Silencing LncRNA SCAMP1 Inhibits Cell Proliferation in Hepatocellular Carcinoma via Activation of p53 Signaling Pathway

Background: Long noncoding RNA secretory carrier membrane protein 1 (SCAMP1) has been recently reported to be an oncogene in several cancers, including glioma and ovarian cancer. However, its clinical signicance and functional role in hepatocellular carcinoma (HCC) remain unknown. Methods: The expression of SCAMP1 was determined in tissue samples and cell lines using quantitative reverse transcription PCR. The clinical signicance of SCAMP1 in HCC was evaluated using chi-squared test, Kaplan-Meier survival, as well as univariate and multivariate analysis. Cell proliferation, cell cycle distribution and apoptosis were evaluated using CCK-8 assay, colony formation assay, Flow cytometry analysis. Related protein expression levels were measured by western blot analysis. Results: We found SCAMP1 expression levels were remarkably up-regulated in HCC tissues compared with that in matched adjacent tissues. Increased SCAMP1 expression was signicantly correlated with large tumor size, advanced TNM stage and poor survival prognosis in HCC. Knockdown of SCAMP1 signicantly inhibited cell proliferation, induced G0/G1 phase arrest and apoptosis in HepG2 and SNU-182 cells. More importantly, knockdown of SCAMP1 downregulated the expression levels of CDK4, Cyclin D1 and Bcl-2, while upregulated the expression levels of p21, p53 and Bax. Conclusions: In summary, our study claried the oncogenic role of SCAMP1 in HCC, and provided a potential therapeutic target for HCC treatment.


Introduction
As the major subtype of liver cancer, hepatocellular carcinoma (HCC), accounting for almost 80% of all primary liver cancers, is clinically characterized by high prevalence, high mortality rate and drug resistance [1,2]. At present, signi cant advances have been made in traditional treatments, including surgical resection, chemotherapy or radiotherapy for HCC, but the ve-year survival rate is generally below 50% because of the absence of speci c indicator in the early stage [3][4][5]. Therefore, identi cation of new therapeutic targets is of great importance for developing effective therapeutic strategy for HCC.
Long non-coding RNAs (lncRNAs) are a group of endogenous transcripts with more than 200 nucleotides in length that exhibit limited or no protein-coding potential [6]. Accumulating evidence has indicated that lncRNAs are involved in almost every aspect of biological processes, including proliferation, cell cycle, apoptosis and invasion [7][8][9]. It has been reported that aberrantly expressed lncRNAs function as oncogenes or tumor suppressor genes associated with the occurrence and development of various cancers [10,11]. For example, the expression of SAMMSON was upregulated in HCC tissues, and patients with high levels of SAMMSON had signi cantly lower overall rate within ve years after admission [12]. Upregulation of PDPK2P was clinically associated with a larger tumor embolus, low differentiation, and poor survival, as well as functionally promoted HCC cell proliferation and metastasis [13]. On the contrary, LINC01554 was downregulated in HCC, and its expression was correlated with tumor recurrence, TNM stage and long-term survival rate of patients [14]. In addition, RUNX1-IT1 [15], EPB41L4A-AS2 [16] and TPTEP1 [17] exert tumor suppressive properties by inhibiting HCC cell growth and proliferation.
Recently, a lncRNA transcript secretory carrier membrane protein 1 (SCAMP1) functions as a carrier and participates in post-Golgi recycling pathway [18], which has been reported to be dysregulated and correlated with the development of various tumors [19,20]. For instance, Wang et al [21] identi ed that SCAMP1, as one of the key lncRNAs, is a prognostic biomarker of pancreatic cancer by performing bioinformatic analysis. Functionally, inhibition of SCAMP1 signi cantly restrained the cell proliferation, migration and invasion, as well as promoted apoptosis in glioma cells [22]. SCAMP1 depletion attenuated cell viability and promoted apoptosis in pediatric renal cell carcinoma under oxidative stress [23]. Similarly, Song et al [24] found that SCAMP1 was highly expressed in ovarian cancer, which promoted ovarian cancer cell invasion and angiogenesis. Nevertheless, the biological function and molecular mechanism of SCAMP1 in HCC have not been fully elucidated. Thus, this study was performed to investigate the expression, clinical signi cance, biological effects and related molecular mechanisms of SCAMP1 in HCC.

Clinical tissue samples
Tumor tissues and matched adjacent non-cancer tissues were collected from 76 cases of HCC patients who received hepatectomy at Qilu Hospital of Shandong University (Shandong, China) between January 2009 and December 2012 with informed consent from all patients. Before surgery, none of HCC patients received any chemotherapy, radiation therapy or immunotherapy and targeted therapy. The basic demographic and clinicopathological features for HCC patients were described in Table 1. All tissue samples were immediately frozen in liquid nitrogen and stored at − 80 °C for further analysis. This study was approved by Institutional Ethics Committee of Qilu Hospital of Shandong University (Shandong, China) based on the Declaration of Helsinki. Cell counting Kit-8 (CCK-8) assay After 48 h transfection, cells were seeded into 96-well plates at a density of 3 × 10 3 cells per well and cultured overnight. Next day, a total of 10 µL of CCK-8 solution (Dojindo, Kumamoto, Japan) was added to cells in each well at 0, 24, 48 and 72 h, respectively. After another 2 h incubation at 37 °C, the absorbance was measured at a wavelength of 450 nm by a microplate reader.

