Long non-coding RNA SNHG7 facilitates pancreatic cancer progression by regulating the miR-146b-5p/Robo1 axis

Abstract

Long non-coding RNA (lncRNA) small nucleolar RNA host gene 7 (SNHG7) plays a crucial role in the progression of pancreatic cancer (PC). SNHG7 is upregulated in PC; therefore, the purpose of the present study was to investigate the role and underlying mechanism of SNHG7 on PC progression. In the present study, the mRNA expression levels of SNHG7, microRNA(miR)-146b-5p and roundabout homolog 1 (Robo1) were measured via reverse transcription-quantitative PCR. Moreover, cell viability and apoptosis were assessed by MTT and flow cytometry assays, respectively. The ability of cells to migrate and invade was evaluated by Transwell assays. In addition, dual-luciferase reporter, RNA immunoprecipitation and RNA pull-down assays were conducted to assess the interaction between miR-146b-5p and SNHG7 or Robo1. The protein expression of Robo1 was measured via western blotting. Furthermore, mouse xenograft models were established to further investigate the effect of SNHG7 on PC progression in vivo. The results indicated that SNHG7 was highly expressed in PC tissues and cells. It was also found that SNHG7 was sponged by miR-146b-5p and that Robo1 was a target of miR-146b-5p. Moreover, it was demonstrated that SNHG7 knockdown inhibited cell proliferation, migration and invasion, as well as tumorigenesis and apoptosis of PC cells in vitro and in vivo by regulating miR-146b-5p. The results also suggested that miR-146b-5p overexpression inhibited the progression of PC cells by modulating Robo1. Furthermore, silencing of SNHG7 downregulated Robo1 expression by sponging miR-146b-5p. Collectively, the present results indicate that SNHG7 promotes PC progression by sponging miR-146b-5p and upregulating Robo1.

Keywords: cancer progression; long non-coding RNA small nucleolar RNA host gene 7; microRNA-146b-5p; pancreatic cancer; roundabout homolog 1.

Figure 1

SNHG7 is significantly upregulated in PC tissues and correlates with the pathological characteristics of tumor size, distant metastasis, lymph node metastasis and TNM stage of patients with PC. Expression of SNHG7 in (A) PC tissues or (B) different tumor stages was measured by reverse transcription-quantitative PCR. (C) Association between overall survival rate and the expression of SNHG7. ^*P<0.05 vs. the respective normal groups. SNHG7, small nucleolar RNA host gene 7.

Figure 2

SNHG7 knockdown impairs cell proliferation, migration and invasion, but induces apoptosis in SW1990 and AsPC-1 cells. (A) Expression of SNHG7 in human pancreatic cancer cell lines PANC-1, BxPC-3, SW1990 and AsPC-1, and human pancreas normal ductal epithelial cell line HPDE was detected via reverse transcription-quantitative PCR. SNHG7 expression was detected in SW1990 and AsPC-1 cells transfected with (B) sh-control and sh-SNHG7, or (C) pcDNA-NC and pcDNA-SNHG7. SW1990 and AsPC-1 cells were transfected with sh-control, sh-SNHG7 or cultured in normal conditions. Cell viability in (D) SW1990 and (E) AsPC-1 cells was evaluated by MTT assay. Apoptotic rate in (F) SW1990 and (G) AsPC-1 cells was assessed via flow cytometry. (H) Migratory and (I) invasive abilities in transfected cells were measured by Transwell assay. Scale bar, 100 µm. ^*P<0.05 vs. their respective normal groups. sh, short hairpin RNA; SC, (standard conditions); SNHG7, small nucleolar RNA host gene 7; OD, optical density.

Figure 3

miR-146b-5p is a direct target of SNHG7. (A) Complementary sequences between miR-146b-5p and SNHG7, and the MUT sequences of SNHG7. Luciferase activity of SNHG7 WT or SNHG7 MUT reporter in (B) SW1990 and (C) AsPC-1 cells transfected with miR-146b-5p or miR-control was assessed via dual-luciferase reporter. (D) Enrichment of SNHG7 in anti-Ago2 or anti-IgG labeled SW1990 cells transfected with miR-146b-5p or miR-control was evaluated by RIP assay. (E) Enrichment of SNHG7 in SW1990 cells transfected with Bio-miR-146b-5p or Bio-NC was analyzed by RNA pull-down assay. (F) Enrichment of SNHG7 in anti-Ago2 or anti-IgG labeled AsPC-1 cells transfected with miR-146b-5p or miR-control was evaluated by RIP assay. (G) Enrichment of SNHG7 in AsPC-1 cells transfected with Bio-miR-146b-5p or Bio-NC was analyzed by RNA pull-down assay. Transfection efficiency of miR-146b-5p probe was examined in (H) SW1990 and (I) AsPC-1 cells transfected with miR-control or miR-146b-5p. miR-146b-5p expression was measured in SW1990 and AsPC-1 cells transfected with (J) miR-control and miR-146b-5p, or (K) inhibitor-control and miR-146b-5p inhibitor. (L) Expression of miR-146b-5p in cells transfected with sh-control, sh-SNHG7, pcDNA-control or pcDNA-SNHG7 were detected via reverse transcription-quantitative PCR. (M) Correlation between SNHG7 and miR-146b-5p was analyzed by Pearson test. ^*P<0.05 vs. their respective normal groups. WT, wild-type; MUT, mutant; SNHG7, small nucleolar RNA host gene 7; miR, microRNA; sh, short hairpin RNA; NC, negative control; Ago2, argonaute-2.

