BANCR positively regulates the HIF-1α/VEGF-C/VEGFR-3 pathway in a hypoxic microenvironment to promote lymphangiogenesis in pancreatic cancer cells

Abstract

The aim of the present study was to explore the effects of BRAF-activated non-protein coding RNA (BANCR) on pancreatic microlymphangiogenesis in pancreatic cancer (PC) and its molecular mechanism under hypoxic conditions. Reverse transcription-quantitative PCR (RT-qPCR) was used to detect the expression of BANCR in SW1990 and PANC-1 PC cell lines under normoxic and hypoxic conditions. Subsequently, the expression of BANCR in the PC cells was knocked down using small interfering RNAs (siRNAs). Western blotting and RT-qPCR analyses were performed to detect the expression of hypoxia-inducible factor (HIF-1α), VEGF-C and VEGFR-3 in the transfected cells. In addition, the transfected PC cells were co-cultured with human lymphatic endothelial cells and the lymphatic microvessel density (MLVD) was detected under normal and hypoxic conditions. Furthermore, HIF-1α expression in the PC cells was knocked down using siRNAs, and VEGF-C and VEGFR-3 mRNA expression in the HIF-1α knockdown cells was detected using RT-qPCR. The results showed that the expression of BANCR in the SW1990 and PANC-1 PC cell lines was significantly higher than that in human pancreatic duct endothelial cells. Additionally, the expression of BANCR was significantly increased in PC cells under hypoxic conditions compared with normoxic conditions. The MLVD of PC cells under hypoxic conditions was significantly higher compared with that under normoxic conditions, and the MLVD in the si-BANCR group was lower than that in the si-NC group, indicating that si-BANCR downregulated MLVD. These results indicate that BANCR positively regulated the expression of HIF-1α in PC cells at the transcriptional and translational levels. Finally, the expression levels of VEGF-C and VEGFR-3 in PC cells were significantly reduced when BANCR or HIF-1α expression was knocked down. In conclusion, the results demonstrate that the expression of BANCR in PC cells was significantly increased under hypoxic conditions and suggest that BANCR promoted tumor cell lymphangiogenesis by upregulating the HIF-1α/VEGF-C/VEGFR-3 pathway, which plays an important role in the process of PC lymph node metastasis.

Keywords: hypoxic microenvironment; long non-coding RNA; lymphangiogenesis; pancreatic cancer.

Figure 1.

BANCR mRNA expression in PANC-1 and SW1990 pancreatic cells and HPDCs. ^*P<0.05 vs. HPDCs. BANCR, BRAF-activated non-protein coding RNA; HPDCs, human pancreatic ductal epithelial cells.

Figure 2.

Relative BANCR expression in PANC-1 and SW1990 pancreatic cancer cells under normoxic and hypoxic conditions. ^*P<0.05 vs. the respective normoxia group. BANCR, BRAF-activated non-protein coding RNA.

Figure 3.

Effect of BANCR knockdown on MLVD in PANC-1 and SW1990 pancreatic cancer cells under normoxic and hypoxic conditions. (A) Representative images (magnification, ×100) and (B) quantitative analysis of MLVD in si-BANCR and si-NC transfected PANC-1 cells under normoxic and hypoxic conditions. (C) Representative images (magnification, ×100) and (D) quantitative analysis of MLVD in si-BANCR and si-NC transfected SW1990 cells under normoxic and hypoxic conditions. ^*P<0.05 vs. the respective normoxia group. BANCR, BRAF-activated non-protein coding RNA; MLVD, lymphatic microvessel density; si, small interfering; NC, negative control.

Figure 4.

Effect of BANCR knockdown on HIF-1α protein and mRNA expression in PANC-1 and SW1990 PC cells under hypoxic conditions. (A) Western blots and (B) quantitative analysis of HIF-1α expression in PC cells. (C) HIF-1α mRNA expression levels in the treated PC cells. ^*P<0.05 vs. the respective si-NC group. BANCR, BRAF-activated non-protein coding RNA; HIF, hypoxia-inducible factor; PC, pancreatic cancer; si, small interfering; NC, negative control.

Figure 5.

BANCR and HIF-1α upregulate the expression of VEGF-C and VEGFR-3 in PC cells. (A) Western blots, (B) quantitative western blot analysis and (C) RT-qPCR analysis of the VEGF-C and VEGFR-3 expression levels in SW1990 PC cells transfected with si-BANCR or si-NC. (D) Western blots, (E) quantitative western blot analysis and (F) RT-qPCR analysis of the VEGF-C and VEGFR-3 expression levels in SW1990 PC cells transfected with si-HIF-1α or si-NC. ^*P<0.05 and ^**P<0.01 vs. the respective si-NC group. BANCR, BRAF-activated non-protein coding RNA; PC, pancreatic cancer; RT-qPCR, reverse transcription-quantitative PCR; si, small interfering.