Colony formation assay
After 48 h transfection, cells were seeded into six-well plates at a density of 500 cells per well and cultured for consecutive two weeks to form cell colony. Subsequently, cell colonies were washed twice with PBS and xed with 4% paraformaldehyde for 30 min. After stained with crystal violet for 20 min, the colonies were photographed and counted under a light microscope.
Flow cytometry assay

Western blot analysis
Total protein samples were extracted with RIPA lysis buffer (Beyotime, Shanghai, China) and quanti ed with a BCA protein assay kit (Beyotime). Equal amount of protein samples was separated on 10% SDSpolyacrylamide gel electrophoresis and transferred onto PVDF membranes (Millipore, Billerica, MA, USA).
The membranes were blocked with 5% non-fat milk and then incubated with primary antibodies against CDK4, Cyclin D1, p21, p53, Bcl-2, Bax and GAPDH (all from Abcam, Cambridge, MA, USA) overnight at 4 °C. After washed with PBS twice, the membranes were incubated with horseradish peroxidaseconjugated secondary antibodies (Cell Signaling Technology) for 2 h at room temperature. The protein bands were visualized using ECL reagent (Millipore) with GAPDH as the internal control.

Statistical analysis
Experimental data were analyzed by GraphPad Prism 6.0 Software (GraphPad Inc., San Diego, CA, USA) and expressed as mean ± standard deviation (SD) from three biological replicates of each experiment.
The associations between SCAMP1 expression and clinicopathological features were studied using chisquare test. Overall survival curves were plotted based on Kaplan-Meier method and estimated by the logrank test. Cox regression analysis was used to determine the independent factors that in uenced survival.
Paired t test was used to analyze differences between two types of tissues. One-way ANOVA and Tukey test were used to analyze differences among different cell transfection groups. The values of all p less than 0.05 were considered to be statistically signi cant level.

SCAMP1 was upregulated in HCC tissues and cell lines
Using quantitative reverse transcription PCR, we determined the expression level of SCAMP1 in tumor tissues and adjacent non-cancer tissues derived from 76 cases of HCC patients. As shown in Fig. 1A, SCAMP1 expression level was signi cantly upregulated in HCC tissues compared with adjacent tissues.
In addition, we detected the expression level of SCAMP1 in HCC cell lines. Consistently, elevated expression of SCAMP1 was also observed in two HCC cell lines (HepG2 and SNU-182), in comparison with human liver epithelial-3 cell line THLE-3 (Fig. 1B).
High level of SCAMP1 was associated with poor prognosis of HCC patients Based on the expression level of SCAMP1 in HCC tissues, we next analyzed the clinical signi cance of SCAMP1 in HCC. At rst, 76 cases of HCC patients were divided into two subgroups (high/low SCAMP1 level) using the median of the cohort as a cut-off value. The correlation analysis between SCAMP1 expression and clinicopathological features of HCC patients was displayed in Table 1, which indicated that high expression of SCAMP1 was positively correlated with large tumor size (p = 0.003) and advanced TNM stage (p = 0.006). Through survival analysis, it was found that patients with the high expression of SCAMP1 had a shorter overall survival time than those with low SCAMP1 expression (Fig. 2,

SCAMP1 knockdown suppressed cell proliferation, induced G0/G1 phase arrest and apoptosis in HCC cells
The increased expression of SCAMP1 was correlated with large tumor size led us to hypothesize that SCAMP1 might promote the HCC cell proliferation in vitro. To this end, SCAMP1 expression was stably depleted in HepG1 and SNU-182 cells with different speci c siRNAs. The knockdown e ciency was veri ed using quantitative reverse transcription PCR (Fig. 3A). The results of CCK-8 assay showed that SCAMP1 knockdown signi cantly inhibited the proliferation of HepG1 (Fig. 3B) and SNU-182 (Fig. 3C) cells. We observed that si-SCAMP1#1 transfection showed stronger suppressive effects on SCAMP1 expression and cell growth and thus selected it for the subsequent experiments. Next, we further con rmed the suppressive effects of SCAMP1 knockdown on cell proliferation by the results of colony formation assay (Fig. 3D). Considering the close relationship between cell proliferation and cell cycle progression/apoptosis status, we next performed ow cytometry analysis to investigate the effects of SCAMP1 knockdown on HCC cell cycle distribution and apoptosis. As depicted in Fig. 4A, the percentage of cells at G0/G1 phase was signi cantly increased, while cells at S and G2/M phase were accordingly decreased in si-SCAMP1#1 transfection group compared with si-NC group in both HepG2 and SNU-182 cells. Similarly, SCAMP1 knockdown remarkedly promoted cell apoptosis, including early and late apoptosis in both HepG2 and SNU-182 cells (Fig. 4B).

