LncRNA SNHG3 Promotes Proliferation and Metastasis of Non-Small-Cell Lung Cancer Cells Through miR-515-5p/SUMO2 Axis

Abstract

Lung cancer is a global disease and a major cause of cancer-related mortality worldwide. Accumulated studies have confirmed the essential role of long non-coding RNAs (lncRNAs) in the occurrence and development of cancers. Meanwhile, there have been reports concerning the role of Small Nucleolar RNA Host Gene 3 (SNHG3) in various cancers. However, there are so far few studies on the function and mechanism of SNHG3 in lung cancer. In the present study, SNHG3 was found to be highly expressed in lung cancer tissues and cells. Downregulation of SNHG3 could inhibit cell proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) process. In addition, SNHG3 was found to have the ability to bind to miR-515-5p. Furthermore, Small Ubiquitin Like Modifier 2 (SUMO2) was identified to be the downstream target of miR-515-5p, which was negatively correlated with miR-515-5p expression. SNHG3 could positively regulate SUMO2 expression by sponging miR-515-5p. In addition, the rescue experiment showed that simultaneous transfection of miR-515-5p or SUMO2 siRNA could reverse the effect of SNHG3 expression on cell proliferation and metastasis. Collectively, our study demonstrates that SNHG3 can act on miR-515-5p in the form of competitive endogenous RNA (ceRNA) to regulate SUMO2 positively and thus affect the proliferation and metastasis of NSCLC cells. Findings in our study support that SNHG3/miR-515-5p/SUMO2 regulatory axis may become a potential therapeutic target for lung cancer.

Keywords: NSCLC; SNHG3; SUMO2; lncRNA; miR-515-5p.

Figure 1.

Abnormal high expression of SNHG3 in lung cancer tissues and cells. A, Online analysis of the difference of SNHG3 mRNA expression in lung adenocarcinoma and normal tissues using Starbase database. B, Quantitative PCR analysis of SNHG3 in 15 pairs of NSCLC tissues and corresponding adjacent tissues. C, Quantitative analysis of the relative expression of SNHG3 in various cells using PCR. Data are the means ± SD of 3 independent experiments. *P < 0.05, **P < 0.01 compared with adjacent tissues group or BEAS-2B cell group.

Figure 2.

Effect of SNHG3 in the regulation of proliferation and metastasis of lung cancer cells. A, Quantitative PCR detection of the silencing effect of siRNA on SNHG3 in A549 and HCC827 cells. B and C, Detection of the proliferation of tumor cells transfected with siRNA using CCK-8. D and E, Detection of the migration and invasion ability of tumor cells transfected with siRNA using Transwell cell migration assay. The cells were stained with DAPI and observed under fluorescence microscope (Magnification, 200×; Scale bar, 50 µm). F and G, Quantification of the relative migration and invasion ability of cells in each group by histogram through counting under different visual fields. H, Analysis of the relative expression of EMT related proteins after siRNA transfection using western blot; GAPDH was used as the internal reference. si, siRNA; si#1 and si#2, two different siRNA duplexes against SNHG3. One-way ANOVA with Tukey post hoc analysis. *P < 0.05 and **P < 0.01.

Figure 3.

Subcellular localization analysis of SNHG3 in lung cancer cells. A, Online prediction of SNHG3 localization by lncLocator and Locate-R. B, Detection of the localization of SNHG3 in A549 and HCC827 lung cancer cells using FISH assay (Green: SNHG3; Blue: DAPI; Red: F-actin; Scale bar, 20 µm). C, Quantitative PCR analysis of the relative content of KLF2 and CDKN1A mRNA after siRNA transfection. *P < 0.05, **P < 0.01 compared with NC siRNA group.

Figure 4.

The sponge effect of SNHG3 on miR-515-5p. A, Quantitative PCR detection of the regulatory effect of the overexpression of various miRNAs on SNHG3 in HCC827 cells. B, Online analysis of potential targets of SNHG3 and miR-515-5p using Starbase database. C, Construction of the wild-type and mutant-type luciferase reporter gene vectors containing the predicted target SNHG3 of miR-515-5p; and the schematic diagram of potential targets of SNHG3 with deletion mutation and miR-515-5p. D, Detection of the direct targeting effect of miR-515-5p on SNHG3 by luciferase activity assay. E, Quantitative PCR detection of the regulatory effect of overexpressing SNHG3-WT or SNHG3-MUT on the relative content of miR-515-5p in HCC827 cells. *P < 0.05, **P < 0.01 versus control.

Figure 5.

Inhibitory effect of miR-515-5p on the proliferation and metastasis of lung cancer cells. A, Detection of cell proliferation ability in each group using CCK-8 following the rescue experiment by overexpressing miR-515-5p and simultaneous transfection of SNHG3 in HCC827 cells. B and C, Detection of the ability of cell migration and invasion by Transwell assay (Magnification, 200×; Scale bar, 50 µm). *P < 0.05, **P < 0.01 versus control.

Figure 6.

Positive regulation of LncRNA SNHG3 on the expression of SUMO2 by sponging miR-515-5p. A, Prediction of the potential targets of miR-515-5p by PITA, RNA22, miRmap, microT, PicTar and TargetScan. B, Analysis of the correlation of miR-515-5p and SNHG3 with SUMO2 based on data from TCGA. C, Diagrammatic sketch of SUMO2 mRNA 3′UTR possessing the target site of miR-515-5p and its target deletion mutation. D, Detection of the direct targeting effect of miR-515-5p on SUMO2 3′UTR using luciferase activity assay. E, Luciferase activity assay was used to detect the effect of SNHG3 on miR-515-5p targeting of SUMO2. F, Sequence similarity of SUMO2 and SUMO3 proteins. G, Western blot detection of the effect of overexpressing miR-515-5p and SNHG3 on the relative expression of SUMO2. *P < 0.05, **P < 0.01 versus control.

Figure 7.

Regulatory effect of LncRNA SNHG3 on the proliferation and metastasis of lung cancer cells through miR-515-5p/SUMO2. A, CCK-8 detection of the inhibitory effect of miR-515-5p or SUMO2 siRNA on the cell proliferation-promoting of overexpressing SNHG3 in lung cancer H1270 cells. B and C, Transwell detection of the inhibitory effect of miR-515-5p or SUMO2 siRNA on the cell migration-promoting of overexpressing SNHG3 in lung cancer H1270 cells (Magnification, 200×; Scale bar, 50 µm). D, Western blot detection of the effect of overexpressing SNHG3 and simultaneous transfection of miR-515-5p or SUMO2 siRNA on the expression of EMT related proteins in lung cancer H1270 cells. *P < 0.05, **P < 0.01 versus control.