The pseudogene-derived long non-coding RNA SFTA1P suppresses cell proliferation, migration, and invasion in gastric cancer

Abstract

Pseudogenes were once regarded as transcriptionally inactive and without specific molecular function. However, current evidence shows that pseudogene-derived long non-coding RNAs (lncRNAs) may be crucial regulators of human cancer development, including gastric cancer (GC). In the present study, we report that a pseudogene-derived lncRNA named surfactant associated 1, pseudogene (SFTA1P), which is 693-nt long, was significantly down-regulated in GC tissues compared with that in the adjacent normal tissues. In addition, decreased SFTA1P expression was strongly correlated with advanced tumor lymph node metastasis (TNM) stage, larger tumor size, lymphatic metastasis, and poor prognosis of patients with GC. Moreover, gain-of-function experiments revealed that the overexpression of SFTA1P inhibits cell proliferation, migration, and invasion, thus verifying the tumor inhibitory role of SFTA1P in GC. Furthermore, we investigated the potential action mechanism of SFTA1P. Our results showed that down-regulation of SFTA1P may be associated with decreased TP53 expression. In summary, our work suggests that the pseudogene-derived lncRNA SFTA1P functions as a tumor suppressor in GC and thus may act as a potential diagnostic and therapeutic target of GC.

Keywords: SFTA1P; gastric cancer; lncRNA; pseudogene.

Figure 1. Relative SFTA1P expression in GC tissues and its clinical significance

(A) Relative expression of SFTA1P in human GC tissues (n=68) compared with corresponding non-tumor tissues (n=68). SFTA1P expression was examined by qRT-PCR and normalized to GAPDH expression (shown as Δc_t). (B) Results are presented as the fold-change in tumor tissues relative to normal tissues, and SFTA1P expression was classified into two groups. (C,D) Kaplan–Meier progression-free survival and overall survival curves according to SFTA1P expression level. Error bars indicate mean ± S.E.M. *P-value <0.05, **P-value <0.01.

Figure 2. SFTA1P inhibits GC cell proliferation in vitro

(A) qRT-PCR analysis of SFTA1P expression in the normal gastric epithelium cell line (GES1) and GC cells. (B) qRT-PCR analysis of SFTA1P expression in empty vector, pCDNA-SFTA1P transfected GC cells. (C,D) MTT assays were used to determine the viability of pCDNA-SFTA1P transfected GC cells. (E) Colony formation assays were performed to determine the proliferation of pCDNA-SFTA1P transfected GC cells. Colonies were counted and captured. (F,G) Proliferating BGC823 and SGC7901 cells were labeled with EdU. The Click-it reaction revealed EdU staining (red). Cell nuclei were stained with DAPI (blue). Representative images and data based on three independent experiments. Error bars indicate mean ± S.E.M. *P-value <0.05, **P-value <0.01.

Figure 3. Effect of SFTA1P on GC cell apoptosis and cell cycle regulation in vitro

(A,B) Flow cytometry was used to detect the cell cycle regulation. The bar chart represented the percentage of BGC823 and SGC7901 cells in G₀/G₁, S, or G₂/M phase, as indicated. (C,D) Flow cytometry was used to detect the apoptotic rates of cells. Error bars indicate mean ± S.E.M. *P-value <0.05, **P-value <0.01. Abbreviations: LR, early apoptotic cell; UR, terminal apoptotic cell.

Figure 4. SFTA1P overexpression inhibits GC cells migration and invasion

(A) Transwell assays were used to investigate the changes in migratory abilities of SFTA1P overexpression cells. (B) Transwell assays were used to investigate the changes in invasive abilities of SFTA1P overexpression cells. Error bars indicate mean ± S.E.M. *P-value <0.05, **P-value <0.01.

Figure 5. SFTA1P overexpression inhibits tumorigenesis of GC cells in vivo

(A) Empty vector or pCDNA-SFTA1P were transfected into SGC7901 cells, which were injected in the nude mice (n=5), respectively. Tumors formed in pCDNA-SFTA1P group were dramatically smaller than the control group. (B) qRT-PCR was performed to detect the average expression of SFTA1P in xenograft tumors. (C) Tumor weights were represented as means of tumor weights ± S.D. (D) Tumor volumes were calculated after injection every 3 days. Points, mean (n=5); bars indicate S.D. (E) The tumor sections were under H&E staining and immunohistochemical (IHC) staining using antibodies against Ki-67. Error bars indicate mean ± S.E.M. *P-value <0.05, **P-value <0.01.

Figure 6. Potential targets involved in SFTA1P tumor suppressor function

(A) qRT-PCR analysis of TP53 expression in empty vector, pCDNA-SFTA1P transfected GC cells. (B) The expression level of TP53 protein using Western blot analysis. Error bars indicate mean ± S.E.M. *P-value <0.05, **P-value <0.01.