Overexpression of SSR2 promotes proliferation of liver cancer cells and predicts prognosis of patients with hepatocellular carcinoma

Abstract Signal Sequence Receptor Subunit 2 (SSR2) is a key endoplasmic reticulum gene involved in protein folding and processing. Previous studies found that it was upregulated in several cancers, but its precise role in hepatocellular carcinoma (HCC) remains unclear. To have a better understanding of this gene in HCC, we examined the expression of SSR2 in HCC tissues by analysing The Cancer Genome Atlas (TCGA) data and immunohistochemistry. We also assessed the association between SSR2 expression and clinicopathological characteristics of HCC patients and patient survival. Potential function of SSR2 was predicted through GSEA and protein–protein interaction analysis. MTT, flowcytometry, transwell and a nude mice xenograft model were employed to investigate the biological functions in vivo and in vitro. The results showed that the expression of SSR2 was significantly increased in HCC tissues, and SSR2 expression was associated with several clinical characteristics. In addition, patients with higher SSR2 expression had poorer survival. Enrichment analysis suggested that SSR2 was probably involved in biological process or signalling pathways related to G2/M checkpoint, passive transmembrane transporter activity, ATF2_S_UP. V1_UP and ncRNA metabolic process. Further experimental study showed that SSR2 knockdown inhibited cell proliferation, migration and invasion ability and promoted apoptosis and cell cycle arrest in vitro. Moreover, downregulation of SSR2 also repressed the growth of HepG2 cells in vivo. In conclusion, our study suggests that SSR2 may act as an oncogene in HCC.


| INTRODUC TI ON
Hepatocellular carcinoma (HCC) is the dominant primary liver cancer, and accounts for 90% of patients with liver cancer. 1 There are approximately 850,000 new liver cancer cases and 800,000 deaths worldwide each year. 2 For early HCC, surgical resection and liver transplantation are recommended treatments. 3 However, due to the difficulty in early diagnosis, a majority of patients are ineligible to surgical resection upon diagnosis. Multikinase inhibitors sorafenib and lenvatinib are the standard therapies for patients with advanced HCC, but with limited clinical efficacy. 4 Therefore, novel biomarkers are necessary for timey diagnosis and effective treatment in HCC patients.
The signal sequence receptors (SSRs) are glycosylated endoplasmic reticulum (ER) membrane receptors associated with protein translocation within the ER membrane. 5 Signal sequence receptor subunit 2 (SSR2), as one of the important subunits of SSR, was abundant in mammals. Studies have shown that SSR2 regulated the neural differentiation and zebrafish embryogenesis. 6,7 In recent years, SSR2 has been implicated in the progression of gastric cancer 8 and melanoma. 9,10 In 2020, Hong et al. reported that SSR2 promoted the HCC metastasis by modulating epithelial-mesenchymal transition (EMT). 11 However, a comprehensive role of SSR2 in HCC remains unclear.
In this study, we aimed to reveal the significance of SSR2 in HCC by using a variety of bioinformatics tool, in vitro experiments and a nude mice xenograft model.

| SSR2 differential expression and survival analysis in the TCGA database
Boxplots and scatter plots were employed to generate differential expressions of SSR2 in TCGA using disease state (tumour or normal, TNM, pathological stage, histological stage and vascular invasion) as the variables. Statistical ranking for SSR2 expression above or below the median value was defined into SSR2-high or SSR2-low, respectively. The relationship between clinicopathological parameters and SSR2 was analysed with the Wilcoxon signed-rank sum test and logistic regression. Cox regression and the Kaplan-Meier method were used to analyse the association between clinicopathological characteristics and the overall survival were then employed for statistical analysis of survival data and subsequential visualization, respectively.

