Upregulation of SNTB1 Correlates with Poor Prognosis and Promotes Cell Growth in Colorectal Cancer

Background: Colorectal cancer (CRC) is one of the most highly malignant tumors and has a complicated pathogenesis. A preliminary study identied syntrophin beta 1 (SNTB1) as a potential oncogene in CRC. However, the clinical signicance, biological function, and underlying mechanisms of SNTB1 in CRC are unknown. Thus, the present study aimed to investigate the function of SNTB1 in CRC. Methods: The expression prole of SNTB1 in CRC samples was evaluated by database analysis, cDNA array, tissue microarray, Quantitative real-time PCR (qPCR), and immunohistochemistry. SNTB1 expression in human CRC cells was silenced using short hairpin RNAs and its mRNA and protein levels were assessed by qPCR and western blotting, respectively. Cell proliferation, colony formation, cell cycle and apoptosis were determined by the cell counting, colony formation, and ow cytometry assays, respectively. A xenograft nude mouse model of CRC was established for validating the roles of SNTB1 in vivo. Immunohistochemistry was used to score the expression of SNTB1 in tissue samples. The isobaric tags for relative and absolute quantication (iTRAQ) was used to analyze the differentially expressed proteins after knockdown of SNTB1 in CRC cells. Results: SNTB1 expression was increased in CRC tissue compared with adjacent noncancerous tissues and the increased expression was associated with shorter overall survival of CRC patients. Silencing of SNTB1 suppressed cell viability and survival, induced cell cycle arrest and apoptosis in vitro, and inhibited the growth of CRC cells in vivo. Further elucidation of the regulation of STNB on CRC growth by iTRAQ analysis identied 210 up-regulated and 55 down-regulated proteins in CRC cells after SNTB knockdown. A PPI network analysis identied protein kinase N2 (PKN2) as a hub protein and was upregulated in CRC cells after SNTB1 knockdown. Western-blot analysis further conrmed that SNTB1 knockdown signicantly up-regulated

In preliminary published work, using a gene expression pro le microarray to screen for differential expressed genes (DEGs) between paired CRC and noncancerous tissue, we found the level of syntrophin beta 1 (SNTB1) to be upregulated in CRC tissue (GEO ID: GSE113513). However, the clinical signi cance, biological function, and underlying regulating mechanisms of SNTB1 remain largely unknown in most of cancers, including CRC, which encouraged us to further explore the role of SNTB1 in CRC.
SNTB1 is a member of syntrophin gene family which consists of ve homologous isoforms, α1, β1, β2, γ1 and γ2 [5][6][7]. Two pleckstrin homology (PH) domains, a PDZ domain, and a conserved syntrophin unique (SU) region constitute the structure of SNTB1 and are sites of interaction with other proteins [8]. SNTB1 is associated with dystrophin and dystrophin-related proteins, which is one of the members of syntrophin gene family and mainly expressed in skeletal and smooth muscle, liver and kidney and expressed at low levels in many other tissues, including colorectal tissue [5]. Loss and reduction of SNTB1 is closely associated with Duchenne muscular dystrophy and Becker's muscular dystrophy [6]. The functional variants of SNTB1 can cause other abnormalities such as acute pancreatitis, oral cancer and severe myopia [7,9,10]. Whether SNTB1 has a pathological role in CRC is unknown.
In the present study, bioinformatics analysis of online databases, cDNA array, and tissue microarray (TMA) were used to evaluate the expression pro le of SNTB1 and the relationship between SNTB1 expression and clinical pathological parameters in CRC. Moreover, a series of in vitro and in vivo experiments using representative CRC cell lines and xenograft nude mice models were conducted to examine the functional role of SNTB1.Furthermore, the underlying mechanisms of SNTB1 on tumor growth were explored.

