Long non-coding RNA SNHG5 affects the invasion and apoptosis of renal cell carcinoma by regulating the miR-363-3p-Twist1 interaction

Abstract

Non-coding RNA dysregulation is associated with many human diseases, including cancer. This study explored the effects of lncRNA SNHG5 on clear cell renal cell carcinoma (ccRCC). We found that lncRNA SNHG5 is upregulated in human ccRCC tissues and that lncRNA SNHG5 inhibition reduced ccRCC cell invasion and promoted apoptosis in vitro. Bioinformatics database searching revealed that lncRNA SNHG5 is predicted to regulate the interaction between miR-363-3p and Twist1. We further verified a ccRCC biomarker panel, which consists of lncRNA SNHG5, miR-363-3p, and Twist1 in ccRCC tissue samples. The direct SNHG5-miR-363-3p and Twist1-miR-363-3p interactions were confirmed via dual-luciferase reporter assays. Additionally, functional assays demonstrated that SNHG5 promotes cell invasion and inhibits apoptosis, while miR-363-3p inhibits cell invasion and promotes apoptosis via an interaction with Twist1. Furthermore, we found that Twist1 promotes tumor metastasis by regulating matrix metalloproteinase (MMP)2 and MMP9 levels. Together, these results suggest that lncRNA SNHG5 may predict ccRCC patient clinical outcome and serve as a novel anti-ccRCC therapeutic target.

Keywords: Twist1; ccRCC; lncRNA SNHG5; metastasis; miR-363-3p.

Figure 1

Effect of lncRNA SNHG5 on metastasis and apoptosis of RCC cells. (A) qRT-PCR results of lncRNA SNHG5 in human ccRCC tissues and non-tumor tissues. (B) The relative expression of lncRNA SNHG5 was determined in the non-malignant (HK-2 and SW-13) and malignant (786-O, Caki1, and ACHN) cell lines. (C) The expression of lncRNA SNHG5 in 786-O cells, cells treated with shRNA-NC, and cells treated with lncRNA SNHG5-shRNA was detected by qRT-PCR. (D) Transwell assay was used to determine the metastasis/invasion ability of the cells described for (C). (E) Apoptosis by the three cell groups was detected by flow cytometry. *P<0.05, **P<0.01.

Figure 2

The miR-363-3p targeted the expression of lncRNA SNHG5 in 786-O cells. A. qRT-PCR results of miR-363-3p in human ccRCC tissues and non-tumor tissues. B. The expression of miR-363-3p in 780-O cells, cells treated with shRNA-NC, and cells treated with lncRNA SNHG5-shRNA was detected by qRT-PCR. C. The binding sites of miR-363-3p and lncRNA SNHG5-WT were detected using a luciferase reporter assay. *P<0.05, **P<0.01.

Figure 3

Knockdown of lncRNA snhg5 inhibited RCC cell invasion and promoted cell apoptosis by targeting miR-363-3p. A. The effects of lncRNA SNHG5 on cell invasion via targeting of miR-363-3p. B. The apoptosis rates of cells treated with shRNA-NC, SNHG5-shRNA, mimic-NC+SNHG5-shRNA, and miR-363-3p-mimics+SNHG5-shRNA were detected by flow cytometry. **P<0.01, ***P<0.001.

Figure 4

miR-363-3p affects the invasion and apoptosis of 786-O cells via its interaction with Twist1. A. Twist1 protein expression levels were increased in cells treated with the miR-363-3p inhibitor compared to those treated with NC and were decreased in cells treated with the miR-363-3p mimic. B. A potential binding site for miR-363-3p in Twist1 is shown, and binding was detected by a luciferase reporter assay. C, D. The effects of inhibition of Twist1 in 786-O cells on invasion and apoptosis were determined. E. The expression of MMP2 and MMP9 protein were measured and quantitated (right panel) following control shRNA treatment of shRNA downregulation of miR-363-3p in 786-O cells (The original western blot images reference to Supplementary Figure 1). *P<0.05, **P<0.01, ***P<0.001.