Decreased expression of the long non-coding RNA FENDRR is associated with poor prognosis in gastric cancer and FENDRR regulates gastric cancer cell metastasis by affecting fibronectin1 expression

Abstract

Background: FENDRR is a long non-coding RNAs (lncRNA) that binds to polycomb repressive complexe 2 (PRC2) to epigenetically regulate the expression of its target gene. The clinical role of FENDRR in carcinomas remains yet to be found.

Method: Real-time polymerase chain reaction (PCR) was used to examine FENDRR expression in gastric cancer cell lines/tissues compared with normal epithelial cells/adjacent non-tumorous tissues. Cell proliferation assays, Wound healing assays, and in vitro and in vivo invasion and migration assays were performed to detect the biological effects of FENDRR in gastric cancer cells. Real-time PCR, western-blot and immunohistochemistry were used to evaluate the mRNA and protein expression of fibronectin1 (FN1). Secreted matrix metalloproteinase (MMP) activities were detected and characterized using gelatin zymography assay.

Results: FENDRR was downregulated in gastric cancer cell lines and cancerous tissues, as compared with normal gastric epithelial cells and adjacent noncancerous tissue samples. Low FENDRR expression was correlated with deeper tumor invasion (p < 0.001), higher tumor stage (p = 0.001), and lymphatic metastasis (p = 0.007). Univariate and multivariate analyses indicated that low FENDRR expression predicted poor prognosis. Histone deacetylation was involved in the downregulation of FENDRR in gastric cancer cells. FENDER overexpression suppressed invasion and migration by gastric cancer cells in vitro, by downregulating FN1 and MMP2/MMP9 expression.

Conclusion: Low expression of the lncRNA FENDRR occurs in gastric cancer and is associated with poor prognosis. Thus, FENDRR plays an important role in the progression and metastasis of gastric cancer.

Figure 1

Decreased expression ofFENDRRin gastric cancer cells; histone deacetylation is involved inFENDRRdownregulation. (A)FENDRR expression is examined by qRT-PCR in 158 paired human gastric cancer tissues and adjacent noncancerous tissues (Wilcoxon signed-rank test, p < 0.05). Data are represented as log₂ fold change (cancer/normal), with “<−1” indicating underexpression, and “>1” indicating overexpression. The patients were divided into a low FENDRR expression group (79) and a high FENDRR expression group (79) according to the median value of relative FENDRR expression (2.7-fold, noncancerous/tumors) (B) Real-time PCR analysis of FENDRR expression in normal gastric epithelial cell line (GES-1) and gastric cancer cells. Experiments were performed in triplicate. Bars: SD; *p < 0.05. (C) qPCR analysis of FENDRR expression levels following the treatment of BGC823 and MGC803 cells with TSA. Experiments were performed in triplicate. Bars: SD; *p < 0.05. (D) qPCR analysis of FENDRR expression levels following the treatment of BGC-823 and MGC-803 cells with si-HDAC1 and si-HDAC3. Experiments were performed in triplicate. Bars: SD; *p < 0.05.

Figure 2

The prognostic significance ofFENDRRin gastric cancer patients. (A) Kaplan–Meier analysis of disease-free survival (DFS) based on FENDRR expression in all 158 patients. (B), (C), and (D) Kaplan–Meier analysis of DFS based on FENDRR expression in gastric cancer patients in stages I-II (B), III (C) and IV (D). (E) Kaplan–Meier analysis of overall survival (OS) based on FENDRR expression in all 158 patients. (F), (G) and (H) Kaplan–Meier analysis of OS based on FENDRR expression in gastric cancer patients in stages I-II (F), III (G) and IV (H).

Figure 3

Effects ofFENDRRon gastric cancer cell migration and invasion in vitro and in vivo. MGC803 cells were transfected with pcDNA-FENDRR, and BGC823 cells were transfected with si-FENDRR. (A) qPCR analysis of FENDRR expression levels following the treatment of MGC803 cells with empty vector and pcDNA-FENDRR (left panel), and the treatment of BGC823 cells with scrambled siRNA and si-FENDRR (right panel). Experiments were performed in triplicate. Bars: SD; **p < 0.01. (B) Forty-eight hours after transfection, MTT assays were conducted to determine the proliferation of MGC803 and BGC823 cells. Experiments were performed in triplicate. Bars: SD. (C) Colony-formation growth assays were conducted to determine the proliferation of MGC803 and BGC823 cells. The colonies were counted and photographed. Experiments were performed in triplicate. Bars: SD. (D) Wound healing assays were used to investigate the migratory ability of gastric cancer cells. Experiments were performed in triplicate. Bars: SD; **p < 0.01. (E) and (F) Transwell assays were used to investigate the changes in the migratory and invasive abilities of gastric cancer cells. Experiments were performed in triplicate. Bars: SD; *p < 0.05 and **p < 0.01. (G) MGC803 cells transfected with pcDNA-FENDRR and empty vector were separately injected into the tail veins of athymic mice. Lungs were harvested from the mice in each experimental group, and the numbers of tumor nodules visible on lung surfaces were counted. The assay was independently conducted twice. Bars: SD; *p < 0.05 and **p < 0.01. (H) Visualization of the entire lung, and hematoxylin and eosin (HE)-stained lung sections.

Figure 4

FENDRRregulates FN1 expression and MMP2/MMP9 activity. (A) Expression of cell adhesion molecules (FN1, integrin, CD44, ICAM-1, E-cadherin, N-cadherin and vimentin) as detected using qRT-PCR after FENDRR was overexpressed in MGC803 cells or (B) blocked in BGC823 cells. (C) After FENDRR is overexpressed in MGC803 cells, western blot analysis shows that the FN1 protein level is diminished, as compared to the level in the control group. (D) The FN1 protein level is elevated after FENDRR expression is blocked in BGC823 cells, as compared to the level in the control group. (E) and (F)FENDRR regulates the enzymatic activity of MMP2/MMP9 in gastric cancer cells. All the above experiments were performed in triplicate. Bars: SD; *p < 0.05 and **p < 0.01.

Figure 5

FN1 promotes cells mobility and regulates MMP2/MMP9 activity in MGC803 and BGC823 cells; and is involved in theFENDRR-mediated inhibition of metastasis. (A) and (B) FN1 knockdown inhibits the migration and invasion potential of MGC803 and BGC823 cells. (C) FN1 regulates the enzymatic activity of MMP2/MMP-9 in gastric cancer cells. (D) The expression of FN1 protein in BGC-823 cells was analyzed by Western blotting. (E) MMP2/MMP9 expression in BGC823 cells is analyzed by gelatin zymography. (F) Migration and invasion analyses of BGC823 cells is shown. All experiments were performed in triplicate. Bars: SD; *p < 0.05, **p < 0.01.

Figure 6

FN1 expression in gastric cancer tissues and cell lines. FN1 mRNA levels in gastric cancer tissues (A) and cell lines (B) were analyzed by qRT-PCR. (C) FN1 protein levels in gastric cancer tissues (T) and adjacent noncancerous tissues (ANT) were analyzed by immunohistochemistry. (D) Analysis of the relationship between FENDRR expression (△Ct value) and FN1 mRNA level (△Ct value) in 40 gastric cancer tissues. Bars: SD; *p < 0.05, **p < 0.01.