Silencing of long non-coding RNA SNHG15 suppresses proliferation, migration and invasion of pancreatic cancer cells by regulating the microRNA-345-5p/RAB27B axis

Abstract

Pancreatic cancer (PC) is the seventh most common cause of cancer-associated mortality worldwide. The current study aimed to investigate the function and molecular mechanism underlying long non-coding (lnc)RNA SNHG15 in PC tissues and cells. Relative expression levels of lncRNA SNHG15, miR-345-5p and RAB27B in PC cells and tissues were examined by performing reverse transcription-quantitative PCR. The association between SNHG15, miR-345-5p and RAB27B was validated using a Dual-luciferase reporter assay. Proliferation, invasion and migration of PC cells were analysed by conducting MTT, wound healing and Transwell assays. Western blotting was performed to detect the relative expression of the RAB27B protein. The relative expression level of lncRNA SNHG15 and RAB27B was elevated, but that of miR-345-5p was decreased in PC. Silencing of SNHG15 suppressed the proliferation, invasion and migration of PC cells in vitro and suppressed tumour growth in xenograft mice in vivo. miR-345-5p was the target gene of SNHG15 and suppressed cell proliferation, migration and invasion in PC. Furthermore, miR-345-5p targeted RAB27B. The use of miR-345-5p inhibitor or overexpression of RAB27B reversed the suppressive effect of the small interfering RNA si-SNHG15-1 exerted on the proliferation, invasion and migration of PC cells. Silencing of SNHG15 inhibited the proliferation, invasion and migration of PC cells by mediating the miR-345-5p/RAB27B axis, thereby implying its potential as a prognostic marker and target for PC therapy.

Keywords: RAB27B; SNHG15; long non-coding RNA; microRNA-345-5p; pancreatic cancer.

Figure 1

Dysregulation of long non-coding RNA SNHG15 in pancreatic cancer. (A) Relative expression of SNHG15 in PAAD tissues compared with non-cancerous tissues from The Cancer Genome Atlas database. ^*P<0.05, vs. N. (B) Relative expression of SNHG15 in tumour tissues and adjacent non-cancerous tissues. ^**P<0.01, vs. adjacent non-cancerous tissues. (C) Relative expression of SNHG15 in tumours at Tumour Node Metastasis stages I/II and III/IV was determined by RT-qPCR. ^**P<0.01, vs. I/II. (D) Relative expression of SNHG15 in HPDE6, BXPC-3 and PANC-1 cells determined by RT-qPCR. ^**P<0.01, vs. HPDE6. PAAD, pancreatic adenocarcinoma; N, non-cancerous tissues; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Figure 2

Knockdown of long non-coding RNA SNHG15 inhibits proliferation, migration and invasion of pancreatic cancer cells. (A) Relative expression of SNHG15 in BXPC-3 and PANC-1 cells after transfection with si-SNHG15-1, si-SNHG15-2 and si-NC was detected by reverse transcription-quantitative polymerase chain reaction. ^**P<0.01, vs. si-NC. (B) Cell viability in BXPC-3 and PANC-1 cells was detected by 3-(4, 5-Dimethyl-2-Thiazolyl)-2, 5-Diphenyl-2-H-Tetrazolium Bromide assay. ^*P<0.05, ^**P<0.01, vs. si-NC. (C) Wound-healing rate of BXPC-3 and PANC-1 cells was detected by wound-healing assay. ^**P<0.01, vs. si-NC. (D) Number of invading cells was detected by Transwell assay. ^**P<0.01, vs. si-NC. (E) The growth of tumour xenograft in mice injected with PANC-1 cells transfected with Lv-si-SNHG15-1 or Lv-si-NC. ^**P<0.01, vs. Lv-si-NC. (F) The expression levels of SNHG15, miR-345-5p and RAB27B in tumour xenograft after injection with Lv-si-SNHG15-1 or Lv-si-NC. ^**P<0.01, vs. Lv-si-NC. si-, small interfering RNA-; NC, negative control; miR, microRNA; Lv, lentivirus vector.

