LncRNA CASC9 facilitates papillary thyroid cancer development and doxorubicin resistance via miR-28-3p/BCL-2 axis and PI3K/AKT signaling pathway

Abstract

Background: Papillary thyroid cancer (PTC) is a malignant tumor that poses a serious threat to human health. LncRNA CASC9 serves as an oncogene in numerous tumors. The purpose of this study was to explore the mechanism of lncRNA CASC9 regulating doxorubicin (Dox) resistance in PTC.

Methods: The expression of CASC9, miR-28-3p and BCL-2 in PTC tissues or dox-resistant cells was determined by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot (WB). CCK-8, colony formation assay, flow cytometry and transwell assay were used to measure the semi-inhibitory concentration (IC50) of dox, cell proliferation, apoptosis and migration, respectively. Dual luciferase reporter gene assays were performed to verify the targeting relationship between miR-28-3p and CASC9 or BCL-2. Rescue experiments were applied to verify the mechanism of CASC9. Finally, the role of CASC9 was verified by xenograft modeling in vivo.

Results: We discovered that CASC9 was enhanced in PTC tissues, cells and Dox-resistant cells (BCPAP/Dox and K1/Dox). Furthermore, CASC9 inhibition markedly restrained the proliferation, migration and facilitated apoptosis of Dox cells. In vivo experiments also showed that silencing of CASC9 inhibited tumor growth. Meanwhile, knockdown of CASC9 sensitized PTC cells to Dox. CASC9 enhanced tumor progression by activating the PI3K/AKT signaling pathway. Furthermore, bioinformatics analysis identified miR-28-3p as a downstream target of CASC9. MiR-28-3p inhibitor reversed the impact of CASC9 knockdown in BCPAP/Dox and K1/Dox. Further studies showed that CASC9 positively regulated BCL-2 expression through miR-28-3p. miR-28-3p weakened Dox resistance, proliferation, migration and accelerated apoptosis of PTC cells via BCL-2.

Conclusion: CASC9, as an oncogenic lncRNA, has a promotional effect on Dox resistance and PTC progression via miR-28-3p/BCL-2 axis and PI3K/AKT signaling pathway.

Keywords: BCL-2; Doxorubicin; Papillary thyroid cancer; lncRNA CASC9; miR-28-3p.

Fig. 1

LncRNA CASC9 is upregulated in PTC cancer tissues and cell lines. (A) The expression of CASC9 in 60 PTC tissues and paired normal tissues; (B) The expression of CASC9 in PTC cell lines including BCPAP, TPC-1, K1 and the corresponding Dox-resistant cell lines compared with normal thyroid follicular epithelial cell line NTHY-ORI 3 − 1; (C) The IC50 of BCPAP, BCPAP/Dox, TPC-1, TPC-1/Dox, K1 and K1/Dox cells to Dox. ***P < 0.001

Fig. 2

CASC9 knockdown inhibited Dox-resistance, cell proliferation and metastasis in BCPAP/Dox and K1/Dox. (A) Knockdown efficiency of CASC9 using small interfere RNA in BCPAP/Dox and K1/Dox; (B) IC50 of BCPAP/Dox and K1/Dox after CASC9 knockdown; (C) Colony ability of BCPAP/Dox and K1/Dox after CASC9 knockdown and Dox treatment; (D) Flow cytometry was used to test the apoptotic rate in BCPAP/Dox and K1/Dox after CASC9 knockdown and Dox treatment; (E) Transwell assay was used to measure the migration ability in BCPAP/Dox and K1/Dox after CASC9 knockdown and Dox treatment. **P < 0.01; ***P < 0.001

Fig. 3

LncRNA CASC9 acts in PTC by activating the PI3K/AKT signaling pathway. (A) WB analysis showed the detection of phosphorylated PI3K, AKT levels in PTC Dox cells after treatment with silencing lncRNA CASC9 or PI3K/AKT pathway activator 740 Y-P. (B) Quantification of phosphorylated PI3K, AKT levels.  **P＜0.01, **P＜0.001

Fig. 4

miR-28-3p is a potential target of CASC9. (A) The potential binding site between CASC9 and miR-28-3p was predicted by Starbase; (B) Luciferase assay was used to confirm the binding relationship between CASC9 and miR-28-3p; (C) The expression of miR-28-3p in 60 PTC tissues and paired normal tissues; (D) qRT-PCR was used to measure the expression of miR-28-3p in BCPAP, BCPAP/Dox, K1 and K1/Dox cells. (E) Spearman’s correlation analysis for expression of CASC9 and miR-28-3p in 60 PTC tissues. **P < 0.01; ***P < 0.001

Fig. 5

miR-28-3p inhibitor reversed the effects of CASC9 knockdown in PTC cell lines. (A) The expression of miR-28-3p after CASC9 knockdown or/and miR-28-3p inhibited with Dox treatment; (B-E) IC50, Colony ability, apoptotic rate and migration ability of BCPAP/Dox and K1/Dox after different treatment as indicated. **P < 0.01; ***P < 0.001

Fig. 6

CASC9 regulates BCL-2 expression via miR-28-3p. (A) The binding region of 3’ UTR of BCL-2 and miR-28-3p; (B) Luciferase assay was used to confirm the binding relationship between miR-28-3p and BCL-2; (C) BCL-2 protein expression in PTC patient tissues and adjacent normal tissues; (D) BCL-2 protein expression in NTHY-ORI 3 − 1, BCPAP, BCPAP/Dox, K1 and K1/Dox cells; (E) BCL-2 protein expression after CASC9 knockdown or/and miR-28-3p inhibited. **P < 0.01; ***P < 0.001

Fig. 7

miR-28-3p plays an important role in Dox-resistance, cell proliferation and metastasis of Dox-resistant via regulating BCL-2. BCPAP/Dox and K1/Dox cells were transfected with miR-NC, miR-28-3p, miR-28-3p + NC, miR-28-3p + BCL-2 while treated with Dox. (A) BCL-2 expression in different groups; (B-E) IC50, Colony ability, apoptotic rate and migration ability of BCPAP/Dox and K1/Dox after different treatment as indicated. **P < 0.01; ***P < 0.001

Fig. 8

Knockdown of CASC9 inhibits tumor growthin vivo. (A) Representative pictures of tumors. (B) Volume changes of tumors. (C) Weight change of tumors. **P < 0.01; ***P < 0.001