Overexpression of SNHG12 is associated with advanced staging of GC and indicates poor prognosis in GC patients.
Previous studies have shown that SNHG12 plays a critical role in GC progression, however, the role of SNHG12 in regulating GC metastasis is yet unclear. To address this issue, we first determined the expression of SNHG12 in human GC samples and GC cell lines by qRT-PCR assays. As shown in Fig. 1a, SNHG12 was markedly highly expressed in tumor tissues than in the matched normal gastric epithelial tissues, suggesting that SNHG12 acted as an oncogene in GC. Further investigation in GC cell lines demonstrated that compared with the GES-1 normal gastric epithelial cell line, SNHG12 was significantly highly expressed in HS-746T, MGC-803, SGC-7901, AGS, and HGC-27 but poorly expressed in MKN-45 (Fig. 1b). Based on their SNHG12 expression pattern, MGC-803 and AGS were selected for further experiments. Based on their SNHG12 expression pattern, MGC-803 and AGS were selected for vitro experiments. FISH assays showed that the fluorescence intensity of SNHG12 in GC tissues was much higher than that in matched normal gastric epithelial tissues (Fig. 1c).
Since peritoneal metastasis still resides as one of the main forms of disease progression, we additionally investigated the expression of SNHG12 in peritoneal metastases obtained from our sample pool by FISH, as shown in Fig. 1c and the fluorescence intensity of SNHG12 in GC peritoneal metastasis tissues and GC tissues was much higher than that in primary tumor and matched normal gastric epithelial tissues, suggesting a possible underlying role of SNHG12 in the progression of peritoneal carcinomatosis from GC.
In order to verify the clinical significance of SNHG12, we analyzed the correlation between the expression levels of SNHG12 and the clinicopathological characteristics of the 54 GC patients. The results showed that higher SNHG12 expression was significantly related to depth of tumor invasion, extent of lymph node metastasis and the TNM staging of GC patients (Table 1). Moreover, Kaplan-Meier analysis indicated that patients with high SNHG12 expression had poorer survival outcome, and the respective 5-year disease free survival (DFS) of the high and low SHNG12 expression groups was 72.2% vs. 94.4% respectively (p = 0.0112) (Fig. 1d) while the corresponding 5-year overall survival (OS) was 72.2% and 94.4% respectively (p = 0.0266) (Fig. 1e). Further analysis of the nature of disease progression revealed that most of the patients with poor survival showed disease progression involving the peritoneum.
SNHG12 promotes GC cell migration and EMT in vitro.
To verify the role of SNHG12 in GC metastasis, loss-and-gain assays were conducted in GC cells by shRNA and pCDH-CMV-Human vector. The knockdown and overexpression efficiencies of SNHG12 in GC cells were validated by RT-qPCR (Additional file3 figure S1). Since sh-SNHG12-2 manifested the optimal knockdown efficiency, it was used for the subsequent investigations. Transwell assays indicated that knockdown of SNHG12 significantly suppressed GC cell migration (Fig. 2a). Conversely, overexpression of SNHG12 promoted GC cell migration (Fig. 2b).
Epithelial-Mesenchymal Transition (EMT), a process in which epithelial cells transdifferentiated into motile mesenchymal cells, endows cancer cells with high invasion and mobility. We therefore investigated the effects of SNHG12 on EMT of GC cells. As shown in Fig. 2c, knockdown of SNHG12 induced morphological changes in GC cells, from a spindle-shaped mesenchymal appearance to a cobble-like and spherical epithelial phenotype. Western blotting (WB) results showed that knockdown of SNHG12 decreased the expression of the mesenchymal markers N-cadherin, vimentin and elevated the epithelial marker E-cadherin expression, while overexpression of SNHG12 demonstrated the opposite effects (Figs. 2d,e).
SNHG12 promotes GC metastasis in vivo.
