AT), Exon Skip (ES), Mutually Exclusive Exons (ME), and Retained Intron (RI) [8]. More importantly, the expression of Specific Splicing Factors (SFs) could regulate AS events [9,10].
Oncofetal splicing factor MBNL3 could promote tumorigenesis and indicates poor prognosis of hepatocellular carcinoma patients. The knockdown of MBNL3 almost completely abolishes hepatocellular carcinoma tumorigenesis [11]. Therefore, understanding the roles of splicing factors and splicing events during tumorigenesis would open new avenues for targeted therapies.
Small nuclear ribonucleoprotein polypeptide A (SNRPA1) is a spliceosome component responsible for the processing of pre−mRNA into mRNA. It is a necessary factor for male reproductive ability and the defects of spliceosome could affect the differentiation of human spermatogonia [12].
SNRPA1 regulates the expression of CDK1, PIK3R1, VEGFC, and MKI67 in colorectal cancer. It can be recruited to laser−induced DNA damage sites to prevent R−loop−induced DNA damage [13,14]. In another study, SNRPA1 and TCF7L2 were found to bind to the insertion allele of rs386772267, a genetic insertion which is associated with the increased risk of pancreatic cancer [15]. SNRPA1 could interact with certain structural splicing enhancer, which is enriched near cassette exons with increased inclusion in highly metastatic cells of breast cancer to promote cassette exon inclusion. This interaction enhances metastatic lung colonization and cancer cell invasion [16].
Although several studies have reported this gene [13][14][15][16], the prognostic value and potential mechanism of SNRPA1 as a splicing factor in liver cancer remain unclear. As a SF associated with hepatocellular carcinoma, it will be important to understand the function and regulatory genes of SNRPA1. In this study we explored the SNRPA1 expression in liver cancer and the related signaling pathways through the Gene Set Enrichment Analysis

Data acquisition and preprocessing
The Level 3 expression data were downloaded from The

Analysis of the role of SNRPA1 as a splicing factor
The 'upset' function in the 'UpSet' R package was used to visualize the interactive AS events between the seven AS types, to clearly show quantitative results of multiple interactive sets. The prognostic relationship between the PSI value of AS events and overall survival rates (follow-up time >30 days) were performed using the univariate Cox proportional hazards regression model.

Correlation analysis of AS events and SFs
A total of 404 splicing factors were retrieved from the SpliceAid2 database [26]. Pearson correlation analysis was performed to explore the interaction and correlation between SFs and significant AS events (p<0.05, OS−related ASs

Relationships between different splices and cancers
For further analysis of the relationships between different splices and cancers, we downloaded the genes of interest in different cancers on the TCGA SpliceSeq database. They

The relationships between PSI values of SNRPA1, SCP2 and clinical analysis
We used the PSI values of SNRPA1 and SCP2 to analyze the differences in grade, stage, and T classification, among subgroups via the Kruskal Wallis tests. We then drew the survival curves according to PSI value (median level as a cut-off point) via Kaplan−Meier analysis (follow-up time >30 days).

SNRPA1 expression in CPTAC database
To find out whether the genes identified from the TCGA database also are of prognostic significance in protein level, Tandem mass spectra were searched against Human Uniprot database concatenated with reverse decoy database.  Figure 1A) in liver cancer. A paired comparison between normal and liver cancer tissue from the same patients also showed a significant up−regulation (p=4.08e−16, Figure 1B). SNRPA1 expression level showed a positive correlation with survival status (p=0.035, Figure   1C). The raw data of the survival analysis were shown in Supplementary Table S1. Furthermore, significant differences were observed in SNRPA1 expression based on histological grade and T classification ( Figure 1D-1F

SNRPA1 gene set enrichment analysis in liver cancer
To identify the potential mechanisms of SNRPA1 expression on liver cancer prognosis, we conducted the GSEA (GO and KEGG pathway enrichment analysis) between low and high SNRPA1 expression groups ( Table   3). The GO and KEGG analyses results showed processes and pathways associated with AS. "RNA splicing", "small nuclear ribonucleoprotein complex", "RNA splicing via transesterification reactions", "spliceosomal complex" and "mRNA processing" were enriched in GO analysis.
"Spliceosome", as well as some carcinogenesis and development associated pathways, like "DNA replication", "base excision repair", "RNA degradation" and "cell cycle" were enriched in KEGG analysis. These related results have been shown in Figure 2. Our results suggested that SNRPA1 is related to other gene functions through alternative splicing.

Alternative splicing profiles of liver cancer in TCGA
As       Figure   6A) in liver cancer. Our clinical samples results were consistent with the CPTAC database. SNRPA1 protein expression level was significantly up−regulated in tumor tissues compared with normal tissues (p=0.029, Figure 6B).

Discussion
Liver cancer is one of the most common cancers worldwide.
Although significant improvement in diagnosis and treatment has been witnessed in clinical practice, the prognosis of liver cancer remains considerably unfavorable [29].

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