Differential expressed genes analysis
In prior work, we screened DEGs on 14 pairs of CRC primary lesions and surrounding non-cancerous tissues (GEO Submission: GSE113513) [11]. Among these DEGs, 8 of DEGs including SNTB1 were selected for further investigation due to the limited amount of published data regarding their involvement in carcinogenesis. In this study, mRNA expression of SNTB1in CRC and control tissues was analyzed thorough TCGA (https://cancergenome.nih.gov/) [12].

Cell lines and culture
Both human CRC cell lines HCT116 and RKO were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). HCT116 cells were cultured in M5′A medium (KeyGEN, Jiangsu, China) and RKO cells were cultured in MEM-alpha medium (Thermo Fisher Scienti c) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scienti c), 1% penicillin-streptomycin (Hyclone, Logan, UT, USA).
All cells were cultured at 37°C in a humidi ed atmosphere with 5% CO 2 . Short tandem repeat genotyping was performed for examination of mycoplasma contamination of cells using qPCR. Lentiviral transduction and high-content screening for cell growth High-content screening (HCS) was performed to assess the growth of CRC cells. Brie y, control shRNA lentivirus and shRNA lentivirus targeting 8 DEGs (the sequences of shRNAs are listed in Supplement  Table S1) were constructed by Shanghai GeneChem (Shanghai, China). HCT116 cells were seeded in 12well plates for 16h prior to lentivirus transduction and then transduced by adding the shRNA lentiviral particles (multiplicity of infection: 10) with GFP into the cell culture medium according to the protocol of the manufacturer. At the end of transduction, 2 x 10 3 cells in 100 µL of complete medium were reseeded into 96-well plates. Cellomics Array Scan VTI HCS (Thermo Fisher Scienti c) was used to monitor the cell growth over 5 days. Image analysis was performed using HCS Studio Cell Analysis Software (Thermo Fisher Scienti c).

Quantitative real-time PCR (qPCR) analysis
The total RNA was isolated from cells using RNAiso Plus reagent (Takara, Beijing, China). Reverse transcription into complementary DNA (cDNA) was ampli ed according to the manufacturer's instructions using the PrimeScript RT reagent kit (Takara). Tissue cDNA array containing 79 primary CRC and 15 noncancerous colorectal tissues (Cat#: cDNA-hcola095su01) was purchased from Shanghai Outdo Biotech Company (Shanghai, China) and the levels of mRNA encoding SNTB1 and GAPDH were detected using an ABI 7500 Fast Real-Time PCR System (Applied Biosystems) and the SYBR Premix Ex Tag (Takara). The conditions for qPCR were as follows: pre-denaturation (95℃ for 10minutes), denaturation (95℃ for 15 seconds), annealing and extension (60℃ for 60 seconds) for a total 40 cycles.

Western-blot analysis
Cells were harvested and lysed in RIPA lysis buffer (Thermo Fisher Scienti c) containing 1 mM phenylmethylsulfonyl uoride (PMSF) and protease inhibitors. The Pierce BCA Protein Assay Kit (Thermo Fisher Scienti c) was used to measure concentrations of total protein. Equal amount of total protein lysate was separated on 10% SDS-polyacrylamide gel and transferred to PVDF membranes (Millipore, Bedford, MA, USA). Next the membranes were blocked with 5% skim milk in TBST at room temperature for 2 h and incubated overnight at 4 ℃ with primary antibodies (1:1000), and followed by incubation with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:2000). Proteins were visualized using an ECL imager (Thermo Fisher Scienti c, USA) and band intensities were quanti ed using ImageLab software. The expression of GAPDH was used as a control. Three independent experiments were performed for each assay.

CCK-8 assay
Transduced cells were re-seeded into 96-well plates (2,000 cells per well) and cultured in at 37°C and 5% CO 2 for the indicated time points. Cell Counting Kit-8 reagent (10 ul; Abbkine, Wuhan, Hubei, China) was added to each well, plates were incubated for an additional 2 h at 37°C, and the optical density (OD) was measured at a wavelength of 450 nm. The cell viability was calculated based on the OD for each group.