To further investigate the metastatic potential of SNHG12 in vivo, a peritoneal metastasis mice model was constructed. Significant differences were noted in the body weight of the subjects: After 9 days, it was noted that the control mice (NC) weighed more than the sh-SNHG12 mice, most probably due to possible mass formation (Fig. 3a). After 1 month, the mice were sacrificed and the anatomical dissection findings were as follows: in the NC mice, there was notable inflammatory adhesion in the mesentery with nodular formations in the mesentery and intestinal surface (around 6 to 8 masses in each mouse) while in the sh-SNHG12 mice, the abdominal cavity was clear with rare nodular findings (3 masses obtained from 3 of the subjects) (Figs. 3b,c). FISH hybridization of the masses obtained from all the subjects were further investigated and the SNHG12 signals were higher in the NC group (Fig. 3d).
SNHG12 acts as a competitive endogenous RNA for miR-218-5p to regulate YWHAZ expression in GC cells.
FISH assays showed that SNHG12 is mainly located in cytoplasm (Fig. 4a). Further cytoplasmic and nuclear RNA purification assays further confirmed that the majority of SNHG12 transcripts were detected in cytoplasm instead of nucleus (Fig. 4b). This result suggested that SNHG12 mainly exerted its function at the post-transcriptional level and may sponge miRNAs to regulate downstream molecules. Bioinformatics databases (Starbase and miRcode) indicated that miR-218-5p exhibited the complementary binding sites with the 3’-UTR of SNHG12, suggesting the direct sponging of miR-218-5p by SNHG12 (Additional file3 figure S2). Moreover, YWHAZ was chosen as the putative downstream genes of SNHG12 and miR-218-5p by using the databases TargetScan Human 7.2 and Starbase (Additional file3 figure S3). The regulation of miR-218-5p was implemented in the MGC-803 and AGS cell lines by using miR-218-5p mimics for overexpression and inhibitors for suppression assays. Compared with miR-NC groups, the expression of SNHG12 and YWHAZ was suppressed in miR-218-5p mimics group while overexpressed in inhibitors group (Fig. 4c). We further investigated whether miR-218-5p could directly bind to the 3’-UTR of SNHG12 and YWHAZ and dual-luciferase reporter assays indicated a significant reduction in luciferase activities after the co-transfection of miR-218-5p-mimics and a wild-type SNHG12 reporter vector or a wild-type YWHAZ reporter vector, but this reduction was not observed upon transfection with mutant 3’-UTR of SNHG12 reporter vector or mutant 3’-UTR of YWHAZ reporter vector (Figs. 4d, e, f and g). To further elucidate the relationship between SNHG12, miR-218-5p and YWHAZ, pCDH-CMV-SNHG12 or miR-218-5p mimics were transfected into MGC-803 and AGS cells, qRT-PCR assays indicated the expression of SNHG12 and YWHAZ was significantly increased or decreased respectively. On the other hand, when MGC-803 and AGS cells were co-transfected with pCDH-CMV-SNHG12 and miR-218-5p mimics, both of the above effects could be inverted (Fig. 4 h). In parallel, sh-SNHG12-2 or si-YWHAZ or miR-218-5p inhibitors were transfected into MGC-803 and AGS cells, the relative expression of SNHG12 and YWHAZ was decreased or increased respectively. Sh-SNHG12-2 or si-YWHAZ and miR-218-5p inhibitors were co-transfected into cells, both of the above trends could be inverted (Figs. 4i, j). RIP assays on Ago2, a component of the RNA-induced silencing complex (RISC), were conducted and the results revealed that SNHG12, miR-218-5p and YWHAZ could bind to Ago2, knockdown SNHG12 in MGC-803 and AGS cells led to the increase enrichment of YWHA (Fig. 4k). These results suggested that SNHG12 may compete with YWHAZ for miR-218-5p containing Ago2-based RISC.
SNHG12 activates β-catenin via YWHAZ encoded protein stabilizing β-catenin and reducing its ubiquitination degradation.
