YY1-modulated long non-coding RNA SNHG12 promotes gastric cancer metastasis by activating the miR-218-5p/YWHAZ axis

Abstract

Long non-coding RNA (lncRNA) small nucleolar RNA host gene 12 (SNHG12) plays important roles in the pathogenesis and progression of cancers. However, the role of SNHG12 in the metastasis of gastric cancer (GC) has not yet been thoroughly investigated. In the present study, we demonstrated that SNHG12 was upregulated in GC tissues and cell lines. In addition, the expression level of SNHG12 in GC samples was significantly related to tumor invasion depth, TNM stage and lymph node metastasis and was associated with disease-free survival (DFS) and overall survival (OS) in GC patients. In vivo and in vitro assays indicated that SNHG12 promotes GC metastasis and epithelial-mesenchymal transition (EMT). Bioinformatics and mechanistic analyses revealed that SNHG12 can directly target miR-218-5p to regulate YWHAZ mRNA, forming an SNHG12/miR-218-5p/YWHAZ axis and decreasing the ubiquitination of β-catenin. In addition, SNHG12 stabilizes CTNNB1 mRNA by binding with HuR, thus activating the β-catenin signaling pathway. Further analysis also revealed that the transcription factor YY1 negatively modulates SNHG12 transcription. In conclusion, SNHG12 is a potential prognostic marker and therapeutic target for GC. Negatively modulated by YY1, SNHG12 promotes GC metastasis and EMT by regulating the miR-218-5p/YWHAZ axis and stabilizing CTNNB1 via activation of the β-catenin signaling pathway.

Keywords: SNHG12; YWHAZ; gastric cancer; metastasis; miR-218-5p.

Figure 1

SNHG12 is overexpressed in GC tissues and cell lines and indicates poor prognosis in GC. (A) SNHG12 expression in 54 pairs GC tissues and corresponding adjacent non-cancerous epithelial tissues. (B) qRT-PCR assays show the relative SNHG12 expression in GC cell lines normalized to GES-1. (C, D) ISH assays showing SNHG12 expression in normal tissues and tumor tissues, magnification ×200. Histogram show the ISH scores of SNHG12 in tumor tissues and adjacent non-cancerous epithelial tissues. (E, F) Kaplan-Meier analysis showing the 5-year DFS and OS of GC patients with high SNHG12 expression or low SNHG12 expression. Scale bar, 20 µm. Significant results were presented as **P<0.01, ***P<0.001.

Figure 2

SNHG12 promotes GC migration and EMT in vitro and vivo. (A) The efficiencies of SNHG12 knockdown in MGC-803 and AGS cells detected by qRT-PCR. (B, C) Transwell assays showing the effects of SNHG12 knockdown on GC cell migration. (D) The efficiencies of SNHG12 overexpression in MGC-803 and AGS cells detected by qRT-PCR. (E, F) Transwell assays showing the effects of SNHG12 overexpression on GC cell migration. Magnification ×200, Scale bar 20 µm. Significant results were presented as *P<0.05, **P<0.01, ***P<0.001. (G) Morphological change of the cells with stable SNHG12 knockdown (sh-SNHG12) compared with mock control cells (NC). Magnification ×200, Scale bar 100 µm. (H, I) WB assays exhibit change of EMT markers among stable SNHG12 knockdown cells (sh-SNHG12-1 and (sh-SNHG12-2), stable SNHG12 overexpressed cells (OE-SNHG12) and mock control cells (NC). (J) Body weight of the animal subjects were recorded every 3 days for 1 month. (K, L) Obvious metastatic formations in the control group (NC) as compared with the SNHG12 knockdown group (sh-SNHG12). (M) ISH and IHC assays showing SNHG12, N-cadherin, and E-cadherin intensity between NC group samples and sh-SNHG12 group samples. Magnification ×200 and ×400, Scale bar 20 µm. Histogram show the ISH scores of SNHG12 and IHC scores of N-cadherin and E-cadherin in NC group samples and sh-SNHG12 group samples.