Colony formation assay
Transduced cells were seeded into 12-well plates at a density of 500 cells per well and cultured at 37°C and 5% CO 2 for 10-14 days. Cells were xed in 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet (Solarbio, Beijing, China) for 20 min at room temperature. Colonies were manually counted.
Each assay was performed in triplicate.

Cell cycle and apoptosis analysis
For the cell cycle assay, transduced cells were collected and xed with 70% ethanol at 4℃ overnight. The xed cells were centrifuged at 2000 rpm for 3 min and washed, followed by incubation with a mixture of FxCycle PI/RNase Staining Solution (Thermo Fisher Scienti c) for 30 min at room temperature. FACS (Fluorescence activated Cell Sorting; Becton Dickinson, CA, USA) was used to analyze cell cycle progression using Mod tLT version 3.0 (Verity Software House). For the apoptosis assay, transduced cells were washed twice with ice-cold PBS and incubated with Annexin-V-AbFlour™ 647 Apoptosis Detection Kit solution (Abbkine, Wuhan, China). The apoptotic rate was analyzed using FACS.

In vivo experiments
A xenograft nude mouse model was constructed to investigate the effects of SNTB1 knockdown on tumor growth. Male BALB/c nude mice (6-8 week old) were obtained from Shanghai Laboratory Animal Center at the Chinese Academy of Sciences and raised in a speci c pathogen-free facility at Fujian University of Traditional Chinese Medicine (Fujian, China). All animal procedures were approved by the Committee of Fujian University of Traditional Chinese Medicine (Approval No: FJTCM IACUC 2019050). HCT116 cells or RKO cells were transduced with a lentivirus encoding anti-SNTB1 shRNA (sh-SNTB1) or control shRNA (sh-Ctrl). Cells (1 × 10 6 in 100 µL PBS containing 50% Matrigel) were injected subcutaneously into the anks of nude mice (n = 6). Tumor volume was determined every other day using measurements obtained with a vernier caliper and the following formula: 1/2 (larger diameter×smaller diameter 2 ). At the end of the experiment, mice were anesthetized with iso urane. An IVIS Spectrum liveanimal imaging system (PerkinElmer; Santa Clara, CA, USA) was used to capture tumor images. Mice were sacri ced for tissue collection for further use. Signal intensity was quanti ed as the number of photons within the region of interest per second.

Immunohistochemistry
Tissues were xed with 4% paraformaldehyde at 4°C overnight, embedded with para n, cut into 5µmthick sections, and mounted onto slides. The slides were dehydrated by graded ethanol. Antigens were retrieved by using microwave heating for 20 min in sodium citrate-hydrochloric acid buffer. Tissue sections were incubated with an antibody against SNTB1 (1:100; Thermo sher Scienti c) or PCNA (1:800; Genetex). Background staining was assessed by omitting the primary antibody. The intensity of staining was evaluated using a scoring system described in detail in the Section "Tissue microarray array". The overall staining score was calculated by multiplying the intensity score and percentage score together.

TUNEL assay
Apoptotic cells in tissue sections were detected using terminal deoxynucleotidyl transferase dUTPnick end labeling (TUNEL) staining according to the manufacturer's instruction. The percentage of TUNELpositive cells and staining intensity were evaluated using a scoring system described in detail in the Section "Tissue microarray array".
Isobaric tags for relative and absolute quanti cation (iTRAQ) analysis and protein identi cation.
iTRAQ was used to identify differential expressed proteins (DEPs) [15,16]. SDS-PAGE electrophoresis was rst carried out for protein quanti cation. The protein samples were cysteine-blocked and digested, protein labelling and mass spectrometry (MS) analysis were performed. Two-dimensional liquid chromatography-mass spectrometry (2D-LC-MSMS) analysis including reversed-phase chromatographic separation (Agilent Technologies, Santa Clara, CA, USA) and reversed-phase chromatography on a TripleTOF (AB SCIEX, Framingham, MA, USA) was conducted. Proteins were classi ed as differentially expressed if their expression differed at least 1.5-fold between the two conditions and if the difference was associated with P < 0.05. These proteins were identi ed using volcano plots and hierarchical clustering plots.
To enrich the biological groups and KEGG pathway, the identi ed proteins were submitted to Omicsbean (http://www.omicsbean.cn/) software. The signi cantly enriched gene ontology (GO) categories were reported using a right-sided hypergeometric test, which compares the background set of GO annotations in the whole genome of homo sapiens. The false discovery rate (FDR) was controlled by the Bonferroni step-down test to correct the p-value [17,18].
To better understand the protein-protein interactions among the differentially expressed proteins of each group, we constructed protein-protein interaction (PPI) networks through Omicsbean. Proteins were then grouped based on their GO annotations with p-value < 0.05 [19].