Wnt/β-catenin signaling pathway is well-established in cancer cell invasiveness and EMT. YWHAZ encoded protein, 14-3-3ζ, can interact β-catenin to increase its expression via decreasing its ubiquitination. According to previous studies, we determined the focused on the effects of SNHG12 on regulating β-catenin signaling activity via YWHAZ encoding protein, 14-3-3ζ. As shown in Figs. 5a, b, YWHAZ knockdown led to a decrease of β-catenin at protein level, while no obvious change at RNA level. In parallel, SNHG12 knockdown resulted in the decrease of β-catenin at both of RNA and protein levels (Figs. 5c, d). The nuclear expression of β-catenin dramatically decreased when SNHG12 knockdown, while, its nuclear expression increased when SNHG12 overexpressed (Fig. 5e). Moreover, Co-IP assays validated the interaction of YWHAZ protein and β-catenin in GC cells (Fig. 5f), and IP assays proved the ubiquitination level increased in YWHAZ or SNHG12 knockdown group, compared with mock control group (Figs. 5 g, h). Furthermore, TOPFLASH and FOPFLASH reporters were constructed to verify whether SNHG12 expression modulated the activation of β-catenin pathway, and as expected, the overexpression of SNHG12 in MGC-803 and AGS cells resulted in the remarkable increase in TOP/FOP reporter activity (Fig. 5i), suggesting activation of β-catenin-dependent transcription.
SNHG12/miR-218-5p/YWHAZ axis positively regulates GC cell metastatic potential via β-catenin pathway
To further understand the involvement of miR-218-5p/YWHAZ/β-catenin pathway in regulating metastatic potential of GC cells induced by SNHG12, transwell assays were performed in MGC-803 and AGS cells transfected with pCDH-CMV-SNHG12 or/and miR-218-5p mimics, respectively. Figures 6a, b showed that migrated cell count was significantly increased or decreased in MGC-803 and AGS cells transfected with pCDH-CMV-SNHG12 or miR-218-5p mimics only, however, when GC cells were co-transfected with pCDH-CMV-SNHG12 and miR-218-5p mimics, the increased migrated cells induced by SNHG12 overexpression could be inverted by overexpressing miR-218-5p and had no significant difference from control groups. In addition, we observed the abnormal expression of EMT-related proteins, β-catenin and YWHAZ encoded protein induced by SNHG12 overexpression was reversed after introduction of miR-218-5p mimics (Fig. 6c). Similarly, transwell assays showed that migrated cell count was significantly decreased or increased in MGC-803 and AGS cells transfected with sh-SNHG12/si-YWHAZ or miR-218-5p inhibitors only. On the other hand, when GC cells were co-transfected with sh-SNHG12/si-YWHAZ and miR-218-5p inhibitors, the decreased migrated cells induced by SNHG12 knockdown could be inverted by inhibiting miR-218-5p and had no significant difference from control groups (Figs. 6d,e,g,h). Therefore, abnormal expression of EMT-related proteins, β-catenin and YWHAZ encoded protein induced by SNHG12 knockdown was reversed after introduction of miR-218-5p inhibitors (Fig. 6f).
Transcription factor YY1 modulates SNHG12 expression
To further elucidate the mechanism underlying SNHG12 overexpression in GC, we investigated the involvement of transcription factors in regulating the transcription of SNHG12. JASPAR and PROMO databases were used to analyze the potential TFs that may bind to the region of SNHG12 promotor, and the transcription factor YYI showed similar affinity to the binding site on the promotor of SNHG12. Ch-IP assays results showed that site1 (+ 232 to + 237) and site2 (+ 1357 to + 1362) regions in the SNHG12 promotor might mediate YY1 binding to the endogenous SNHG12 promotor (Fig. 7a,b). Initially, RT-qPCR was performed to determine YY1 expression in GC cell lines and tissues. As shown in Fig. 7c, compared with GES-1, YY1 was relatively poorly expressed in most of GC cell lines (MGC-803, AGS, MKN-28, HS-746T, and SGC-7901) except for MKN-45 and HGC-27. Moreover, YY1 expression was significantly lower in GC tissue samples than that in paired normal gastric epithelial tissues (Fig. 7d). Furthermore, the results from transwell assays indicated that YY1-silencing led to significant decrease in the migration of MGC-803 and AGS cells (Fig. 7e). Nevertheless, upon treatment with si-YY1, the expressions of SNHG12 and YWHAZ increased while that of miR-218-5p decreased (Fig. 7f), which was verified by RT-qPCR. Thus, these results suggested that low expressed YY1 in GC promotes the transcription of SNHG12.