MicroRNA-340-5p modulates cisplatin resistance by targeting LPAATβ in osteosarcoma

Abstract

MicroRNAs (miRNAs) play an important role in drug resistance and modulate the efficiency of chemotherapy. A recent study indicated that miR-340 functions as a tumor suppressor in various types of cancer. However, the role of miR-340 in chemotherapy has not been reported yet. In this study, we found that miR-340 enhanced cisplatin (CDDP)-induced cell death. Induction of miR-340-5p expression decreased the IC50 of CDDP and increased the apoptosis of CDDP-resistant MG-63 and Saos-2 cells. Moreover, miR-340-5p decreased the accumulation of MRP1 and MDR1. We further explored the mechanism underlying the promoting effects of miR-340-5p on CDDP-induced cell death. We identified a potential target of miR-340 in the 3' untranslated region of lysophosphatidic acid acyltransferase (LPAATβ) using the online program Targetscan (http://www.microrna.org). Luciferase reporter assays showed that miR-340 binds to the 3'UTR of LPAATβ. Enforced expression of miR-340-5p decreased the accumulation of LPAATβ in both MG-63 and Saos-2 cells. Silencing LPAATβ decreased the IC50 of CDDP and increased the apoptosis of CDDP-resistant MG-63 and Saos-2 cells, which is consistent with the effect of miR-340-5p on CDDP-induced cell death. Moreover, induced expression of LPAATβ compromised the effects of miR-340-5p on CDDP-induced cell death and accumulation of MRP1 and MDR1. Taken together, our data indicated that miR-340-5p enhanced the sensitivity to CDDP by targeting LPAATβ.

Figure 1. miR-340-5p is down-regulated in cisplatin (CDDP)-resistant osteosarcoma cells. The expression of miR-340-5p was examined in MG-63, Saos-2, MG-63/CDDP, and Saos-2/CDDP cells. U6 served as an internal reference. The relative expression of miR-340-5p was calculated using the 2^-ΔΔCT method. All experiments were repeated three times. Data are reported as means±SD. **P<0.05 compared to its respective control (Student’s t-test).

Figure 2. miR-340-5p enhanced sensitivity of cisplatin (CDDP) in osteosarcoma cells. A, Relative RNA expression of miR-340-5p in MG-63/CDDP and Saos-2/CDDP cells transfected with miR-340-5p or normal control (NC). B, miR-340-5p decreased IC₅₀ of CDDP in MG-63/CDDP and Saos-2/CDDP cells. C, miR-340-5p increased CDDP-induced apoptosis in MG-63/CDDP and Saos-2/CDDP cells. Data are reported as means±SD. **P<0.01 (Student’s t-test). D, miR-340-5p decreased the expression of MRP1 and MDR1 in MG-63/CDDP and Saos-2/CDDP cells exposed to CDDP.

Figure 3. A, Schematic diagram of interaction between miR-340-5p and LPAATβ. B, Dual luciferase assay of cells co-transfected with luciferase reporter constructs and miR-340-5p or normal control (NC). C, miR-340-5p did not affect mRNA expression of LPAATβ in MG-63/CDDP and Saos-2/CDDP cells. Data are reported as means±SD. **P<0.01 (Student’s t-test). D, miR-340-5p decreased protein expression of LPAATβ in MG-63/CDDP and Saos-2/CDDP cells.

Figure 4. LPAATβ was up-regulated in cisplatin (CDDP)-resistant osteosarcoma cells. The expression of LPAATβ was examined in MG-63, Saos-2, MG-63/CDDP, and Saos-2/CDDP cells using western blot analysis. GAPDH served as loading control. Upper: representative image of western blot analysis. Lower: histogram represents the densitometry data for at least 3 independent experiments. Data are reported as means±SD. **P<0.01, compared to its respective control (Student’s t-test).

Figure 5. Effect of silencing LPAATβ on cisplatin (CDDP) resistance. A, Relative protein expression of LPAATβ in MG-63/CDDP and Saos-2/CDDP cells transfected with siRNA or normal control (NC). B, Silencing LPAATβ decreased IC₅₀ of CDDP in MG-63/CDDP and Saos-2/CDDP cells. C, Silencing LPAATβ increased CDDP-induced apoptosis in MG-63/CDDP and Saos-2/CDDP cells. Data are reported as means±SD. **P<0.01, compared to its respective control (Student’s t-test). D, Silencing LPAATβ decreased the expression of MRP1 and MDR1 in MG-63/CDDP and Saos-2/CDDP cells exposed to CDDP.

Figure 6. Expression of LPAATβ attenuated inhibitory effects of miR-340-5p on cisplatin (CDDP) resistance. A, Expression of LPAATβ in MG-63/CDDP and Saos-2/CDDP cells co-transfected with miR-340-5p and pcDNA-LPAATβ or pcDNA3.0. B, Expression of LPAATβ compromised inhibitory effects of miR-340-5p on IC50 of CDDP in MG-63/CDDP and Saos-2/CDDP cells. C, Expression of LPAATβ attenuated promoting effects of miR-340-5p on CDDP-induced apoptosis in MG-63/CDDP and Saos-2/CDDP cells. Data are reported as means±SD. **P<0.01 (Student’s t-test). D, Expression of LPAATβ compromised inhibitory effects of miR-340-5p on expression of MRP1 and MDR1 in MG-63/CDDP and Saos-2/CDDP cells exposed to CDDP.