LncRNA-HAGLR motivates triple negative breast cancer progression by regulation of WNT2 via sponging miR-335-3p

Abstract

Background: Triple negative breast cancer (TNBC) is a group of highly heterogeneous mixed breast cancer at the level of gene expression profile. Therefore, it is of great clinical significance to explore the molecular mechanism of TNBC and find a targeted therapeutic approach from the molecular level.

Methods: Long non-coding RNA (lncRNA) HAGLR expression level was measured by and qRT-PCR in TNBC tissues and cell lines. EdU, MTT, wound healing and Transwell assays were performed to explore the role of HAGLR on the malignancy of TNBC cells. Luciferase assay was used to clarify the binding between miR-335-3p with HAGLR and WNT2. The tumor formation experiment in nude mice was used to explore the function of HAGLR in vivo.

Results: HAGLR was increased in TNBC tissues and cell lines. Silencing of HAGLR inhibited viability, proliferation, migration, and invasion of BT549 cells. Furthermore, HAGLR acted as a sponge of miR-335-3p and inhibited its expression. And miR-335-3p directly targeted WNT2. Functionally, forced expression of miR-335-3p or knockdown of WNT2 removed the promoted effects of lncRNA HAGLR on TNBC development. In vivo tumorigenesis experiments indicated HAGLR accelerated tumor growth via miR-335-3p/WNT2 axis.

Conclusion: Our study revealed that HAGLR promoted the growth of TNBC, which was mediated by miR-335-3p/WNT2 axis.

Keywords: WNT2; lncRNA HAGLR; miR-335-3p; triple negative breast cancer; tumor progression.

Figure 1

The expression of lncRNA HAGLR in TNBC tissues and cells. We collected 40 samples of patients diagnosed with TNBC. (A) The expression of HAGLR in para-carcinoma and cancer tissues was detected by qRT-PCR. (B) qRT-PCR analysis for HAGLR level in normal breast cell MCF10A and TNBC cell lines MDA-MB-231 and BT549. (C) According to the mean level of HAGLR in Figure 1A, 40 TNBC patients was divided into low (n = 20) and high expression group (n = 20). Kaplan-Meier curves indicated 5-year survival rate of TNBC patients. (D) Another 40 TNBC patients were collected, which includes grade 0 to grade IV of TNBC (n = 8 for each grade), and qRT-PCR was used to test HAGLR level in different grades. Data are mean ± SD; ^*P < 0.05, ^**P < 0.01.

Figure 2

Deletion of HAGLR inhibited the viability, proliferation, migration and invasion of BT549 cells. Small interfering RNA of HAGLR (si-HAGLR) and its Scramble were transfected into BT549 cells. (A) The knockdown efficiency of si- HAGLR was determined by qRT-PCR. (B) CCK8 assay was used to test viability of BT549 cells. (C) EdU assay was to detected proliferation of BT549 cells. Scale bar, 100 μm. (D) Wound healing assay was to evaluate migration of BT549 cells. Scale bar, 100 μm. (E) Transwell assay was to examine invasion of BT549 cells. Scale bar, 50 μm. Data are mean ± SD; ^*P < 0.05, ^**P < 0.01.

Figure 3

HAGLR acted as a sponge of miR-335-3p and inhibited its expression. (A) MiRanda database showing the binding sites of miR-335-3p with HAGLR, and the mutant sequence of miR-335-3p. (B) Wild type and mutant miR-335-3p was transfected into HEK293 cells with or without HAGLR, and luciferase assay was to evaluate the binding between miR-335-3p and HAGLR. (C) BT549 cells were transfected with HAGLR plasmid or si-HAGLR or its NC, the mRNA level of miR-335-3p was detected using qRT-PCR. (D) The expression of miR-335-3p in para-carcinoma and cancer tissues was detected by qRT-PCR. (E) qRT-PCR analysis for miR-335-3p level in normal breast cell MCF10A and TNBC cell lines MDA-MB-231 and BT549. Data are mean ± SD; ^*P < 0.05, Abbreviation: ns: no statistical significance.

Figure 4

WNT2 was a directed target of miR-335-3p. (A) The binding bases of miR-335-3p and WNT2 from Targetscan. (B) Wild type and mutant WNT2 was transfected into HEK293 cells with or without miR-335-3p, and luciferase assay was to evaluate the binding. BT549 cells were transfected with miR-335-3p or AMO-miR-335-3p, (C) the mRNA level and (D) the protein level of WNT2 was detected. (E–F) The expression of WNT2 in TNBC tissues and cells was determined by qRT-PCR. Data are mean ± SD; ^*P < 0.05, Abbreviation: ns: no statistical significance.

Figure 5

HAGLR promoted TNBC growth through miR-335-3p/WNT2 axis. HAGLR was transfected into BT549 cells with miR-335-3p or si-WNT2. (A) The transfection efficiency was detected using qRT-PCR. (B) CCK8 assay for cell viability of BT549 cells. (C) EdU assay for cell proliferation of BT549 cells. Scale bar, 100 μm. (D) Wound healing assay for cell migration of BT549 cells. Scale bar, 100 μm. (E) Transwell assay for cell invasion of BT549 cells. Scale bar, 50 μm. Data are mean ± SD; ^*P < 0.05 vs pcDNA3.1, ^#P < 0.05 vs HAGLR.

Figure 6

HAGLR promoted TNBC growth in vivo. 30 mice were divided into two group randomly, BT549 cells was subcutaneously injected into nude mice. 1 week later, we injected lentivirus packaged HAGLR or NC into tumors. (A) Tumor volume was measured every 7 days. (B) Tumors was isolated after 28 days of BT549 cells injection, and photos for representative tumors. (C) The mRNA of HAGLR, miR-335-3p, and WNT2 in isolated tumors were detected by qRT-PCR. Data are mean ± SD; ^*P < 0.05.