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Upregulation of cystatin SN promotes hepatocellular carcinoma progression and predicts a poor prognosis

Abstract Cystatin SN, a specific cysteine protease inhibitor, is thought to be involved in various malignant tumors. Therefore, we evaluated the role of cystatin SN in hepatocellular carcinoma (HCC). Notably, cystatin SN was elevated in tumorous samples and cells. Moreover, overexpression of cystatin SN was correlated with tumor diameter and TNM stage. Cox multivariate analysis displayed that cystatin SN was an independent prognosis indicator and that high cystatin SN level was associated with a dismal prognosis. Moreover, cystatin SN enhancement facilitated the proliferation, migratory, and invasive potential of Huh7 and HCCLM3 cells, whereas cystatin SN knockdown caused the opposite effect. Cystatin SN also modulated the epithelial‐mesenchymal transition progression through the PI3K/AKT pathway. In vivo cystatin SN promoted HCCLM3 cell growth and metastasis in xenograft mice model. Thus, cystatin SN was involved in HCC progression and could be a latent target for HCC treatment.

unclear. Therefore, it is imperative to further unravel the unknown molecular mechanisms of HCC.
Cystatin SN (CST1) is a specific inhibitor of cysteine proteases.
Various research have inferred that CST1 acts as an imperative role in inflammation and tumorigenesis. For instance, Upregulation of CST1 promotes gastric carcinoma cells proliferation and inhibits cathepsin activity (Choi et al., 2009). Moreover, raised CST1 expression promotes malignant progression of breast carcinoma and forecasts a poor prognosis (Dai et al., 2017). In colorectal cancer and pancreatic cancer, CST1 is considered as a new tumor biomarker (Jiang, Liu, Liu, Tan, & Wu, 2015;Kim et al., 2013;Yoneda et al., 2009). Meanwhile, CST1 may be used as an independent prognosis factor for patients with esophageal cancer surgery (Y. F. Chen et al., 2013). However, the roles and mechanisms of CST1 in HCC have not been clarified.
Accordingly, in this study, we used microarray analysis (G. Yang et al., 2016) to verify the expression profile of CST1 in HCC. We then explored the role of CST1 in the promotion of HCC, both clinically and experimentally. Overall, our outcomes displayed that CST1 was upregulation in HCC. High CST1 expression promoted HCC cells proliferation, migration, and invasion, thereby leading to poor prognosis.

| HCC sample collection
The samples of HCC specimens and their matched samples were gathered from surgically resected specimens from patients with HCC between April 2009 and May 2013. All patients signed written informed consent and received ethical approval to use human liver tissue for resection. The diagnosis of HCC was referred to World Health Organization criteria.

| Immunoblotting analysis
Immunoblotting assays were executed following our previous study (Han et al., 2017). Briefly, the protein sample was loaded, separated, and transferred to NC membranes, then blocked at 25°C for 1 hr, and incubated with anti-CST1 antibodies (1:1,000; Novus Biologicals, Littleton, CO) or anti-glyceraldehyde phosphate dehydrogenase antibodies. Horseradish peroxidase-conjugated secondary antibodies were used at 25°C. After sufficient washing, protein blots were detected.
PureLink™ RNA Kit (Thermo Fisher Scientific) was used to extract total RNA from tissues and cells. High Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific) was applied for reverse transcription.
Cells in log-phase growth were inoculated in 96-well plates at a concentration of 5 × 10 3 cells/well, and cultured overnight in DMEM for attachment. Cell viability was measured with CCK8 reagent (CK04-01; Kyushu, Japan).

| Colony formation assay
Cells in log-phase growth were inoculated in six-well plates at a concentration of 1,000 cells/well, and cultured for 14 days, then settled in methanol for 20 min, and stained with 1% crystal violet in methanol for 20 min. Finally, the plates were captured and then calculated the colonies.

| Scratched wound assay
Scratched wound assay was executed as previously performed (Li et al., 2016). Cells in log-phase growth were cultured in six-well plates and grown to convergence. Next, rinsing the cells with polymerase chain reaction (PBS) three times, and a wound in the monolayer was made by scratching the well with a 10-μl pipette tip.
Pictures were photographed at 0 and 24 hr on a microscope (Nikon, Tokyo, Japan).

| Transwell experiment
Transwell experiment was executed followed by our previous study  using Matrigel-coated membranes (BD Biosciences, Franklin Lakes, NJ). Cells in log-phase were inoculated in 24-well dishes at a concentration of 4 × 10 4 cells/insert. DMEM with a low concentration of FBS was added and then moved to wells injected with DMEM containing a high concentration of FBS. When the cells were cultured for 24-48 hr. Then the cells that passed through the chamber filter were settled in methanol, then the membrane filter stained with 0.5% crystal violet. Next, the cells were calculated and photographed on a microscope (×100 magnification, five microscopic fields/filter).

