SNHG17, as an EMT-related lncRNA, promotes the expression of c-Myc by binding to c-Jun in esophageal squamous cell carcinoma

Abstract

Dysregulation of long noncoding RNA SNHG17 is associated with the occurrence of several tumors; however, its role in esophageal squamous cell carcinoma (ESCC) remains obscure. The present study demonstrated that SNHG17 was upregulated in ESCC tissues and cell lines, induced by TGF-β1, and associated with poor survival. It is also involved in the epithelial-to-mesenchymal transition (EMT) process. The mechanism underlying SNHG17-regulated c-Myc was detected by RNA immunoprecipitation, RNA pull-down, chromatin immunoprecipitation, and luciferase reporter assays. SNHG17 was found to directly regulate c-Myc transcription by binding to c-Jun protein and recruiting the complex to specific sequences of the c-Myc promoter region, thereby increasing its expression. Moreover, SNHG17 hyperactivation induced by TGF-β1 results in PI3K/AKT pathway activation, promoting cells EMT, forming a positive feedback loop. Furthermore, SNHG17 facilitated ESCC tumor growth in vivo. Overall, this study demonstrated that the SNHG17/c-Jun/c-Myc axis aggravates ESCC progression and EMT induction by TGF-β1 and may serve as a new therapeutic target for ESCC.

Keywords: SNHG17; EMT; c-Myc; esophageal squamous cell carcinoma; metastasis.

FIGURE 1

SNHG17 is elevated in ESCC tissues and cell lines and associated with a poor prognosis. A, GEO database (GSE20347) analysis showed the expression of SNHG17 in 17 ESCC tissues and matched para‐tumorous tissues. B, Expression level of SNHG17 in 182 ESCA tumor samples and 13 normal controls from TCGA database. C, Expression of SNHG17 in ESCC cell lines and pools, as shown by qRT‐PCR. Pools: average expression in 10 normal tissues was used as the normal control. D, Expression of SNHG17 in ESCC tissues and matched para‐normal tissues was detected by qRT‐PCR. Heatmap shows the expression of SNHG17 in each pair of samples. E, Kaplan‐Meier curves of SNHG17 in ESCC patients for OS. Error bars are shown as mean ± SD. *P < .05, **P < .01, ***P < .001

FIGURE 2

SNHG17 boosts the proliferation, lessens apoptosis, and elevates migration, invasion, and EMT of ESCC cells in vitro. A, Expression of SNHG17 was detected by qRT‐PCR after transfection with shSNHG17 for 48 h in Eca109 and Kyse150 cells. B, C, MTS and colony forming assays were performed to evaluate Eca109 and Kyse150 cell proliferation after knocking down SNHG17. D, Apoptosis of ESCC cells was determined by flow cytometry apoptosis analysis under SNHG17 knockdown. E, Cell migration and invasion ability were analyzed by transwell assays (magnification: ×100). F, G, qRT‐PCR and western blot were performed to examine the expression levels of E‐cadherin and N‐cadherin in ESCC cells. Error bars are shown as mean ± SD, **P < .01

FIGURE 3

SNHG17 is upregulated in TGF‐β‐induced Eca109 cells and involved in TGF‐β1‐mediated EMT. A, Expression of SNHG17 was assessed in Eca109 treatment with TGF‐β1. B, The hallmarks of EMT were detected in TGF‐β1‐treated Eca109 cells. C, Eca109 cells were transfected with siNC and siSNHG17 for 24 h. Then, cells were transfected again and treated without or with 10 ng/mL TGF‐β at 6 h after transfection. Images were acquired at 48 h after the treatment, and representative images of spindle‐like shape cells are shown. Red square areas are magnified in the right panel. D, E, Eca109 cells were transfected with siNC and siSNHG17 for 24 h. The cells were then transfected again and treated without or with 10 ng/mL TGF‐β1 at 6 h after transfection. At 48 h after treatment, qRT‐PCR, and western blot assessed the mRNA and protein expression of EMT‐related markers, respectively. Data are presented as mean ± SD. **P < .01

FIGURE 4

SNHG17 upregulates the expression of c‐Myc and medicates the PI3K/AKT pathway in ESCC. A, Hierarchically clustered heatmap of upregulated and downregulated genes in Eca109 cells after transfection with sh‐SNHG17 or sh‐NC. B, Expression levels of c‐Myc, CLDN4, IL7R, JAK3, and AREG under SNHG17 silencing or overexpression were identified by qRT‐PCR. C, Association analysis of the correlation between c‐Myc and SNHG17 expression (r = .695, P < .01). D, Protein expression of c‐Myc, pPI3K, PI3K, p‐AKT, and AKT under SNHG17 knockdown or overexpression, as detected by western blot. E, ISH and IHC staining results showed that the expression of c‐Myc was high in the same ESCC paraffin‐embedded sample with high SNHG17 expression, and vice versa. Data are presented as mean ± SD. **P < .01