| Analysis of DEGs between SSR2-high and SSR2-low expression GC groups
DEGs between SSR2-high and SSR2-low patients from TCGA-LIHC datasets were determined by the DESeq2 (3.8) package, and the unpaired Student's t-test was employed for statistical analysis.
Genes with the adjusted p-value <0.05 and the absolute FC > 1.5 were considered to be statistically significant. The DEGs between SSR2-high and SSR2-low patients were presented in volcano plot and heat map.

| GSEA enrichment analysis
An ordered list of SSR2-related DEGs was firstly generated based on their association with SSR2 expression by GSEA method. 13 The expression level of SSR2 was a phenotype label. The number of gene set permutations were 1500 times for each analysis. The statistical significance of pathways was set as a normal p-value <0.05 and an FDR q-value < 0.25. Statistical analysis and graphical plotting were conducted using R package cluster Profiler. The diagnostic performance of SSR2 was estimated using receiver operating characteristic (ROC) curves.

| Protein-protein interaction (PPI) network construction
To investigate the protein interactions between SSR2 and other proteins, a SSR2-related PPI network was established via the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database (https://strin g-db.org/) with PPI pairs interaction score > 0.9. 14 2.6 | Cell culture and transfection HepG2 and Huh-7 cell lines were purchased from Cell Bank of Shanghai Institutes for Biological Sciences (Chinese Academy of Sciences) and cultured in Dulbecco's minimal essential medium (Gibco, USA) supplemented with 10% FBS (Gibco, Rockville, US).
Cells were maintained at an incubator with the humidified atmosphere (37°C, 5% CO 2 ). As for transfection of small interference RNAs, both cells were firstly seeded in 6-well plates and then trans-   Substrate was finally carried out for signal detection.

| Cell proliferation assay
For cellular proliferation analysis, Cell Counting Kit-8 (CCK-8) was employed as described previously. 16 In brief, the transfected HepG2 cells were plated in 96-well plates at 2 × 103 per well. After culture for 1, 2, 3, 4 and 5 days, CCK-8 was added to each well, and the data were recorded at the optical density (450 nm) on Infinite F50 (Tecan, China). At least three individual experiments were performed.

| Cell cycle and apoptosis assay
Cell cycle and apoptosis assay were performed on cells transfected with siSSR2 or siCtrl to determine whether SSR2 regulates the growth phase and apoptosis of liver cancer cells. Cells were trypsinized, centrifuged at 300 × g (1000 rpm) for 5 min and resuspended in complete medium to form a cell suspension (1 × 10 6 cells/ml) and fixed with 70% ice-cold ethanol for 30 min. Then, the cells centri-

| Statistical analysis
Results were expressed as the mean ± standard deviation. SPSS
In 50 paracancerous and 374 HCC samples in TCGA-LIHC dataset, the expression of SSR2 was significantly higher in HCC (***p < 0.001) ( Figure 1B). Meanwhile, there was significant difference between the expression of SSR2 in 50 HCC and matched paracancerous samples (***p < 0.001) ( Figure 1C). Differential expression of SSR2 was found in 17 paired HCC and adjacent tissues and normal adjacent tissues by immunohistochemistry analysis ( Figure 1D).

| Identification of DEGs associated with SSR2 in HCC
To identify the DEGs associated with SSR2 in HCC, 187 HCC SSR2-high samples were compared with 188 SSR2-low controls, and a total of 952 DEGs, covering 649 upregulated genes and 303 downregulated genes, were determined to be statistically significant ( Figure 1E, Table S1). The heatmap of the relative expression values for the top 20 DEGs between SSR2-high and SSR2-low cohorts were also showed in Figure 1F.

| Association between SSR2 expression and clinicopathological parameters
To clarify the role and significance of SSR2 in HCC progression, a total of 368 HCC samples with SSR2 expression data and all the patients' characteristics were analysed from TCGA-LIHC. As shown in Figure 2A and B and  Figure S1. All the results above suggested that HCCs with high SSR2 expression were more likely to progress to a more advanced stage than those with low SSR2 expression.