Figure 3

miR-345-5p is the target gene of SNHG15. (A) The binding sequence between SNHG15 and miR-345-5p was predicted by StarBase ver2.0. (B) Relative expression of miR-345-5p was detected by RT-qPCR. ^**P<0.01, vs. si-NC. (C) The interaction between SNHG15 and miR-345-5p in BXPC-3 and PANC-1 cells was validated by dual-luciferase reporter assay. ^**P<0.01, vs. miR-NC. (D) Relative expression of miR-345-5p was detected by RT-qPCR in tumour tissues and adjacent non-cancerous tissues. ^**P<0.001, vs. adjacent non-cancerous tissues. (E) Correlation analysis of SNHG15 and miR-345-5p in tumour tissues. (F) Relative expression of miR-345-5p in HPDE6, BXPC-3 and PANC-1 cells detected by RT-qPCR. ^**P<0.01, vs. HPDE6. miR, microRNA; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; si-, small interfering RNA-; NC, negative control; WT, wild type; MUT, mutant.

Figure 4

miR-345-5p inhibits proliferation, migration and invasion of pancreatic cancer cells. (A) Relative expression of miR-345-5p was detected by reverse transcription-quantitative polymerase chain reaction. ^**P<0.01, vs. miR-NC, ^##P<0.01, vs. inhibitor NC. (B) The cell viability was determined by 3-(4, 5-Dimethyl-2-Thiazolyl)-2, 5-Diphenyl-2-H-Tetrazolium Bromide assay in BXPC-3 and PANC-1 cells. ^**P<0.01, vs. miR-NC. (C) Wound-healing rate of BXPC-3 and PANC-1 cells was determined by wound-healing assay. ^**P<0.01, vs. miR-NC. (D) Number of invasion cells in BXPC-3 and PANC-1 cells was detected by Transwell assay. ^**P<0.01, vs. miR-NC. miR, microRNA; NC, negative control.

Figure 5

miR-345-5p can target RAB27B. (A) The binding site and mutant sites between miR-345-5p and RAB27B was predicted by miRDB. (B) Dual-luciferase reporter assay was used to confirm the targeting association between miR-345-5p and RAB27B in BXPC-3 and PANC-1 cells. ^**P<0.01, vs. miR-NC. (C) Relative expression of RAB27B in PAAD tissues compared with non-cancerous tissues from The Caner Genome Atlas database. ^*P<0.05, vs. non-cancerous tissues (N). (D) RT-qPCR was used to detect the expression of RAB27B in tumour tissues and adjacent non-cancerous tissues. ^**P<0.01, vs. adjacent non-cancerous tissues. (E) The correlation between SNHG15 and RAB27B in PC tissues was analysed by Pearson's correlation analysis. (F) The association between RAB27B and miR-345-5p in pancreatic cancer tissues was analysed by Pearson's correlation analysis. (G) Relative expression of RAB27B in HPDE6, BXPC-3 and PANC-1 cells was detected by RT-qPCR. ^**P<0.01, vs. HPDE6. miR, microRNA; NC, negative control; PAAD, pancreatic adenocarcinoma; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; WT, wild type; MUT, mutant.

Figure 6

Long non-coding RNA SNHG15 regulates cell proliferation, migration and invasion through miR-345-5p/RAB27B axis. (A) Protein expression of RAB27B was detected by western blotting. ^**P<0.01 vs. si-NC. ^##P<0.01 vs. miR-NC. ^&&P<0.01 vs. pcDNA-NC. (B) Cell viability was determined by 3-(4, 5-Dimethyl-2-Thiazolyl)-2, 5-Diphenyl-2-H-Tetrazolium Bromide assay in PANC-1 cells. ^*P<0.05, ^**P<0.01, vs. si-NC. ^#P<0.05, vs. si-SNHG15-1. (C) Cell migration was determined by wound-healing assay. ^**P<0.01, vs. si-NC. ^##P<0.01, vs. si-SNHG15-1. (D) Cell invasion in PANC-1 cells was detected by Transwell assay. ^**P<0.01, vs. si-NC. ^##P<0.01, vs. si-SNHG15-1. miR, microRNA; NC, negative control; si-, small interfering RNA-.