FIGURE 5

SNHG17 binds to transcription factor c‐Jun. A, Luciferase reporter assay suggested that SNHG17 regulates the transcription of c‐Myc. B, Subcellular localization of SNHG17 in Eca109 and Kyse150 cells. U6 and GAPDH were used as positive controls for nuclear RNA and cytoplasmic RNA, respectively. C, TFs for the c‐Myc promoter predicted by PROMO and UCSC databases. D, The interaction strength between SNHG17 and c‐Jun was predicted using the catRAPID algorithm (http://s.tartaglialab.com/page/catrapid_group). E, CatRAPID fragment module prediction of the interaction profile between c‐Jun protein and SNHG17. F, Whole cell lysates and nuclear fraction were incubated with in vitro transcribed RNA of sense (S) and antisense (AS) SNHG17. Validation of SNHG17 and c‐Jun interaction by RNA pull down. G, RIP assays verified the direct interaction of SNHG17 with c‐Jun. IgG antibody served as the negative control. H, Lack of the c‐Jun‐binding domain in SNHG17 involving the truncation mutants. Immunoblot analysis for c‐Jun in protein samples pulled down by full‐length SNHG17 and the SNHG17 truncated mutant (Δ101‐400). GAPDH was used as a negative RNA control. I, Immunoblot of His‐tagged c‐Jun (WT vs domain truncation mutants) retrieved by biotinylated SNHG17; the structure of c‐Jun is shown below. The data are shown as mean ± SD, **P < .01

FIGURE 6

SNHG17 recruits c‐Jun, which regulates the expression of c‐Myc by binding to its promoter region. A, qRT‐PCR for mRNA levels of c‐Jun and c‐Myc in the transfection of Eca109 and Kyse150 cells. B, Western blot assays for protein levels of c‐Jun and c‐Myc in transfected Eca109 and Kyse150 cells. C, Predicted c‐Jun binding site in the −1000 bp region of the c‐Myc promoter using JASPAR (E1: −17 to −30 bp; E2: −103 to −116 bp; and E3: −757 to 770 bp). D, ChIP‐qPCR assays were conducted to show direct binding between c‐Jun and c‐Myc promoter regions in Eca109 (the P1 region contained E1 and E2 sites, the P2 region contained the E3 site). E, Verified c‐Myc promoter sequences were added into a pGL3 vector. The activity of the c‐Myc promoter containing or lacking c‐Jun‐binding elements was detected as luciferase reporter assays. F, G, Interaction between SNHG17, c‐Jun, and c‐Myc was confirmed by luciferase reporter assays. H, ChIP‐qPCR analysis revealed that SNHG17 fortified the enrichment of c‐Jun in the c‐Myc promoter through the region of SNHG17 binding to c‐Jun. IgG was the normalized control for qRT‐PCR analysis. I, Expression of c‐Myc was measured by qRT‐PCR assay in SNHG17, SNHG17‐MUT, or si c‐Jun and SNHG17 co‐transfected Eca109 and Kyse150 cells. J, qRT‐PCR for mRNA levels of c‐Myc in the transfection of Eca109 cells. K, L, qRT‐PCR and western blot assays showed the expression of Twist1 in the different groups of Eca109 cells of overexpressing c‐Myc/c‐Myc + si c‐Jun/c‐Myc + si c‐Jun + SNHG17. Data are shown as the mean ± SD, **P < .01, ***P < .001

FIGURE 7

SNHG17/c‐Jun/c‐Myc axis promotes ESCC cells growth and metastasis in vitro and in vivo. A, B, MTS and colony formation assays were performed to examine the effect of the SNHG17/c‐Jun/c‐Myc axis on cell proliferation ability of Eca109 and Kyse150 cells. C, Transwell assay was performed for evaluating the effect of the SNHG17/c‐Jun/c‐Myc axis on cell invasion and metastatic ability of Eca109 and Kyse150 cells in each group. D, E, Eca109 cells stably overexpressing SNHG17 were subcutaneously injected into nude mice and xenografts. The growth curve and final tumor weight of xenografts from mice in groups with different treatments (n = 8). F, qRT‐PCR analysis showed that the expression of SNHG17 and c‐Myc in xenograft tissues with overexpression of SNHG17. G, Western blot assays were performed to measure c‐Myc, E‐cadherin, N‐cadherin, Twist1, SNAI2, and Ki67 expression levels in xenografts of mice with different treatments. H, IHC staining of c‐Myc, N‐cadherin, and Ki‐67 in the xenografts of mice in the 2 groups. The results are shown as the mean ± SD. ***P < .001