The effect of splicing MST1R in gastric cancer was enhanced by lncRNA FENDRR

Abstract

Gastric cancer (GC) poses a serious threat to human health worldwide. Serine/arginine rich splicing factor 1 (SRSF1) has been reported to serve regulatory roles during the tumorigenesis of GC. In addition, the macrophage stimulating 1 receptor (MST1R) signaling pathway was found to participate in the progression of GC. However, the association between MST1R and SRSF1 in the tumorigenesis of GC remains unclear. The expression levels of MST1R and the recepteur d'origine nantais (RON) Δ160 splicing variant were analyzed in cells using western blotting and immunofluorescence staining. Co-immunoprecipitation assays were used to investigate the interaction between SRSF1 and MST1R. A Cell Counting Kit-8 assay was performed to analyze cell viability. Flow cytometry and Transwell assays were used to determine cell apoptosis and invasiveness levels. The potential interaction between SFSR1 and long non-coding RNAs (lncRNAs) was investigated with an online bioinformatics tool. The findings of the present study revealed that the expression levels of MST1R and RON Δ160 were significantly upregulated in GC Kato III cells. SRSF1 was found to be regulated by the lncRNA FOXF1 adjacent non-coding developmental regulatory RNA (FENDRR). The knockdown of SRSF1 or FENDRR downregulated the expression levels of MST1R in Kato III cells. In addition, the expression levels of RON Δ160 were markedly downregulated in Kato III cells following the knockdown of FENDRR. Meanwhile, SRSF1 directly bound to MST1R, while this phenomenon was partially reversed by FENDRR short interfering RNA. FENDRR could interact with SRSF1 in Kato III cells and the knockdown of FENDRR also induced the apoptosis of GC cells. In conclusion, the findings of the present study suggested that the lncRNA FENDRR may function as an oncogene during the progression of GC by regulating alternative splicing of MST1R and SRSF1 expression levels. lncRNA FENDRR may serve as a potential marker for the diagnosis or target for the treatment of GC.

Keywords: FOXF1-AS1; MST1R; SRSF1; gastric cancer.

Figure 1

MST1R and RON Δ160 are upregulated in Kato III cells. (A) The protein expression levels of MST1R in GES-1, Kato III and MKN-45 cells were determined by western blot. The relative expression was quantified by normalizing to β-actin. (B) The expression levels of RON Δ160 in GES-1, Kato III and MKN-45 cells were investigated by immunofluorescence staining. Green fluorescence indicates RON Δ160. Blue fluorescence indicates DAPI. (C) Electrophoresis of MST1R, RON Δ160 and β-actin polymerase chain reaction products on the agarose gel showed a unique band with expected sizes for each gene for GES-1, Kato III and MKN-45 cells. DNA expression was quantified by normalizing to β-actin. MST1R, macrophage stimulating 1 receptor; RON Δ160, recepteur d'origine nantais. ^**P<0.01 vs. GES-1 cells.

Figure 2

Knockdown of SRSF1 significantly inactivates MST1R and RON Δ160 in Kato III cells. (A) Kato III cells were transfected with NC, siRNA-SRSF1-1, siRNA-SRSF1-2 or siRNA-SRSF1-3 for 24 h and then western blotting was used to detect the efficiency of transfection. (B) The relative protein expression of SRSF1 was quantified by normalizing to β-actin. (C) The expression of RON Δ160 in Kato III cells was measured by immunofluorescence staining. (D) The protein expression level of MST1R in Kato III cells was determined by western blot. The relative expression levels of MST1R were quantified by normalizing to β-actin. (E) Electrophoresis of MST1R, RON Δ160 and β-actin polymerase chain reaction products on the agarose gel showed a unique band with expected sizes for each gene for Kato III cells. DNA expression was quantified by normalizing to β-actin. SRSF1, serine/arginine rich splicing factor 1; MST1R, macrophage stimulating 1 receptor; RON Δ160, recepteur d'origine nantais; NC, non-coding control; siRNA, short interfering RNA. ^**P<0.01 vs. control.

Figure 3

Silencing of FENDRR suppresses the expression of MST1R and altered the distribution of RON Δ160 in Kato III cells. (A) Kato III cells were transfected with NC or FENDRR siRNA for 24 h. The efficiency of transfection was measured by reverse transcription-quantitative PCR. (B) The protein expression levels of MST1R and SRSF1 in Kato III cells were determined by western blot. The relative protein expression levels of MST1R and SRSF1 were quantified by normalizing to β-actin. (C) The expression level of RON Δ160 in Kato III cells was determined by immunofluorescence staining. (D) Electrophoresis of MST1R, RON Δ160 and β-actin polymerase chain reaction products on the agarose gel showed a unique band with expected sizes for each gene for Kato III cells. DNA expression was quantified by normalizing to β-actin. FENDRR, FOXF1 adjacent non-coding developmental regulatory RNA; SRSF1, serine/arginine rich splicing factor 1; MST1R, macrophage stimulating 1 receptor; RON Δ160, recepteur d'origine nantais; NC, non-coding control; siRNA, short interfering RNA. ^**P<0.01 vs. NC.

Figure 4

SRSF1 binds to MST1R. (A) Equal amounts of total lysates were applied to co-immunoprecipitation with IgG, anti-SRSF1 or anti-MST1R magnetic beads. Immunoprecipitation analysis of Kato III cells transfected with NC or siRNA- FENDRR . (B) The enrichment of SRSF1 was detected by RNA pull-down. FENDRR, FOXF1 adjacent non-coding developmental regulatory RNA; SRSF1, serine/arginine rich splicing factor 1; MST1R, macrophage stimulating 1 receptor; NC, non-coding control; siRNA, short interfering RNA; IgG, immunoglobulin G; ctrl, control. ^**P<0.01 vs. probe-ctrl.

Figure 5

Silencing of FENDRR suppresses the growth of gastric cells via MST1R. (A) Kato III cells were transfected with pcDNA3.1 (NC) or MST1R OE. The expression of MST1R in Kato III cells was determined by reverse transcription-quantitative PCR. (B) Cells were treated with NC, siRNA-FENDRR or FENDRR + MST1R OE for 72 h. Cell viability was then tested. (C) The apoptosis rate in Kato III cells was measured by flow cytometry after double staining with Annexin V and PI. (D) Cell migration and (E) invasion were assessed by transwell assay. FENDRR, FOXF1 adjacent non-coding developmental regulatory RNA; MST1R, macrophage stimulating 1 receptor; OE, overexpression vector; NC, non-coding control; siRNA, short interfering RNA; PI, propidium iodide. ^**P<0.01 vs. control, ^##P<0.01 vs. siRNA-FENDRR.

Figure 6

Silencing of FENDRR inhibits the tumorigenesis of gastric cancer cells in vitro via inactivation of PI3K/Akt signaling. (A) The protein expression levels of MST1R, caspase-3, p-Akt, Akt, p-ERK and ERK in Kato III cells were determined by western blot. (B) The relative protein expression of MST1R was quantified by normalizing to β-actin. (C) The ratio of cleaved caspase-3/procaspase-3 was calculated. (D) The ratio of p-Akt/Akt was calculated. (E) The ratio of p-ERK/ERK was calculated. FENDRR, FOXF1 adjacent non-coding developmental regulatory RNA; MST1R, macrophage stimulating 1 receptor; OE, overexpression vector; NC, non-coding control; siRNA, short interfering RNA; ERK, extracellular signal related kinase; p, phosphorylated ^**P<0.01 vs. NC; ^##P<0.01 vs. siRNA-FENDRR.