Long non-coding RNA SNHG17 may function as a competitive endogenous RNA in diffuse large B-cell lymphoma progression by sponging miR-34a-5p

Abstract

We investigated the functional mechanism of long non-coding small nucleolar host gene 17 (SNHG17) in diffuse large B-cell lymphoma (DLBCL). lncRNAs related to the prognosis of patients with DLBCL were screened to analyze long non-coding small nucleolar host gene 17 (SNHG17) expression in DLBCL and normal tissues, and a nomogram established for predicting DLBCL prognosis. SNHG17 expression in B-cell lymphoma cells was detected using qPCR. The effects of SNHG17 with/without doxorubicin on the proliferation and apoptosis of DoHH2 and Daudi were detected. The effects of combined SNHG17 and doxorubicin were analyzed. The regulatory function of SNHG17 in DLBCL was investigated using a mouse tumor xenotransplantation model. RNA sequencing was used to analyze the signaling pathways involved in SNHG17 knockdown in B-cell lymphoma cell lines. The target relationships among SNHG17, microRNA, and downstream mRNA biomolecules were detected. A higher SNHG17 level predicted a lower survival rate. SNHG17 was highly expressed in DLBCL patient tissues and cell lines. We established a prognostic model containing SNHG17 expression, which could effectively predict the overall survival rate of DLBCL patients. SNHG17 knockdown inhibited the proliferation and induced the apoptosis of B-cell lymphoma cells, and the combination of SNHG17 and doxorubicin had a synergistic effect. SNHG17, miR-34a-5p, and ZESTE gene enhancer homolog 2 (EZH2) had common hypothetical binding sites, and the luciferase reporter assay verified that miR-34a-5p was the direct target of SNHG17, and EZH2 was the direct target of miR-34a-5p. The carcinogenic function of SNHG17 in the proliferation and apoptosis of DLBCL cells was partially reversed by a miR-34a-5p inhibitor. SNHG17 increases EZH2 levels by inhibiting miR-34a-5p. Our findings indicate SNHG17 as critical for promoting DLBCL progression by regulating the EZH2 signaling pathway and sponging miR-34a-5p. These findings provide a new prognostic marker and therapeutic target for the prognosis and treatment of DLBCL.

Fig 1. Screening the lncRNAs related to DLBCL prognosis and survival.

(A, B) Venn diagram of GSE10846 and GSE31312 datasets. (C) Forest diagram of DLBCL patients with good prognosis and low-risk lncRNAs. (D) Forest diagram of DLBCL patients with poor prognosis and high-risk lncRNAs.

Fig 2. SNHG17 is highly expressed in DLBCL patients’ tumor cells and in B-lymphoma cell lines.

(A) Survival analyses of GSE10846 (n = 420) in DLBCL patients with high and low expression of SNHG17.(B, C) Kaplan–Meier survival analysis. (D, E) Uniform and multivariate expression analyses (all bars correspond to the 95% confidence intervals). (F) Nomogram was used to predict the OS of patients. (G) Expression of SNHG17 in DLBCL patients’ tumor and normal tissues.The sequencing data for normal cells and diffuse large B-cell lymphoma were obtained from UCSC XENA, which we have described in the methods section. (H) qPCR shows the expression of SNHG17 in lymphoma cell lines (Daudi, Ramos, DoHH2, Farage, and Raji) and peripheral blood mononuclear cells (PBMC).*P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3. SNHG17 can promote the growth of DLBCL cells in vitro and in vivo.

(AⅠ-Ⅱ) CCK-8 experiment confirmed the proliferation ability of DoHH2 and Daudi cells, and the concentration of DOX was 0.125 μM. (B) Flow cytometry was used to analyze the apoptosis of DoHH2 and Daudi cells. (C-F) DoHH2 cells transfected with sh-NC or sh-SNHG17 were inoculated into the back of nude mice. The tumor planting diagram (C), picture (D), growth curve (E), and weight (F) are shown. (G) Flow cytometry was used to analyze the apoptosis of transplanted tumor cells. The data are expressed as mean ± SEM* P < 0.05, **P < 0.01, ***P < 0.001. n = 4 mice per group.

Fig 4. SNHG17 knockdown enhanced the sensitivity of DLBCL cells to doxorubicin.

Knockdown of SNHG17 enhanced the sensitivity of lymphoma cells to doxorubicin. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5. miR-34a-5p is the direct target of SNHG17; miR-34a-5p inhibitor reverses the inhibitory effect of SNHG17 knockdown on lymphoma cells.

(A) Signal pathways enriched from clinical and cell line lymphoma samples were taken at the Venn diagram intersection. (B) Bar graph of common signal pathways in clinical and cell line samples. (C) Venn diagram of miRNA binding to SNHG17 and EZH2 at the same time. (DⅠ-Ⅱ) Expression of miR-34a-5p and miR-101-3p in lymphoma. (E) Binding sites of miR-34a-5p and SNHG17 were predicted by bioinformatics analysis. (FⅠ-Ⅱ) Double luciferase reporter was used to confirm the binding. (G) Expression of miR-34a-5p decreased in lymphoma cells and was negatively correlated with SNHG17. (HⅠ-Ⅱ) miR-34a-5p inhibitor reversed the effect of SNHG17 knockdown on cell proliferation. (I) miR-34a-5p inhibitor reversed the effect of SNHG17 knockdown on cell apoptosis. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 6. miR-34a-5p affects the EZH2 protein pathway.

(A) Binding sites of EZH2 and miR-34a-5p were predicted through bioinformatics analysis. (BⅠ-Ⅱ) Double luciferase reporter experiment confirmed this prediction. (CⅠ-Ⅱ) Expression of EZH2 was increased in lymphoma cells and negatively correlated with miR-34a-5p. (DⅠ-Ⅱ) Overexpression of miR-34a-5p resulted in significant inhibition of the EZH2 signaling pathway. (E, F) miR-34a-5p inhibitor significantly reversed the EZH2 protein inhibition caused by SNHG17 knockdown. *P < 0.05, **P < 0.01, ***P < 0.001.