LINC00504 Promotes the Malignant Biological Behavior of Breast Cancer Cells by Upregulating HMGB3 via Decoying MicroRNA-876-3p

Abstract

Purpose: Long intergenic non-protein coding RNA 504 (LINC00504) is a long non-coding RNA that has an important regulatory role in a variety of human cancers. In this study, LINC00504 expression in breast cancer tissues and cell lines was detected. Studies were also conducted to determine the impact of LINC00504 on the tumor behavior of breast cancer cells. The potential mechanisms underlying the oncogenic role of LINC00504 in breast cancer cells were elucidated in detail.

Methods: Expression of LINC00504 in breast cancer was analyzed by quantitative real-time polymerase chain reaction. The effects of LINC00504 on proliferation, apoptosis, in vitro migration and invasion, and in vivo tumor growth were elucidated using Cell Counting Kit-8 assay, flow cytometry, Transwell assays, and tumor xenograft models, respectively. Bioinformatics analyses in conjunction with RNA immunoprecipitation, luciferase reporter assays, and rescue experiments were conducted to investigate the underlying molecular mechanisms.

Results: LINC00504 was upregulated in breast cancer tissues and cell lines. Knocking down LINC00504 suppressed breast cancer cell proliferation, migration, and invasion and facilitated apoptosis in vitro. In addition, tumor growth in vivo was significantly inhibited by LINC00504 depletion. Regarding the underlying mechanism, LIN00504 could function as a competing endogenous RNA in breast cancer by sponging microRNA-876-3p (miR-876-3p), resulting in the upregulation of high mobility group box 3 (HMGB3). Rescue experiments further revealed that miR-876-3p downregulation or HMGB3 upregulation effectively reversed the inhibitory effects of LIN00504 deficiency on breast cancer cells.

Conclusion: The LIN00504-miR-876-3p-HMGB3 axis shows carcinogenic effects in modulating the biological behavior of breast cancer cells. This pathway may represent an effective target for CRC diagnosis and anticancer therapy.

Keywords: ceRNA; high mobility group box 3; long intergenic non-protein coding RNA 504; therapeutic target.

Figure 1

Loss of LINC00504 restricts the malignant behavior of breast cancer cells in vitro. (A) LINC00504 expression in breast cancer was analyzed using the TCGA and GTEx databases. (B) qRT-PCR was used to measure LINC00504 expression in 57 pairs of breast cancer tissues and corresponding adjacent normal tissues. (C) The expression of LINC00504 in five breast cancer cell lines (BT-474, MCF-7, MDA-MB-231, SKBR3 and T-47D) and in a human immortalized breast epithelial cell line (MCF-10A). (D and E) The correlation between LINC00504 expression and tumor stage or overall survival in breast cancer was analyzed using the TCGA and GTEx databases. (F) qRT-PCR was done to assess the transfection efficiency of si-LINC00504 in MCF-7 and MDA-MB-231 cells. (G) CCK-8 assay was applied to measure the proliferation of MCF-7 and MDA-MB-231 cells after LINC00504 silencing. (H) Apoptosis of si-LINC00504- or si-NC-transfected MCF-7 and MDA-MB-231 cells was examined by flow cytometry. (I and J) Transwell experiments were employed to measure the migration and invasion capacities of MCF-7 and MDA-MB-231 cells after si-LINC00504 or si-NC transfection. **P < 0.01.

Figure 2

LINC00504 functions as a miR-876-3p sponge in breast cancer. (A and B) lncLocator and lncATLAS were used to predict the subcellular location of LINC00504. (C) The subcellular distribution of LINC00504 in MCF-7 and MDA-MB-231 cells was determined by subcellular fractionation followed by qRT-PCR analysis. (D) Schematic diagram indicating the predicted miRNAs targeting LINC00504. (E) qRT-PCR was used to detect the expression of miR-205-5p, miR-129-5p, and miR-876-3p in MCF-7 and MDA-MB-231 cells after LINC00504 depletion. (F) MiR-876-3p expression in 57 pairs of breast cancer tissues and corresponding adjacent normal tissues as analyzed by qRT-PCR. (G) The relationship between miR-876-3p and LINC00504 expression in 57 breast cancer tissues as determined by Pearson’s correlation analysis. (H) The binding sites between LINC00504 and miR-876-3p were predicted by bioinformatics analysis. (I) Luciferase reporter assay was conducted to study the molecular interaction of LINC00504 and miR-876-3p in breast cancer cells. MCF-7 and MDA-MB-231 cells were co-transfected with LINC00504-wt or LINC00504-mut and miR-876-3p mimic or miR-NC, followed by the evaluation of luciferase activity at 48 h post-transfection. (J) RIP assay was carried out in MCF-7 and MDA-MB-231 cells, followed by qRT-PCR to measure the enrichment of LINC00504 and miR-876-3p associated with Ago2. **P < 0.01.