| Immunofluorescence assay
Immunofluorescence was executed as previously described (Lan et al., 2018). Cells in log-phase growth were inoculated on cover glasses and cultured overnight in DMEM for attachment. Then, settled in 4% paraformaldehyde for 15 min. Next, permeabilized the cells for 30 min in 0.5% Triton X-100 at 25°C for 30 min, then incubated with special primary antibodies at 4°C overnight, whereafter incubated with fluorescent secondary antibody (Thermo Fisher Scientific) for 60 min at 25°C, and rinsed three times. Finally, the cells were counterstained and pictures were photographed.

| Tumor-bearing mouse model
Male BALB/c nude mice (4-6-week old) were obtained from the experimental animal center of Shanghai Institute (Shanghai, China).
To establish an orthotopic HCC mouse model, 4 × 10 6 cells dissolved in PBS were inoculated subcutaneously into each flank of the nude mice. After 1 week, the subcutaneous neoplasms were resected and diced into 1 mm 3 cubes, then implanted into the livers (left lobes).
Tumors were resected after 4 weeks, and their sizes were measured.
To establish a metastasis model 3 × 10 6 cells were inoculated into the tail veins of each mouse. After 8 weeks, the mice were euthanized, and their lungs were resected. The procedures were executed followed by the protocols authorized by the Institutional Animal Care and Use Committee of Harbin Medical University.

| CST1 expression was increased in HCC tissue
First, we evaluated the CST1 expression at message RNA (mRNA) and protein levels in matched tumorous tissues and adjacent tissues.
The outcomes demonstrated that CST1 was upregulated in HCC tissues in comparison with that in corresponding normal tissues at mRNA and protein levels (Figure 1a,b). Next, to detect the localization of CST1 in HCC, we used immunohistochemical analysis of a recently prepared tissue microarray. As a result, CST1 expression was dramatically upregulated in HCC specimens than their counterparts and was localized in the cytoplasm (Figure 1c-h).

| Upregulation of CST1 was linked to clinicopathologic parameters and predicted dismal prognosis
To explore the relationship between CST1 and parameters of clinicopathological, we concluded the clinicopathological features of 75 patients with HCC. As shown in Table 1, statistical method, Fisher's exact text, demonstrated that overexpression of CST1 in cancerous samples was related to tumor diameter as well as TNM stage. However, the expression of CST1 was not related to age, sex, HBV infection, liver cirrhosis, or α-fetoprotein. There was a potential relationship between CST1 expression and lymph node invasion (p = .073). Furthermore, Kaplan-Meier analysis of 75 patients with HCC showed that higher CST1 expression levels in HCC tissues were dramatically related to reduced overall survival (OS; Figure 1i) and recurrence-free survival (RFS; Figure 1j) for HCC patients after surgery. The univariate and multivariate analysis exposed that high level of CST1 (p = .046), advanced TNM stages (p = .032), and lymph node invasion (p = .030) were independent prognostic factors for overall survival (Table 2). On the whole, these data suggested that CST1 act as a crucial role in HCC progression and might play as a promising prognosis factor.

| Expression of CST1 in HCC cell lines
To confirm the findings from HCC tissues in vitro using HCC cell lines,

| CST1 promoted HCC cell proliferation and carcinogenicity
To examine the effects of CST1 on growth and carcinogenicity of HCC cells, CCK8 assays and colony formation assays were applied. As expected, overexpression of CST1 stimulated Huh7 and HCCLM3 cell proliferation ( Figure 2c). Inversely, knockdown CST1 had the opposite effects. Colony formation assays showed the same outcomes (Figure 2d).

| CST1 facilitated HCC cell migratory and invasive potential
To examine the effects of CST1 on HCC cell migration and invasion,  The epithelial-mesenchymal transition (EMT) has a vital function in cancer metastatic properties; hence, we explored the influence of CST1 on EMT. Our outcomes displayed that E-cadherin expression was inhibited by CST1 overexpression. In contrast, silencing of CST1 expression caused the opposite effect (Figure 3c). Immunofluorescence analysis also confirmed these results (Figure 3e). Many pathways have been reported to be involved in the EMT, such as Wnt/β-catenin and PI3K/AKT signaling (Lan et al., 2018;C. Wang et al., 2015). Thus, we examined the expression and activation of AKT. Immunoblotting analyses displayed that the expression of phospho-AKT increased and decreased after CST1 overexpression and silencing, respectively, but that total AKT levels were unchanged (Figure 3d). These results displayed that CST1 could promote HCC metastatic properties through the EMT, and the PI3K/AKT signaling might be involved in this progression.