Statistics analysis
Data were analyzed using SPSS 22.0 software. For survival analysis, SNTB1 mRNA expression in CRC tissues from cDNA array was classi ed into high or low expression groups based on mean. Kaplan-Meier survival curves were plotted for high-and low-expression groups and the correlation of SNTB1 mRNA expression with overall survival of CRC patients was analyzed using log-rank test. The correlation between SNTB1 mRNA expression and CRC patients' survival in dataset of COAD sourcing from TCGA was analyzed through Kaplan-Meier Plotter (http://kmplot.com/). Student's t-test or Mann-Whitney U was used for comparisons between two groups. One-way ANOVA or Kruskal-Walis H was applied to assess multiple group comparisons. All quantitative data are presented as the mean ± SD. P < 0.05 (two sided) was considered statistically signi cant. All experiments were repeated at least three times.

Results
Identi cation of SNTB1 as a potential target in CRC In a previous experiment to identify potential oncogenes, we evaluated for differential expressed genes (DEGs) within a CRC microarray containing 14 pairs of CRC tissue and its noncancerous surrounding tissue (GEO ID: GSE113513). Among the identi ed DEGs, we focused on 8 up-regulated genes (SNTB1, PLEKHG4, JPH1, CTPS1, LRRC6, LY6G6F, PLCB4 and LRP8; Fig. 1a) that had not been extensively studied in CRC. The potential involvement of these genes in CRC were further explored through high-content screening (HCS) using lentivirus delivered shRNAs into HCT116 cells. Seven of the 8 constructs inhibited cell growth (Fig. 1b, c). Considering the unexplored role of SNTB1in oncogenesis [9,11,20,21], we focused on SNTB1 in these studies. SNTB1 is highly expressed in CRC tissues and is associated with poor prognosis QPCR analysis on a cDNA array of 79 primary CRC and 15 noncancerous colorectal tissues indicated that SNTB1 mRNA expression is up-regulated in CRC tissues (Fig. 2a). Similar results were obtained with online data mining using the GEO (GEO ID: GSE113513) and TCGA (Fig. 2b,c) datasets. In a consistent fashion, immunohistochemical (IHC) evaluation of a tissue microarray (TMA) containing 70 pairs of primary CRC lesions and adjacent noncancerous tissues (Fig. 2d, e) con rmed the up-regulation of SNTB1 protein expression in CRC tissues. Correlation analysis of the CRC patients represented in the utilized cNDA array, showed that higher SNTB1 expression (as determined with QPCR-based cDNA array) correlated with shorter overall survival rate (Fig. 2f). There was no correlation between SNTB1 expression and clinicopathological characteristics of CRC patients (Supplementary Table 5). Online data mining using the dataset of COAD from TCGA also revealed an association between higher SNTB1 expression and shorter overall survival of CRC patients (Fig. 2g).

