LncRNA BANCR Promotes Pancreatic Cancer Tumorigenesis via Modulating MiR-195-5p/Wnt/β-Catenin Signaling Pathway

Abstract

Long noncoding BRAF-activated noncoding RNA has been reported to be tightly associated with tumorigenesis and development in various types of cancers. However, the expression, biological function, and modulatory mechanism of BRAF-activated noncoding RNA in pancreatic cancer remained unclear. In the present work, we explored the carcinogenic activity and underlying mechanism of BRAF-activated noncoding RNA on pancreatic cancer in vitro. We identified that BRAF-activated noncoding RNA was upregulated in pancreatic cancer tissues and cell lines, and BRAF-activated noncoding RNA was related to tumor metastasis and stage. BRAF-activated noncoding RNA reinforces proliferation, invasion, and migration in PANC-1 and SW1990 cells. Moreover, miR-195-5p was downregulated in both PC tissues and cell lines. Our results based on luciferase reporter, RIP-Ago2 and qRT-PCR assays, showed that miR-195-5p was a direct target of BRAF-activated noncoding RNA. Furthermore, miR-195-5p inhibitor abrogated the effects of short-interfering BRAF-activated noncoding RNA on PANC-1 and SW1990 cell growth and invasion in vitro. We further identified that BRAF-activated noncoding RNA played a vital role in activating the Wnt/β-catenin pathway by sponging miR-195-5p. Collectively, our study showed that BRAF-activated noncoding RNA promotes pancreatic cancer tumorigenesis through miR-195-5p/Wnt/β-catenin axis may serve as a potential target for diagnostics and therapeutics in pancreatic cancer.

Keywords: Wnt/β-catenin; lncRNA BANCR; miR-195-5p; pancreatic cancer; tumorigenesis.

Figure 1.

BRAF-activated noncoding RNA is significantly upregulated in PC tissues and cell lines. (A) Relative expression of BANCR in PC tissues (n = 45) and adjacent healthy tissues (n = 45) was analyzed by qRT-PCR. (***P < .01). (B) Relative expression of BANCR in PC tissues from patients with metastasis (n = 27) and from patients without metastasis (n = 18; ***P < .01). (C) Relative expression of BANCR in PC tissues from patients with different clinical stages (stage I: n = 12; stage II: n = 8; stage III: n = 10; stage IV: n = 15; *P < .05, **P < .01, and ***P < .001 vs stage I group). (D) Relative expression of BANCR in human pancreatic ductal cell (HPNE) and PC cell lines (PANC-1, SW1990, HS766T, and CFPAC-1). **P < .01 and ***P < .001 versus HPNE group. BANCR indicates RNA BRAF-activated noncoding RNA; PC, pancreatic cancer; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 2.

BRAF-activated noncoding RNA knockdown significantly inhibits PC cell proliferation, invasion, and migration. (A) PANC-1 and SW1990 cells were transfected with si-BANCR1, si-BANCR2, or their negative controls (si-NC). The relative BANCR levels were determined by qRT-PCR following 48 hours of culture. (B) and (C) MTT assay was used to detect the cell viability of si-BANCR-transfected PANC-1 and SW1990 cells. (D) Colony formation assay was performed to clarify the cell proliferation of PANC-1 and SW1990 cells. (E) Transwell assay was used to detect the invasion capacity in PANC-1 and SW1990 cells. (F) Transwell assay was used to detect the migration capacity in PANC-1 and SW1990 cells. *P < .05 and **P < .01 compared to si-NC group. BANCR indicates BRAF-activated noncoding RNA; PC, pancreatic cancer; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 3.

BRAF-activated noncoding RNA directly targets miR-195-5p. (A) The predicated miR-195-5p binding site of BANCR (CCAT1-Wt) and the designed BANCR-Mut are indicated. (B) Relative expression of miR-195-5p in PC tissues and adjacent healthy tissues was analyzed by qRT-PCR (n = 45, **P < .01). (C) Relative expression of miR-195-5p in PC cell lines and HPNE cells was measured by qRT-PCR. The level of relative miR-195-5p expression was decreased in 4 PC cell lines, PANC-1, SW1990, HS766T, and CFPAC-1, compared to HPNE. *P < .05 and **P < .01 versus HPNE group. (D) MiR-195-5p was negatively correlated with BANCR expression. (E) Luciferase assay was used to verify the binding between miR-195-5p and BANCR. **P < .01 versus miR-NC group. (F-G) Endogenous BANCR pulldown by Ago2 upon overexpression of miR-195-5p was determined using RIP assays. **P < .01 versus miR-NC group. (H-I) PANC-1 and SW1990 cells were transfected with si-BANCR1 and si-BANCR2; the expression of miR-195-5p was examined using qRT-PCR. **P < .01 and ***P < .001 versus si-NC group. BANCR indicates BRAF-activated noncoding RNA; PC, pancreatic cancer; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 4.

BRAF-activated noncoding RNA promotes PC cell proliferation, invasion, and migration via the inhibition of miR-195-5p in pancreatic cancer. Both PANC-1 and SW1990 cells were transfected with miR-NC, miR-195-5p inhibitor, si-BANCR, and miR-195-5p inhibitor + si-BANCR. (A) and (B) Relative expression of BANCR and miR-195-5p was determined by qRT-PCR after treatment. (C) and (D) Cell viability was measured in PANC-1 and SW1990 cells using MTT assay. (E) The proliferation capacity of PANC-1 and SW1990 cells was examined using colony formation assay. (F-G) Cells invasion (G) and migration (H) were measured using Transwell assay, respectively. *P < .05 and **P < .01 versus control group; ^# P < .05 and ^## P < .01 versus si-BANCR group. BANCR indicates BRAF-activated noncoding RNA; PC, pancreatic cancer; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 5.

MiR-195-5p regulates Wnt/β-catenin signaling pathway in PC cells. The protein levels of β-catenin, c-Myc, and cyclinD1 were examined using Western blot analysis. (A) and (B) PANC-1 and SW1990 cells were treatment with miR-NC, miR-195-5p inhibitor, and miR-195-5p inhibitor + si-BANCR. *P < .05 and **P < .01 vs miR NC group; ^## P < .01 versus si-NC group; ^$ P < .01 versus si-BANCR1 group (C and D) PANC-1 and SW1990 cells were treated with miR-NC, miR-195-5p inhibitor, XAV939, and miR-195-5p inhibitor + XAV939. *P < .05 and **P < .01 versus miR NC group; ^## P < .01 versus control group; ^$ P < .01 versus XAV939 group. BANCR indicates BRAF-activated noncoding RNA; PC, pancreatic cancer; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 6.

BANCR regulates Wnt/β-catenin signaling pathway in PC cells. The protein levels of β-catenin, c-Myc, and cyclinD1 were examined using Western blot analysis. (A and B) PANC-1 and SW1990 cells were treated with BANCR overexpression NC, BANCR overexpression, XAV939, and BANCR overexpression + XAV939. *P < .05 and **P < .01 vs BANCR-NC group; ^## P < .01 vs control group; ^$ P < .01 vs XAV939 group. BANCR indicates long non-coding RNA BRAF-activated noncoding RNA; NC, negative control; PC, pancreatic cancer; qRT-PCR, quantitative real-time polymerase chain reaction.