The novel long noncoding RNA RP11-357H14.17 acts as an oncogene by promoting cell proliferation and invasion in diffuse-type gastric cancer

Abstract

Current evidence indicates that long noncoding RNAs (lncRNAs) play pivotal roles in human cancers. The present study aims to assess differentially expressed lncRNAs related to diffuse-type gastric carcinoma (DGC). Next-generation RNA sequencing was carried out to detect aberrantly expressed lncRNAs in DGC. Real-time polymerase chain reaction (RT-PCR) was performed to evaluate RP11-357H14.17 gene expression levels in DGC cell lines/tissues comparatively with normal gastric epithelial cell lines and adjacent normal tissues. The associations of RP11-357H14.17 expression levels with the clinicopathological features were also analyzed. The regulatory effects of RP11-357H14.17 on the biological behaviors of DGC cells were evaluated by MTT, colony formation assays, flow cytometry for apoptosis, wound healing assay, and transwell migration and invasion assays. RP11-357H14.17 expression was remarkably increased in DGC tissues and cell lines compared with normal gastric epithelial cells and adjacent normal tissues. High levels of RP11-357H14.17 were associated with increased tumor size, deeper depth of invasion, lymphatic metastasis, and advanced pathological stage. Further experiments demonstrated that the DGC cells MGC-803 transfected with si-RP11-357H14.17 showed reduced cell proliferation, migration, invasion, enhanced G1 phase arrest and cell apoptosis. These findings suggest that the novel lncRNA RP11-357H14.17 is associated with poor prognosis, and may serve as a potential prognostic biomarker and target for new antineoplastic therapies in human DGC.

Keywords: RP11–357H14.17; diffuse-type gastric cancer; invasion; long noncoding RNA; metastasis; proliferation.

Figure 1

Hierarchical clustering analysis on the most significantly dysregulated cancer-related lncRNAs. Notes: 1T, 2T, 3T, 4T, 5T, 6T and 1N, 2N, 3N, 4N, 5N, 6N represented cancerous and paired non-cancerous tissues of samples DGC1, DGC2, DGC3, DGC4, DGC5 and DGC6, respectively.

Figure 2

Relative RP11–357H14.17 expression levels in diffuse-type gastric cancer tissues and cell lines. Notes: (A) Relative expression of RP11–357H14.17 in 42 pairs of DGC tissues and adjacent normal tissues by qRT-PCR analysis. RP11–357H14.17 levels were calculated by the 2^−Δct method and normalized to glyceraldehyde 3-phosphate dehydrogenase. (B) The fold change of RP11–357H14.17 in DGC relative to the adjacent normal tissues in 42 DGC patients. (C) qRT-PCR detection in 42 pairs of fresh tissues showed RP11–357H14.17 was highly expressed in DGC tissues. (D) Increased RP11–357H14.17 expression in 4 GC cell lines compared with normal gastric epithelial cell line GES-1. Abbreviations: DGC, diffuse-type gastric cancer; GC, gastric cancer; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 3

The effects of RP11–357H14.17 on cell viability. Notes: (A) The relative expression level of RP11–357H14.17 in MGC-803 cell line was significantly decreased by si-RP11–357H14.17, and compared with si-NC. (B) MTT assay showed down-regulation of RP11–357H14.17 inhibited cell proliferation of MGC-803 cell line. (C) Colony-formation assays showed that down-regulation of RP11-357H14.17 signifiantly decreased the colony-forming ability of MGC-803 cell line. (D) Cell cycle analysis determined that down-regulation of RP11–357H14.17 expression promoted G1 phase arrest in MGC-803 cell line. (E) Flow cytometric analysis showed induced cell apoptosis after si-RP11–357H14.17 transfection. The experiments were performed in triplicate. *P<0.05 compared with si-NC, **P<0.01 compared with si-NC. Abbreviations: CON, MGC-803 cell line; si-NC, si-negative control/non-targeting shRNA; KD, si-RP11–357H14.17.

Figure 4

Knock-down RP11–357H14.17 suppresses migration and invasion of DGC cells. Cell migration and invasion were determined by wound healing assay and transwell assay. Notes: (A) Inhibition of migration of MGC-803 cell line by RP11–357H14.17 siRNA. (B) Inhibition of invasion of MGC-803 cell line by RP1–357H14.17 siRNA. Data are shown as mean ± standard deviation. The experiments were all repeated at least three times. **P<0.01 compared with NC. Abbreviations: DGC, diffuse-type gastric carcinoma; CON, MGC-803 cell line; NC, negative control; KD, si-RP11–357H14.17.

Figure 4

Knock-down RP11–357H14.17 suppresses migration and invasion of DGC cells. Cell migration and invasion were determined by wound healing assay and transwell assay. Notes: (A) Inhibition of migration of MGC-803 cell line by RP11–357H14.17 siRNA. (B) Inhibition of invasion of MGC-803 cell line by RP1–357H14.17 siRNA. Data are shown as mean ± standard deviation. The experiments were all repeated at least three times. **P<0.01 compared with NC. Abbreviations: DGC, diffuse-type gastric carcinoma; CON, MGC-803 cell line; NC, negative control; KD, si-RP11–357H14.17.