SNTB1 knockdown inhibits cell proliferation and induces cell apoptosis of CRC cells in vitro
To explore the biological function of SNTB1 on CRC cell growth, three different shRNAs speci c for SNTB1 were encoded within lentiviruses and used for cell transduction. QPCR and Western-blot analysis revealed signi cant down-regulation of SNTB1expression of both mRNA ( Supplementary Fig. 1a) and protein ( Supplementary Fig. 1b, c) levels in HCT116 cells with all three shRNAs. As each of the shRNAs had similar effects on SNTB1 expression and cell growth, subsequent experiments were performed with a single construct referred to as "sh-SNTB1-1".
QPCR and Western-blot analysis con rmed signi cant down-regulation of SNTB1 mRNA and protein levels in both HCT116 and RKO cells after transduced with sh-SNTB1 (Fig. 3a-c). More importantly, SNTB1 knockdown profoundly reduced the viability and colony formation of HCT116 and RKO cells (Fig. 3d, e). Moreover, SNTB1 knockdown markedly increased percentage of cells in the G0/G1 phase, while decreased cell percentage in the S phase in HCT116and RKO cells (Fig. 4a), as well as downregulated the expression of G0/G1-related proteins CDK4 and CyclinD1 in HCT116 cells (Fig. 4b). Flow cytometry with annexin V staining and western-blot analysis demonstrated that SNTB1 knockdown remarkably increased the percentage of apoptotic cells (Fig. 4c) and the ratio of Bax/Bcl-2 expression (Fig. 4d).

SNTB1 knockdown suppresses CRC tumor growth in vivo
We further determined the effects of SNTB1 knockdown on tumor growth in vivo. By evaluating tumor volume (Fig. 5a), intratumoral green uorescent protein (GFP) uorescence (Fig. 5b, c), tumor size (Fig. 5d), and tumor weight (Fig. 5e), we found that SNTB1 knockdown signi cantly suppressed tumor growth of both HCT116 and RKO cells in vivo. IHC analysis and TUNEL assay showed that SNTB1 knockdown signi cantly decreased the expressions of SNTB1 (Fig. 6a) and proliferating cell nuclear antigen (PCNA) (Fig. 6b), and increased cell apoptosis in the HCT116 xenograft (Fig. 6c). SNTB1 knockdown up-regulates PKN2 expression and inhibit the activation of ERK and AKT signaling pathways To further elucidate the underlying mechanism of SNTB1 knockdown on tumor growth suppression in CRC, the isobaric tag for relative and absolute quantitation (iTRAQ) methodology was applied to identify differentially expressed proteins (DEPs) in HCT116 cells after SNTB1 knockdown. As shown in Fig. 7, a total of 265 DEPs were identi ed, including 210up-regulatedand 55down-regulated proteins (fold change≥1.5, P < 0.05). Hierarchical clustering (Fig. 7a) and volcano (Fig. 7b) plots were used to identify DEPs. Analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways showed these proteins to be enriched in multiple signaling pathways (Fig. 7c). A PPI network was constructed (Fig. 7d) and the top 10 hub genes were identi ed on the basis of their degree of connectivity in the PPI network: PKN2, TMX3, DYRK1A, IRAK1, SYNE2, HELZ, GSK3A, MYO5B, and NVL (Supplement Table 6).
As a top hub protein, PKN2 expression was signi cantly up-regulated in HCT116 cells after SNTB1 knockdown. Therefore, we further explored the regulatory effects of SNTB1 knockdown on PKN2 expression and its downstream targets, including ERK and AKT pathways. Consistently, SNTB1 knockdown signi cantly increased the protein pression of PKN2 (Fig. 7e) and inhibited the phosphorylation of ERK (Fig. 7f) and AKT (Fig. 7g). However, SNTB1 knockdown had no signi cant effects on the phosphorylation of c-jun and p38 MAPK in HCT116 cells (Supplement Fig. 2).