Figure 3

SNHG12 acts as a ceRNA for miR-218-5p to regulate YWHAZ expression in GC cells. (A, B) FISH assays and cytoplasmic and nuclear RNA purification assays indicate that SNHG12 is located in GC cell cytoplasm. (C, D, E) qRT-PCR assays showing relative expression of miR-218-5p, SNHG12 and YWHAZ in GC cells transfected with miR-218-5p mimics or inhibitors. (F, G) Luciferase assays revealed the interaction between miR-218-5p and SNHG12. (H, I) Luciferase assays revealed the interaction between miR-218-5p and YWHAZ. (J, K, L, M, N, O) qRT-PCR assays indicated the relative expression of SNHG12 and YWHAZ in GC cells transfected with miR-218-5p mimics or pCDH-CMV-SNHG12 and sh-SNHG12 or si-YWHAZ or miR-218-5p inhibitors. (P, Q, R) RIP assays indicated the binding of SNHG12, miR-218-5p, or YWHAZ with Ago2. Significant results were presented as *P<0.05, **P<0.01, ***P<0.001. Magnification ×200, Scale bar 50 µm. Magnification ×400, Scale bar 20 µm.

Figure 4

SNHG12 increases the expression of β-catenin via YWHAZ stabilizing β-catenin and stabilizing CTNNB1 mRNA. (A, B) The RNA and protein expressions of β-catenin after YWHAZ knockdown were verified by qRT-PCR and WB. (C, D) The RNA and protein expressions of β-catenin after SNHG12 knockdown were verified by qRT-PCR and WB. (E) Co-IP assays showing the interaction between YWHAZ and β-catenin. (F, G) Ubiquitination changes after knockdown YWHAZ and SNHG12 tested by IP in GC cells. (H) RIP assays showing SNHG12 binding with HuR. (I) Interaction probabilities between HuR and CTNNB1 predicted by RPIseq online tool. (J) RIP assays showing CTNNB1 binding with HuR. (K, L) The stability of CTNNB1 after knockdown SNHG12 or HuR. (M, N) The nuclear expression of β-catenin after SNHG12 knockdown and overexpression was tested by western blotting. (O) Luciferase assays showing the effects on TOP/FOP reporter activity in MGC-803 and AGS cells with SNHG12 overexpression. Significant results were presented as *P<0.05, **P<0.01, ***P<0.001. No significantly differences were presented as ns.

Figure 5

SNHG12/miR-218-5p/YWHAZ axis positively regulates GC cell metastatic potential via β-catenin pathway. (A, B) Transwell assays transfected with pCDH-SNHG12 or miR-218-5p mimics or both in MGC-803 and AGS. (C) WB assays showing the expression of EMT-related proteins, YWHAZ encoded protein and β-catenin transfected with pCDH-SNHG12 or miR-218-5p mimics or both in MGC-803 and AGS. (D, E) Transwell assays transfected with sh-SNHG12 or miR-218-5p inhibitors or both in MGC-803 and AGS. (F) WB assays showing the expression of EMT-related proteins, YWHAZ encoded protein and β-catenin transfected with sh-SNHG12 or miR-218-5p inhibitors or both in MGC-803 and AGS. (G, H) Transwell assays transfected with si-YWHAZ or miR-218-5p inhibitors or both in MGC-803 and AGS. (I) WB assays showing the expression of EMT-related proteins, YWHAZ encoded protein and β-catenin transfected with si-YWHAZ or miR-218-5p inhibitors or both in MGC-803 and AGS. Scale bar, 20 µm. No significantly differences were presented as ns.

Figure 6

Transcription factor YY1 regulates the expression of SNHG12. (A, B, C) Bioinformatics analysis and CHIP assay showing YY1 binds to the promoter of SNHG12. (D, E) The expression of YY1 in GC tissues and cell lines. (F, G) Transwell assays showing the effects of the regulation of YY1 on GC metastasis. (H) qRT-PCR assays showing the expression of YY1, SNHG12, YWHAZ, miR-218-5p after YY1 knockdown. (I) Schematic illustration of the mechanism underlying SNHG12 regulation of GC metastasis and EMT. Scale bar, 20 µm. Significant results were presented as *P<0.05, **P<0.01, ***P<0.001.