miR-623 suppresses cell proliferation, migration and invasion through direct inhibition of XRCC5 in breast cancer

Abstract

Background/aims: MicroRNAs (miRNAs) are short, non-coding RNA molecules that control gene expression trough negative translational regulation. MiR-623 is a tumor suppressor, and it's function and mechanism in breast cancer has not been reported.

Results: Exogenous overexpression of miR-623 suppressed cell proliferation, migration and invasion, meanwhile, but promoted cell apoptosis. MiR-623 knockdown displayed opposite results. Overexpression of miR-623 resulted in the downregulation of CDK4/6 as well as the inhibition of the phosphatidylinositol-3-kinase (PI3K)/Akt and Wnt/β-Catenin signaling pathways. MiR-623 knockdown displayed opposite results. Results of the reporter assay revealed that the luciferase activity was decreased in XRCC5-wt cells, suggesting that miR-623 could directly combine with 3' UTR of XRCC5. MiR-623 significantly suppressed XRCC5 expression, which is critical for miR-623-induced proliferation and migration block in breast cancer cells.

Conclusion: miR-623 suppressed cell proliferation, migration and invasion through downregulation of cyclin dependent kinases and inhibition of the phosphatidylinositol-3-kinase (PI3K)/Akt and Wnt/β-Catenin pathways by targeting XRCC5.

Methods: miR-623 was either overexpressed in breast cancer cell lines through exogenous transfection or knocked down by specific siRNA. Cell proliferation, migration and invasion were examined using CCK-8, colony formation and transwell assay. The direct target of miR-623 was verified using luciferase reporter gene assay.

Keywords: XRCC5; breast cancer; microRNA; migration; proliferation.

Figure 1

miR-623 inhibited cell growth. (A) Expression levels of miR-623 were detected in human normal mammary cell line MCF-10A and breast cancer cell lines BT474, MDA-MB-231, MDA-MB-453 and MCF-7 by real-time PCR. MiR-623 expression in MDA-MB-453 cells (B) and MCF7 cells (C) was determined by real-time PCR after transfection. Cell proliferation of MDA-MB-453 (D) and MCF7 (E) after the suppression or overexpression of miR-623 was evaluated by CCK-8 assay. (F–H) Colony formation assay of MDA-MB-453 and MCF7 cells showed that miR-623 knockdown inhibited the proliferation, whereas miR-623overexpression promoted the proliferation in breast cancer cell lines. One of the three repeated experiments with identical results was shown. P values were determined using Student’s t-tests. *P<0.05 compared with O-NC. #P<0.05 compared with siNC.

Figure 2

miR-623 inhibited the expression of cell cycle proteins. The levels of CDK4 and CDK6 in MDA-MB-453 cells (A) and MCF7 cells (C) were detected using western blot assay. The level of CDK4/6 in MDA-MB-453 cells (B) and MCF7 cells (D). GAPDH was the internal control. Relative amounts of proteins normalized to GAPDH were shown. Experiments showing identical results were performed twice. P values were determined using Student’s t-tests. *P<0.05 compared with O-NC. #P<0.05 compared with siNC.

Figure 3

Effects of miR-623 on cell migration and invasion. The migration and invasion of MDA-MB-453 cells (A–C) and MCF7 cells (D–F) were analyzed by transwell migration assays and matrigel invasion assays, respectively. 10% FBS was used as the chemoattractant. Results are represented from three independent experiments. P values were determined using Student’s t-tests. *P<0.05 compared with O-NC. #P<0.05 compared with siNC.

Figure 4

Effects of miR-623 on cell apoptosis. (A–C) The apoptosis of MDA-MB-453 and MCF7 cells was determined using double staining with annexin V/propidium iodide (PI) by flow cytometry. (D–G)The protein levels of apoptosis-related genes in MDA-MB-453 cells and MCF7 cells were detected by western blot assay. GAPDH was the internal control. Relative amounts of proteins normalized to GAPDH were shown. Experiments showing identical results were performed three times. *P<0.05. P values were determined using Student’s t-tests. *P<0.05 compared with O-NC. #P<0.05 compared with siNC.

Figure 5

Effects of miR-623 on PI3K/AKT and Wnt/β-catenin signaling pathways. (A–C) Protein expressions of AKT, p-AKT, mTOR, p-mTOR, Cyclin D1, S6K/P70 in MDA-MB-453 and MCF7 cells were detected by western blot assay. (D–F) Protein expression levels of wnt3, β-catenin, E-cad, Notch1, and HEY1 in MDA-MB-453 and MCF7 cells were detected by western blot assay. GAPDH was the internal control. Relative amounts of proteins normalized to GAPDH were shown. Experiments showing identical results were performed three times. P values were determined using Student’s t-tests. *P<0.05 compared with O-NC. #P<0.05 compared with siNC.

Figure 6

miR-623 inhibited XRCC5 expression and blocked cell growth by targeting XRCC5. (A) miR-623 and XRCC5 binding sites predicted by targetscan. (B) MDA-MB-453 and MCF7 cells were transfected with reporter plasmids followed by transfection with pCMV-miR-623 or pCMV-miR empty vector. The luciferase expression was analyzed as described in the “Material and Methods” section. (C, D) The XRCC5 expression was detected in NC and miR-623 cells using western blot. GAPDH was the internal control. The proliferation of (E) MDA-MB-453 cells and (F) MCF7 cells were confirmed by CCK8 assay to investigate the target of miR-623-induced cell growth block. (G, H) Transwell assay was performed to confirm the cell migration in XRCC5 and XRCC5+miR-623 cells. (I) The expression levels of protein in PI3K/AKT and Wnt/β-catenin signaling pathways were detected by western blot in (J) MDA-MB-453 cells and (K) MCF7 cells *P < 0.05 vs. XRCC5; # P < 0.05 vs. NC. P values were determined using Student’s t-tests.