| PI3K/AKT signaling is involved in CST1-caused HCC progression
To investigate whether PI3K/AKT signaling was involved in HCC progression. We blocked PI3K activity with LY294002 and  documented that the level of phospho-AKT was also decreased by LY294002 treatment (Figure 5e). Therefore, PI3K/AKT signaling pathway might be involved in CST1-induced HCC progression and can promote HCC cell lines proliferation, invasion, and migration. construct an orthotopic tumor-bearing mouse model. After 4 weeks, the volumes and weights of the tumor were increased in HCCLM3-CST1 group than those in the control group (Figure 4a,b). Moreover, immunohistochemical analyses showed that the orthotopic tumor tissues expressed higher levels of Ki-67 in the HCCLM3 overexpression group compared with that in control group (Figure 4c).

| CST1 expression promoted HCC tumor growth and metastasis
Next, we injected the stably transfected HCCLM3 cell line into nude mice through the tail vein so as to examined the effect of CST1 on tumor metastasis in vivo. Necropsies conducted after 8 weeks revealed a significant increase in metastatic modules in CST1overexpressing nude mice than that in control mice (Figure 4d).
Furthermore, western blot analysis showed that E-cadherin expression was decreased and phospho-AKT, but not total AKT, was boosted in the metastatic nodules from the lungs, in the HCCLM3 overexpression group relative to those in the control group (Figure 4e). Conversely, infection of HCV, heavy drinking of alcoholic beverages, and many chronic diseases act as an imperative role in low-incidence areas (McGlynn, Petrick, & London, 2015). Of the two viruses that caused HCC, HBV accounts for nearly 75% of virus-associated HCC, whereas HCV accounts for 15-25% (Perz, Armstrong, Farrington, Hutin, & Bell, 2006).
Cysteine proteases are proteolytic enzymes. They are widely expressed in human organs and have a variety of functions, including inflammatory tissue lesion, immune response regulation, monocyte induction, and enhanced the migratory capacity of cancer cell et al (Koblinski, Ahram, & Sloane, 2000;Lah, Babnik, Schiffmann, Turk, & Skaleric, 1993). Furthermore, lysosomal cathepsins may also activate several important signaling pathways in cells, resulting in cell death.
In our research, we explored the role of CST1 in HCC. The data indicated that CST1 was overexpressed in HCC samples and HCC cells than that in paired noncancerous samples and normal liver cells (L02 and chang), respectively. Additionally, immunohistochemistry showed that CST1 was localized in the cytoplasm, and elevated CST1 level was associated with tumor diameter and TNM stage and predicted a poor  The mechanism through which CST1 contributes to cell progression remains unclear. The EMT is imperative for many physiological and pathological processes, such as embryogenesis, tissue regeneration, and so forth (Kalluri & Weinberg, 2009;Thiery & Sleeman, 2006; J. Yang & Weinberg, 2008). Importantly, we found that upregulation of CST1 dramatically facilitated cell progression via inducing the EMT through the PI3K/AKT signaling. This pathway is often dysfunctional in cancer and acts as a crucial role in modulating cell processes that are characteristic of carcinoma (Thorpe, Yuzugullu, & Zhao, 2015).
Additionally, mutations that activate these enzymes have often been detected in human carcinoma, thereby leading to cancer growth.
PI3K converts PIP2 to PIP3 on the plasma membrane and subsequently phosphorylates and activates AKT (Alessi et al., 1997;Stokoe et al., 1997). Following AKT activation, AKT phosphorylates several downstream effectors, a pathway that regulates a variety of biological processes in different types of cancer, and its excessive activation leads to abnormal cell cycle progression, proliferation, migration, invasion, altered adhesion and motility, inhibition of apoptosis, and induction of angiogenesis (Fruman et al., 2017;Manning & Cantley, 2007). Several publications have showed that PI3K/AKT signaling is involved in mediating the progression of HCC (Samarin et al., 2016;R. Y. Wang et al., 2013), and is involved in oncogenesis of various malignancies of the digestive system (Z. Chen et al., 2019;Gao et al., 2017;. In our outcomes, LY294002 was used for rescue experiments. When HCCLM3-CST1 and Huh7-CST1 cells treated with LY294002, the growth and metastatic properties of these two cells were partly reversed. This consequence suggests that PI3K/AKT signal is involved in CST1-induced HCC progression. In conclusion, we demonstrated the role of CST1 in promoting malignant biological behaviors (proliferation, migration, invasion, etc.) in HCC. However, how does CST1 regulate the progression of HCC through PI3K/AKT signaling and how to induce the occurrence of EMT are a series of problems, which still need further studies to investigate the potential mechanism of CST1's involvement in the occurrence of HCC.

CONFLICT OF INTERESTS
The authors declare that there are no conflict of interests.

AUTHOR CONTRIBUTIONS
Y. C. and D. S. designed the study and wrote the initial draft of the manuscript. Z. L. and L. L. contributed to analysis and interpretation of data and assisted in the preparation of the manuscript. All other authors have contributed to data collection and interpretation and critically reviewed the manuscript. All authors approved the final version of the manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.