Aspirin enhances the sensitivity of hepatocellular carcinoma side population cells to doxorubicin via miR-491/ABCG2

Abstract

Objective: To explore whether aspirin (ASA) enhances the sensitivity of hepatocellular carcinoma (HCC) side population (SP) cells to doxorubicin (Doxo) via miR-491/ATP-binding cassette sub-family G member 2 (ABCG2).Methods: Non-SP and SP cells were isolated from MHCC-97L cell line using flow cytometry analysis and fluorescence-activated cell sorting. Colony formation assay was performed to determine the colony-formation ability of cells. Cell viability of SP cells was determined with the MTT assay. Luciferase reporter assay was applied in confirming the binding between miR-491 and ABCG2.Results: Although the Doxo treatment lowered the colony-formation ability of both non-SP and SP cells, the colony-formation ability of SP cells was 2-fold higher than that of non-SP cells (P<0.05). Doxo slightly inhibited the cell viability of SP cells in a concentration-dependent manner; the addition of ASA dramatically enhanced the inhibitory effect of Doxo on SP cell viability in a concentration-dependent manner (P<0.05). Compared with non-SP cells, the miR-491 expression was significantly decreased in SP cells, which was significantly reversed by ASA (P<0.05). miR-491 directly controlled the ABCG2 expression. In the presence of Doxo, miR-491 inhibitor reduced the inhibitory effect of ASA on the cell viability of SP cells, which was significantly reversed by knockdown of ABCG2 (P<0.05).Conclusion: ASA enhanced the sensitivity of SP cells to Doxo via regulating the miR-491/ABCG2 signaling pathway.

Keywords: ATP-binding cassette sub-family G member 2 (ABCG2); Aspirin (ASA); doxorubicin (Doxo); hepatocellular carcinoma (HCC); miR-491; side population (SP).

Figure 1. Effect of ASA on drug resistance of HCC SP cells

Flow cytometry analysis and fluorescence-activated cell sorting were performed to isolate non-SP and SP cells from the HCC cell line MHCC-97L. (A) The colony-formation ability of non-SP and SP cells was determined in the absence or presence of Doxo (500 ng/ml); ^**P<0.01, compared with Non-SP cells. (B) The cell viability of SP cells treated with different concentrations of ASA (0, 1.25, 2.5, and 5 μmol/ml) for 48 h in the presence of Doxo (500 ng/ml) was analyzed by MTT assay; ^*P<0.05, ^**P<0.01, compared with control.

Figure 2. Decreased miR-491 expression in SP cells was reversed by ASA

ASA (2.5 μmol/ml) was used to treat MHCC-97L SP cells. There were three groups as follows: non-SP group, SP group, and SP + ASA group. (A) The expression of multiple miRNAs (miR-130b-3p, miR-7-5p, miR-491, miR-612, and miR-3650), confirmed by qPCR; ^*P<0.05, ^**P<0.01, compared with non-SP cells; ^#P<0.05, compared with SP. (B) The expression of ABCG2, determined by Western blotting. β-Actin was used as the loading control.

Figure 3. ASA enhanced the sensitivity of SP cells to Doxo via miR-491

The SP cells were divided into five groups (Control, Doxo, Doxo + ASA, Doxo + ASA + NC, and Doxo + ASA + miR-491 inhibitor). The concentration of Doxo was 500 ng/ml, and ASA was 2.5 μmol/ml. (A) The cell viability of SP cells; ^*P<0.05, compared with Doxo; ^#P<0.05, compared with Doxo + ASA + NC. (B) The expression of ABCG2.

Figure 4. miR-491 directly controlled the ABCG2 expression in SP cells

(A) The miR-491-binding site existed in the 3′UTR of ABCG2 mRNA. (B) The effect of miR-491 mimic or inhibitor on the luciferase activity of wild-type ABCG2 3′UTR and mutant ABCG2 3′UTR. (C) The effect of miR-491 mimic or inhibitor on the mRNA and protein expression of ABCG2. ^*P<0.05 and ^**P<0.01, compared with pre-NC or NC.

Figure 5. ASA enhanced the sensitivity of SP cells to Doxo via miR-491/ABCG2

The SP cells were allocated to five groups (Doxo, Doxo + ASA, Doxo + ASA + NC, Doxo + ASA + miR-491 inhibitor, Doxo + ASA + miR-491 inhibitor + si-control, and Doxo + ASA + miR-491 inhibitor + siRNA-ABCG2). The concentration of Doxo was 500 ng/ml, and ASA was 2.5 μmol/ml. (A) The cell viability of SP cells; ^**P<0.01, compared with Doxo; ^##P<0.01, compared with Doxo + ASA + NC; ^&&P<0.01, compared with Doxo + ASA + miR-491 inhibitor + si-control. (B) The protein expression of ABCG2 in SP cells.