Discussion
CRC is one of the most lethal cancers worldwide [1]. Although unhealthy dietary habits, environmental changes, and genetic aberrancies are contributors to the morbidity and mortality of this disease [22], the precise molecular mechanisms of CRC initiation and progression remain elusive. Therefore, the investigation into the molecular determines of CRC has the potential to lead to novel therapeutic targets, and diagnostic and prognostic biomarkers. Herein, we for the rst time describe the potential role of SNTB1 as a oncogene in CRC tissues.
As a modular adapter protein, SNTB1 binds and localizes various transmembrane and intracellular signaling molecules to the membrane [23]. Our current study identi ed the up-regulation of SNTB1 in CRC tissues in both mRNA and protein levels, suggesting that up-regulation of SNTB1 might be a common event during the development of CRC. Moreover, survival analysis based on cDNA array of CRC samples and online public databases indicate that there is a correlation between higher SNTB1 expression and shorter survival of CRC patients. This nding highlights the potential of SNTB1 as a biomarker for disease prognosis.
Previous studies indicate that SNTB1 is involved in the initiation of autophagy in pancreatic cancer cells and has a protective effect on acute pancreatitis [7]. In addition, it has also been reported that SNTB1 is closely related to the occurrence of oral cancer and is considered to be a susceptibility locus for severe myopia [9,10]. However, the functional roles of SNTB1 in CRC are still unknown. In order to further understand the function of SNTB1, we conducted a series of experiments and found that SNTB1 knockdown signi cantly suppressed CRC cell growth in vivo and in vitro by inhibiting cell proliferation and inducing cell cycle progression and cell apoptosis. These ndings suggesting a potential oncogenic activity of endogenous SNTB1 in CRC. However, the effect of SNTB1 overexpression in CRC cell growth should be addressed in future study.
The involvement of SNTB1 in cell proliferation and apoptosis is unknown, we therefore conducted iTRAQ analysis to identify differentially expressed proteins in CRC cells after SNTB1 knockdown. iTRAQ analysis identi ed a total of 265 differentially expressed proteins (including 210 upregulated and 55 downregulated proteins) after knockdown of SNTB1. To identify the downstream targets of SNTB1, a PPI network was constructed. PPI analysis identi ed PKN2 as a hub protein in the PPI network, which was down-regulated in CRC cells after SNTB1 knockdown. Previous studies indicate that PKN2 is involved in cell proliferation by regulating ERK/MAPK or AKT signaling pathways [24,25] and inhibit M2 phenotype polarization of tumor-associated macrophages in CRC cells [26]. Since abnormal activation of MAPK and AKT pathways contribute to multiple cellular processes, including cell survival, cell differentiation, apoptosis, invasion, and in ammation [27][28][29][30], we therefore assessed the regulatory effects of SNTB1 on those multiple signaling pathways. Interestingly, SNTB1 knockdown signi cantly decreased the levels of both p-ERK and p-AKT expression, without effect on p38 MAPK and p-JNK. However, the exactly regulatory role of SNTB1 on PKN2 expression and EKR, AKT pathways activation should be addressed in future studies.

Conclusion
In summary, our current study indicates that SNTB1 expression is upregulated in CRC tissues and correlates with CRC patient survival. In addition, we demonstrate SNTB1 has an essential role in CRC proliferation and apoptosis, which is mediated, in part, through the reduction of PKN2 expression and activation of EKR and AKT pathways. Also, these studies highlight the potential of SNTB1 as a therapeutic target and biomarker for CRC.    (a)The mRNA levels of SNTB1 were determined by qPCR, GAPDH was used as an internal control. *P < 0.05 vs. sh-Ctrl lentivirus. (b and c) The protein levels of SNTB1 were determined by Western-blot analysis. The representative images of SNTB1 and GAPDH were showed (b) and were quantitated using ImageLab software (c). GAPDH was used as an internal control and normalized to GAPDH. *P < 0.05 vs.   using ImageLab software (lower panel). GAPDH was used as an internal control. *P < 0.05 vs. sh-Ctrl. All experiments were performed in triplicate.  SNTB1 knockdown inhibits cell proliferation and induces cell apoptosis in vivo. IHC was performed to detect SNTB1 (a) and PCNA (b) expression in tumor sections, and TUNEL assay (c) was used to determine the apoptotic cells in tissues of both sh-Ctrl and sh-SNTB1. The representative images of IHC analysis or TUNEL assay were taken at a magni cation of ×400 (left panel) and IHC scores were calculated (right panel). P < 0.05 vs. sh-Ctrl group).