F I G U R E 5 Downregulation of SSR2 inhibits the proliferation and promotes apoptosis and cell cycle arrest of HepG2 cells. (A and B)
Statistical result of cell count/fold and growth curve of siCtrl and siSSR2-2 groups. Data are presented as the mean ± SD from three independent experiments. **p < 0.01, ***p < 0.001. (C and D) Apoptosis ratios of siSSR2-2 group were increased compared with those in siCtrl group. Histogram is the average cell apoptosis rate (mean ± SD) of three independent experiments. ***p < 0.001. (E and F) S and G2/M phases of siSSR2-2 groups were decreased compared with those in siCtrl group. Histogram is the average ratio (mean ± SD) of three independent experiments. *p < 0.05, **p < 0.01 by Student's t-test

| PPI network and ROC analysis associated with SSR2
To investigate the interactions between the top DEGs in HCC, the Search Tool for the Retrieval of Interacting Genes/Proteins database (STRING v10.5) was employed to construct a PPI network associated with SSR2 ( Figure 3E). All the interactions between them  (Figure 3F).

| SSR2 promotes liver cancer cell proliferation in vitro
ARCHS 4 database revealed that SSR2 was abundantly expressed in liver cells including HepG2 ( Figure S2). Further research found that both SSR2 mRNA and protein were highly expressed in HepG2 cells than in Huh-7 cells compared with internal reference gene GAPDH. (Figure 4A

| SSR2 depletion inhibits cell migration and invasion in vitro
Downregulation of SSR2 notably inhibited cell migration in the wound assay ( Figure 6A and B). In addition, the number of migrating HepG2 cells in the Transwell assay was significantly decreased in the siSSR2-2 group compared with the siCtrl group ( Figure 6C and F I G U R E 6 Effect of siSSR2-2 transfection on the migration and invasion of HepG2 cells. (A and B) Cell migration after siSSR2-2 transfection analysed by wound healing assay, as compared with siCtrl group. (C and D) Invaded HepG2 cells analysed by Transwell assay, as compared with siCtrl group. The results are presented as means ± SD, and data from three independent experiments are shown. *p < 0.05, *** p < 0.001 compared by t-test with siCtrl-transfected HepG2 cells D, ***p < 0.001). These results suggested that SSR2 played a role in promoting HCC cell migration.  Although the above results improved our understanding of the relationship between SSR2 and HCC, there were some limitations.

| DISCUSS ION
Firstly, the retrospective studies of TCGA-LIHC lacked of some patient information, which may influence the results we have obtained.
Secondly, the possible signalling pathways associated with SSR2 need to be further elucidated.
In this study, we reported that SSR2 was highly expressed in HCC tissues and its high expression was significantly associated with the progression, poor survival, which might promote tumorigenesis through G2/M checkpoint. In vitro study confirmed that SSR2 knockdown via siRNA transfection inhibited cell proliferation, migration, invasion ability and promoted apoptosis, cell cycle arrest.
SSR2 depletion also repressed the tumour growth of HCC cells in vivo. Therefore, SSR2 may become a new biomarker of HCC and has the potential to predict treatment outcomes. The mechanism of SSR2 promoting the progression and metastasis of HCC will be verified in further studies.

| CON CLUS ION
Taken together, our study establishes the role for SSR2 dysregulation in HCC. We fully elucidated its expression profiles, survival analysis and the potential signalling associated with SSR2. Additionally, we report for the first time that the oncogenic activity of SSR2 is attributable to the promotion of HCC cell proliferation, invasion, migration and the inhibition of cell apoptosis. Nonetheless, in this study, we only focus on the cellular function and possible signalling pathways of SSR2 in HCC. Thus, future mechanism of SSR2 is required to uncover. Only by completely elucidating the molecular mechanisms of SSR2 in HCC can we open avenues for utilizing SSR2 to identify novel diagnostic or therapeutic target.

CO N FLI C T O F I NTE R E S T
No potential conflict of interest was reported by the authors.