Figure 3

HMGB3 is a direct target of miR-876-3p and is regulated by LINC00504 in breast cancer cells. (A) Overexpression efficiency of the miR-876-3p mimic in MCF-7 and MDA-MB-231 cells as determined by qRT-PCR. (B–D) CCK-8 assay and flow cytometry were utilized to examine the effects of miR-876-3p upregulation on the proliferation and apoptosis of MCF-7 and MDA-MB-231 cells, respectively. (E and F) The migration and invasion of miR-876-3p-overexpressing MCF-7 and MDA-MB-231 cells as determined by Transwell experiments. (G) The wild-type miR-876-3p binding sequences within the 3ʹ-UTR of HMGB3. The mutant binding sequences are also shown. (H) HMGB3-wt or HMGB3-mut and miR-876-3p mimic or miR-NC was transfected into MCF-7 and MDA-MB-231 cells. After 48 h incubation, luciferase activity was detected to confirm the association between miR-876-3p and HMGB3 3ʹ-UTR. (I and J) The influences of miR-876-3p mimic transfection on HMGB3 mRNA and protein levels in MCF-7 and MDA-MB-231 cells were determined by qRT-PCR and Western blotting, respectively. (K) HMGB3 mRNA expression in 57 pairs of breast cancer tissues and corresponding adjacent normal tissues was measured by qRT-PCR. (L) Pearson’s correlation analysis was done to assess the correlation between miR-876-3p and HMGB3 mRNA levels in 57 breast cancer tissues. (M and N) qRT-PCR and Western blot analysis were performed to detect the expression of HMGB3 mRNA and protein in LINC00504-deficient MCF-7 and MDA-MB-231 cells. (O and P) si-LINC00504 in combination with the miR-876-3p inhibitor or NC inhibitor was introduced into MCF-7 and MDA-MB-231 cell. qRT-PCR and Western blot analysis were done to measure HMGB3 mRNA and protein. (Q) RIP assay was used to determine the association of LINC00504, miR-876-3p, and HMGB3 with Ago2 in MCF-7 and MDA-MB-231 cells. (R) Pearson’s correlation analysis was used to demonstrate the correlation between LINC00504 and HMGB3 mRNA expression in 57 breast cancer tissues. **P < 0.01.

Figure 4

MiR-876-3p inhibition reverses the anticancer effects of si-LINC00504 in breast cancer cells. (A) The knockdown efficiency of miR-876-3p inhibitor was determined in MCF-7 and MDA-MB-231 cells by qRT-PCR. (B and C) si-LINC00504 was co-transfected with the miR-876-3p inhibitor or NC inhibitor into MCF-7 and MDA-MB-231 cells. CCK-8 assay and flow cytometry were performed to measure the proliferation and apoptosis in the different groups, respectively. (D and E) Migration and invasion of the aforementioned cells was evaluated in Transwell experiments. *P < 0.05 and **P < 0.01.

Figure 5

Overexpression of HMGB3 abrogates the impacts of LINC00504 knockdown on breast cancer cells. (A) Western blot analysis was performed to detect HMGB3 protein expression in MCF-7 and MDA-MB-231 cells after pcDNA3.1-HMGB3 or pcDNA3.1 transfection. (B–E) si-LINC00504 in parallel with pcDNA3.1-HMGB3 or pcDNA3.1 was transfected into MCF-7 and MDA-MB-231 cells. CCK-8 assay, flow cytometry and Transwell experiments were done to determine the proliferation, apoptosis, migration, and invasion in the different groups, respectively. *P < 0.05 and **P < 0.01.

Figure 6

Knockdown of LINC00504 suppresses tumor growth of breast cancer cells in vivo. (A) Representative photographs of tumor xenografts obtained from the sh-LINC00504 and sh-NC groups. (B) The growth curves for the tumor xenografts after injection with MCF-7 cells overexpressing sh-LINC00504 and sh-NC. (C) The weight of the tumor xenografts from the sh-LINC00504 and sh-NC groups. (D and E) LINC00504 and miR-876-3p in the tumor xenografts as measured by qRT-PCR. (F) Western blot analysis indicating HMGB3 protein expression in the tumor xenografts. **P < 0.01.