Figure 4

miR-146b-5p inhibitor relieves the constraint effects on cell proliferation, migration and invasion, as well as reduces the effect on apoptosis in SW1990 and AsPC-1 cells caused by SNHG7 silencing. SW1990 and AsPC-1 cells were transfected with sh-control, sh-SNHG7, sh-SNHG7 + inhibitor-control or sh-SNHG7 + miR-146b-5p inhibitor. Expression of miR-146b-5p was measured in (A) SW1990 and (B) AsPC-1 cells by reverse transcription-quantitative PCR. Cell viability was assessed in (C) SW1990 and (D) AsPC-1 cells via MTT assay. Apoptotic rate was evaluated in (E) SW1990 and (F) AsPC-1 cells via flow cytometry. (G) Migratory and (H) invasive abilities were detected via Transwell assay. ^*P<0.05 vs. the respective normal groups. SNHG7, small nucleolar RNA host gene 7; miR, microRNA; sh, short hairpin RNA; OD, optical density.

Figure 5

Robo1 negatively interacts with miR-146b-5p. (A) Complementary binding sites between miR-146b-5p and Robo1 3'UTR, and the MUT sequences of Robo1 3'UTR. Luciferase activity of Robo1 3'UTR WT or Robo1 3'UTR MUT reporter in (B) SW1990 and (C) AsPC-1 cells transfected with miR-146b-5p mimics or miR-control was evaluated via dual-luciferase reporter assay. (D) mRNA and (E) protein expression levels of Robo1 were examined in SW1990 and AsPC-1 cells transfected with vector and Robo1. (F) mRNA and protein expression levels of Robo1 in (G) SW1990 and (H) AsPC-1 cells transfected with miR-control, miR-146b-5p, inhibitor-control or miR-146b-5p inhibitor were detected by reverse transcription-quantitative PCR and western blotting, respectively. (I) Correlation between Robo1 and miR-146b-5p was assessed with a Pearson test. ^*P<0.05 vs. their respective normal groups. 3'UTR, 3'untranslated region; MUT, mutant; WT, wild-type; miR, microRNA; Robo1, roundabout homolog 1; SNHG7, small nucleolar RNA host gene 7.

Figure 6

Robo1 overexpression mitigates the restraint effects on cell proliferation, migration and invasion, and reduces the effect on apoptosis in SW1990 and AsPC-1 cells induced by miR-146b-5p mimics. SW1990 and AsPC-1 cells were transfected with miR-control, miR-146b-5p, miR-146b-5p + vector or miR-146b-5p + Robo1. Expression of Robo1 was measured in (A) SW1990 and (B) AsPC-1 cells by reverse transcription-quantitative PCR. Protein expression of Robo1 was detected in (C) SW1990 and (D) AsPC-1 cells via western blotting. Cell viability in (E) SW1990 and (F) AsPC-1 cells was assessed by MTT assay. Apoptotic rate was evaluated in (G) SW1990 and (H) AsPC-1 cells by flow cytometry. (I) Migratory and (J) invasive abilities in transfected SW1990 and AsPC-1 cells were detected via Transwell assay. ^*P<0.05 vs. the respective normal groups. miR, microRNA; Robo1, roundabout homolog 1; SNHG7, small nucleolar RNA host gene 7; OD, optical density.

Figure 7

SNHG7 silencing downregulates Robo1 expression by targeting miR-146b-5p. (A) Correlation between Robo1 and SNHG7 was determined by Pearson test. SW1990 and AsPC-1 cells were transfected with sh-control, sh-SNHG7, sh-SNHG7 + inhibitor or sh-SNHG7 + miR-146b-5p inhibitor. (B) mRNA expression of Robo1 was measured by reverse transcription-quantitative PCR. Protein expression of Robo1 was detected in (C) SW1990 and (D) AsPC-1 cells via western blotting. ^*P<0.05 vs. the respective normal groups. miR, microRNA; Robo1, roundabout homolog 1; SNHG7, small nucleolar RNA host gene 7; sh, short hairpin RNA.

Figure 8

SNHG7 silencing blocks xenograft tumor growth in vivo. Mice were injected with SW1990 cells transfected with sh-SNHG7 or sh-control. (A) Representative images of PC xenograft tissues are presented. (B) Tumor volume in treated mice. (C) Xenograft tumor weight in treated mice. mRNA expression levels of (D) SNHG7, (E) miR-146b-5p and (F) Robo1 in xenograft tumor were detected via reverse transcription-quantitative PCR. (G) Protein expression of Robo1 in xenograft tumor was measured by western blotting. ^*P<0.05 vs. the respective normal groups. miR, microRNA; Robo1, roundabout homolog 1; SNHG7, small nucleolar RNA host gene 7; sh, short hairpin RNA.