Long non-coding RNA SNHG15 inhibits P15 and KLF2 expression to promote pancreatic cancer proliferation through EZH2-mediated H3K27me3

Abstract

Long non-coding RNA (lncRNA) is emerging as an critical regulator in multiple cancers, including pancreatic cancer (PC). Recently, lncRNA SNHG15 was found to be up-regulated in gastric cancer and hepatocellular carcinoma, exerting oncogenic effects. Nevertheless, the biological function and regulatory mechanism of SNHG15 remain unclear in pancreatic cancer (PC). In this study, we reported that SNHG15 expression was also upregulated in PC tissues, and its overexpression was remarkably associated with tumor size, tumor node metastasis (TNM) stage and lymph node metastasis in patients with PC. SNHG15 knockdown inhibited proliferative capacities and suppressed apoptotic rate of PC cells in vitro, and impaired in-vivo tumorigenicity. Additionally, RNA immunoprecipitation (RIP) assays showed that SNHG15 epigenetically repressed the P15 and Kruppel-like factor 2 (KLF2) expression via binding to enhancer of zeste homolog 2 (EZH2), and chromatin immunoprecipitation assays (CHIP) assays demonstrated that EZH2 was capable of binding to promoter regions of P15 and KLF2 to induce histone H3 lysine 27 trimethylation (H3K27me3). Furthermore, rescue experiments indicated that SNHG15 oncogenic function partially involved P15 and KLF2 repression. Consistently, an inverse correlation between the expression of SNHG15 and traget genes were found in PC tissues. Our results reported that SNHG15 could act as an oncogene in PC, revealing its potential value as a biomarker for early detection and individualized therapy.

Keywords: P15 and KLF2; SNHG15; long noncoding RNA; pancreatic cancer; proliferation.

Figure 1. SNHG15 expression is upregulated in PC tissues and its clinical significance

(A) Relative expression of SNHG15 in human PC tissues (n=48) compared with corresponding adjacent normal tissues (n=48). SNHG15 expression was examined by qPCR and normalized to GAPDH expression (shown as ΔCT). (B) The patients were classified into two groups according to SNHG15 expression. (C-E) The results are presented as relative expression levels in tumor tissues. SNHG15 expression was significantly higher in patients with a larger tumor size, a higher pathological stage, and lymph node metastasis (shown as ΔCT). Bars: s.d, ^*P<0.05, ^**P<0.01.

Figure 2. SNHG15 knockdown inhibits PC cell proliferation in vitro

(A) SNHG15 expression levels of PC cell lines (AsPC-1, BxPC-3 and PANC-1), compared with that in human pancreatic ductal epithelial cells (HPDE6). (B) qRT-PCR analysis of SNHG15 expression in AsPC-1 and BxPC-3 cell lines transfected with SNHG15 siRNAs or the negative control. (C) MTT assays were performed to detect the viability of si-SNHG15 transfected AsPC-1 and BxPC-3 cells. (D) Colony-forming growth assays were performed to determine the proliferation of PC cells. The colonies were counted and captured. (E) Proliferating AsPC-1 and BxPC-3 cells were labeled with Edu. The Click-it reaction revealed Edu staining (red). Cell nuclei were stained with DAPI (blue). The images are representative of the results obtained. ^*P<0.05 and ^**P<0.01.

Figure 3. Knockdown of SNHG15 promotes cell cycle arrest and induces apoptosis in PC cells in vitro

(A) Flow cytometry assays were performed to analysis the cell cycle progression when PC cells transfected with si-SNHG15. The bar chart represented the percentage of cells in G0/G1, S, or G2/M phase, as indicated. All experiments were performed in biological triplicates with three technical replicates. (B) Flow cytometry was used to detect the apoptotic rates of cells. LR, early apoptotic cells; UR, terminal apoptotic cells. (C) Apoptosis in AsPC-1 and BxPC-3 cells after SNHG15 knockdown was detected through TUNEL staining. (D) Western blot analysis of CDK2, CDK4 and cleaved caspase-3 and cleaved caspase-9 after si-NC, si-SNHG15 2#, or si-SNHG15 3# transfection in AsPC-1 and BxPC-3 cells. GAPDH protein was used as an internal control.

Figure 4. Knockdown of SNHG15 inhibits PC cell tumorigenesis in vivo

(A) Empty vector or sh-SNHG15 were transfected into BxPC-3 cells, which were injected in the nude mice (n = 7), respectively. Tumors formed in sh-SNHG15 group were dramatically smaller than the control group. (B) qRT-PCR was performed to detect the average expression of SNHG15 in xenograft tumors (n = 7). (C) Tumor volumes were calculated after injection every four days. Points, mean (n = 7); bars indicate SD. (D) Tumor weights were represented as means of tumor weights±SD. (E) The tumor sections were under H&E staining and IHC staining using antibodies against ki-67. Error bars indicate mean ± standard errors of the mean. ^*P < 0.05, ^**P < 0.01.

Figure 5. SNHG15 epigenetically silences P15 and KLF2 transcription by binding to EZH2

(A) qRT-PCR analysis of SNHG15 nuclear and cytoplasmic expression levels in AsPC-1 and BxPC-3 cells. U6 was used as a nucleus marker, and GAPDH was used as a cytosol marker. (B) RIP experiments were performed in AsPC-1 and BxPC-3 cells, and the coprecipitated RNA was subjected to qRT-PCR for SNHG15. The fold enrichment of SNHG15 in EZH2/SUZ12 RIP is relative to its matched IgG control. (C) The levels of p15, p16, p21, p27, p57, KLF2 and PTEN mRNA were determined by qRT–PCR when knockdown of SNHG15. (D) The p15 and KLF2 protein levels were determined by western blot in SNHG15 knockdown in AsPC-1 and BxPC-3 cells. (E) The p15 and KLF2 expression levels were determined by qRT-PCR in AsPC-1 and BxPC-3 cells transfected with si-EZH2 1# or 2#. (F) ChIP-qRT-PCR of EZH2 occupancy and H3K27me3 binding in the p15 and KLF2 promoters in AsPC-1 and BxPC-3 cells treated with si-SNHG15 3# (48 h) or si-NC; IgG as a negative control. Error bars indicate mean ± standard errors of the mean. ^*P < 0.05, ^**P < 0.01.

Figure 6. Effect of P15 and KLF2 of overexpression on BxPC-3 cell in vitro

(A, B) The mRNA levels and protein levels of P15 and KLF2 in BxPC-3 cells transfected with pCDNA-P15 or pCDNA-KLF2 was detected by qPCR analysis. (C, D) MTT assays and Edu staining assays were used to determine the cell viability. Values represent the mean ± s.d. from three independent experiments. (E) Cell cycle was analyzed by flow cytometry. The bar chart represents the percentage of cells in G1–G0, S, or G2–M phase, as indicated. ^*P < 0.05 and ^**P < 0.01.

Figure 7. SNHG15 negatively regulates expression of P15 and KLF2 by rescue assays

(A, B) MTT and colony formation assays were used to determine the cell proliferation ability for BxPC-3 cells transfected with pCDNA-SNHG15 and pCDNA-P15 and pCDNA-KLF2 and co-transfected with pCDNA-SNHG15 and pCDNA-P15 or pCDNA-SNHG15 and pCDNA-KLF2. (C) qPCR analyzed the P15 and KLF2 mRNA levels in 40 pairs PC tissues and found that there was a significantly negative correlation between SNHG15 and P15 or KLF2. Values represent the mean±s.d. from three independent experiments.