SCAMP1 knockdown activated p53 signaling pathway in HCC cells
To clarify the molecular mechanisms underlying SCAMP1 knockdown regulating HCC cell proliferation, G1/S transition and apoptosis, we analyzed the expression of CDK4/Cyclin D1, associated with G1/S transition and p53 target genes, including p21, p53, Bcl-2 and Bax using western blot analysis. Our results showed that knockdown of SCAMP1 obviously downregulated the expression levels of CDK4, Cyclin D1 and anti-apoptotic Bcl-2, while upregulated the expression levels of p21, p53 and pro-apoptotic Bax in both HepG2 (Fig. 5A) and SNU-182 (Fig. 5B) cells. Taken together, these results indicate that SCAMP1 depletion-mediated inhibition of HCC cell proliferation was associated with the activation of p53 signaling pathway.

Discussion
This study focused on the expression level and role of SCAMP1 in HCC. Using quantitative reverse transcription PCR, we rst showed that SCAMP1 was highly expressed in HCC tissues and cell lines, and SCAMP1 expression was correlated with tumor size, TNM stage and poor prognosis in HCC patients. Consistent with the upregulation of SCAMP1 in HCC, several studies have reported that the expression of SCAMP1 was increased in ovarian cancer [24], glioma [22] and pediatric renal cell carcinoma [23]. In clinical level, Song et al [24] utilized TCGA database to found ovarian cancer patients with high SCAMP1 level had poorer overall survival rates than those with low SCAMP1. The expression level of SCAMP1 was positively correlated with the pathological grades of glioma and higher SCAMP1 expression indicated poorer overall survival [22]. These results indicated that SCAMP1 might be a potential biomarker in clinical application.
Our in vitro experiments showed that SCAMP1 knockdown suppressed cell proliferation, induced cell cycle G0/G1 phase arrest and promoted apoptosis in two HCC cell lines (HepG2 and SNU-182), which suggested that SCAMP1 can be used as an oncogene to promote HCC cell proliferation. In fact, SCAMP1 also plays oncogenic role in other tumors. For instance, inhibition of SCAMP1 signi cantly restrained the cell proliferation, migration and invasion, as well as promoted apoptosis in glioma [22]. SCAMP1 depletion attenuated cell viability and promoted apoptosis under H 2 O 2 treatment in pediatric renal cell carcinoma [23]. Downregulation of SCAMP1 suppressed the pancreatic and gallbladder cancer proliferation, migration, and invasion [25].
In terms of molecular mechanism, our data further demonstrated that SCAMP1 knockdown reduced the expression of CDK4, Cyclin D1 and Bcl-2, but promoted the expression of p21, p53 and Bax in both HepG2 and SNU-182 cells. Three protein families, including cyclin-dependent kinases (CDKs), Cyclins and CDK inhibitors (CDKIs) regulate the progress of the cell cycle in cancer cells, of which CDK4/Cyclin D1 and p21 are the key regulators of G1/S transition [26,27]. Here, SCAMP1 knockdown induced cell cycle G0/G1 phase arrest, which might be correlated with decreased CDK4/Cyclin D1 and increased p21 expression induced by depletion of SCAMP1. P53, the tumor suppressor gene product, can directly regulate the p21 gene, which encodes a universal inhibitor of CDKs, to inhibit the cell cycle progression [28]. In addition to p21, Bcl-2 and Bax are the downstream target genes of p53. As our best knowledge, the tumor suppressor protein p53 has a critical role in regulation of the Bcl-2 family, including antiapoptotic protein Bcl-2 and proapoptotic protein Bax [29,30]. Our ndings revealed that SCAMP1 knockdown induced apoptosis via downregulating Bcl-2 and upregulating Bax expression.
In conclusion, our work demonstrated for the rst time that SCAMP1 was overexpressed in HCC tissues and predicted poor prognosis. Moreover, SCAMP1 positively regulated HCC cell growth and proliferation via activation of p53 signaling pathway. Our data provide new insight into the oncogenic roles of SCAMP1 in HCC progression, which may become a potential therapeutic target for HCC.

Declarations
Ethical approval and consent to participate

Availability of data and materials
All data generated or analyzed during this study are included in this published article.

Funding
Not applicable.

Con ict of interest
The authors declare that they have no con ict of interest. High expression of SCAMP1 was associated with the patient prognosis. Kaplan-Meier analysis of the correlation between SCAMP1 expression and overall survival in 76 patients with HCC. Log-rank tests were used to determine statistical signi cance. Data are the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, compared with si-NC