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.