BRAF-activated long non-coding RNA contributes to cell proliferation and activates autophagy in papillary thyroid carcinoma

Abstract

Long non-coding RNAs (lncRNAs) are novel regulators in cancer biology. BRAF-activated lncRNA (BANCR) is overexpressed in melanoma and has a potential functional role in melanoma cell migration. However, little is known about the role of BANCR in the development of papillary thyroid carcinoma (PTC). In the present study, BANCR expression was examined in six pairs of PTC and matched adjacent normal tissues. The results revealed that BANCR levels were significantly higher in the PTC tissues and PTC IHH-4 cells compared with the normal controls. Knockdown of BANCR in the IHH-4 cells inhibited proliferation and increased apoptosis of the cells in vitro. Further investigation of the underlying mechanisms revealed that BANCR markedly activated autophagy. Overexpression of BANCR inhibited apoptosis in the IHH-4 cells, whereas inhibition of autophagy stimulated apoptosis in the BANCR-overexpressed cells. BANCR overexpression also increased cell proliferation and the inhibition of autophagy abrogated BANCR overexpression-induced cell proliferation. In addition, the overexpression of BANCR resulted in an increase in the ratio of LC3-II/LC3-I, a marker for autophagy, while the knockdown of BANCR decreased the ratio of LC3-II/LC3-I. These results revealed that BANCR expression levels are upregulated in PTC. Additionally, BANCR increases PTC cell proliferation, which could activate autophagy.

Keywords: BRAF-activated long non-coding RNA; autophagy; long non-coding RNA; papillary thyroid carcinoma.

Figure 1

BANCR levels are upregulated in papillary thyroid carcinoma (PTC). Reverse transcription-polymerase chain reaction results showed that BANCR expression was significantly higher in five out of six of the tumor tissues compared with the adjacent normal tissues. The BANCR level in the PTC IHH-4 cell line was also upregulated compared with the mean expression level of the adjacent normal tissues. BANCR expression levels were normalized to β-actin. Data are presented as the mean ± standard deviation (^*P<0.05). BANCR, BRAF-activated long non-coding RNA; N, normal tissue; T, tumorous tissue.

Figure 2

BANCR-knockdown inhibits cell proliferation and increases apoptosis in papillary thyroid carcinoma IHH-4 cells. (A) Following treatment with LV-BANCR-323 and LV-BANCR-540, BANCR expression in the IHH-4 cells was downregulated compared with the cells treated with LV-NC. (B) Cell Counting Kit-8 assays showed that BANCR-knockdown inhibited the proliferation of the IHH-4 cells. (C) Knockdown of BANCR induced apoptosis, which was detected by flow cytometry. The results are presented as the percentage of apoptotic cells in the total number of counted cells. (D) BANCR-knockdown resulted in an increase in the cell population in the G₁ phase. The data represent one of at least three independent experiments. (E) The Transwell migration assays showed that BANCR-knockdown had no significant effect on the migration of IHH-4 cells. The images represent at least three independent experiments. The graph indicates the number of migrated cells per field. The results are presented as the mean ± standard deviation (^*P<0.05). BANCR, BRAF-activated long non-coding RNA; LV-BANCR-323, lentivirus containing shRNA-323; LV-BANCR-540, lentivirus containing short haipin (sh)RNA-540; LV-NC, lentivirus negative control; 7-AAD, 7-amino-actinomycin D; FITC, fluorescein isothiocyanate.

Figure 3

Overexpression of BANCR increases autophagy activation in papillary thyroid carcinoma IHH-4 cells.(A) Following treatment with LV-BANCR, BANCR expression in IHH-4 cells was upregulated compared with the cells treated with LV-NC. (B) Cell Counting Kit-8 assays showed that the overexpression of BANCR promoted cell growth, whereas the inhibition of autophagy inhibited the proliferation of the IHH-4 cells. (C) Overexpression of BANCR inhibited cell apoptosis, whereas autophagy inhibition increased apoptosis in the IHH-4 cells, which was detected by flow cytometry. (D) Overexpression of BANCR decreased the cell population in the G₁ phase, whereas autophagy inhibition increased the cell population in the G₁ phase. The data represent one of at least three independent experiments. (E) Western blotting results revealed that BANCR overexpression resulted in an increase in the ratio of LC3-II/LC3-I, while knockdown of BANCR and treatment with 3-MA decreased the ratio of LC3-II/LC3-I. (F) Reverse transcription polymerase chain reaction results showed that BANCR overexpression resulted in an increase in the LC3 mRNA level, while knockdown of BANCR and treatment with 3-MA decreased LC3 mRNA level. The results are presented as the mean ± standard deviation (^*P<0.05). BANCR, BRAF-activated long non-coding RNA; LV-BANCR, lentivirus containing human full-length BANCR cDNA; LV-BANCR-323, LV containing short hairpin (sh)RNA-323; LV-BANCR-540, LV containing shRNA-540; LV-NC, LV negative control; 3-MA, 3-methyladenine; 7-AAD, 7-amino-actinomycin D; FITC, fluorescein isothiocyanate.