MiR-122 Reverses the Doxorubicin-Resistance in Hepatocellular Carcinoma Cells through Regulating the Tumor Metabolism - PubMed (original) (raw)
MiR-122 Reverses the Doxorubicin-Resistance in Hepatocellular Carcinoma Cells through Regulating the Tumor Metabolism
Chenwei Pan et al. PLoS One. 2016.
Abstract
Doxorubicin (DOX) is one of the most commonly used anticancer drugs in the treatment of hepatoma. However, acquired drug resistance is one of the major challenges for the chemotherapy. In this study, a down-regulation of miR-122 was observed in doxorubicin-resistant Huh7 (Huh7/R) cells compared with its parental Huh7 cells, suggesting miR-122 is associated with the chemoresistance. Meanwhile, luciferase reporter assay proved that the PKM2 is the target of miR-122, and we reported that the glucose metabolism is significantly up-regulated in Huh7/R cells. Importantly, overexpression of miR-122 in Huh7/R cells reversed the doxorubicin-resistance through the inhibition of PKM2, inducing the apoptosis in doxorubicin-resistant cancer cells. Thus, this study revealed that the dysregulated glucose metabolism contributes to doxorubicin resistance, and the inhibition of glycolysis induced by miR-122 might be a promising therapeutic strategy to overcome doxorubicin resistance in hepatocellular carcinoma.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Fig 1. MiR-122 is down-regulated in hepatocellular carcinoma cell lines, and associated with doxorubicin resistance.
(A) The expression of miR-122 was down-regulated in HCC cell lines compared with the normal hepatocytes. *p<0.05 vs. L-O2 cells, t test. (B) The cell viability of Huh7, Hep3B, HepG2, and PLC was measured by MTT assay after they were treated with 0.2 μg/ml doxorubicin or 1.0 μg/ml doxorubicin for 48 h. *p<0.05 vs. Huh7 cells, t test. (C) MiR-122 expression was further down-regulated in Huh7/R cells compared with its parental Huh7 cells. *p<0.05, t test.
Fig 2. Overexpression of miR-122 resensitizes Huh7/R cells to DOX.
(A) Huh7 and Huh7/R cells were treated with different concentrations of doxorubicin for 48 h, and cell viability was determined by MTT assay. The IC50 was determined according to the survival curves. *p<0.05, t test. (B) Huh7 cells were transfected with miR-122 inhibitors or NCO for 24 h followed by the treatment of DOX at different concentrations for another 48 h followed by cell viability analysis. The IC50 was determined according to the survival curves. *p<0.05, t test. (C) Huh7/R cells were transfected with miR-122 mimics or NCO for 24 h followed by the treatment of DOX at different concentrations for another 48 h followed by cell viability analysis. The IC50 was determined according to the survival curves. *p<0.05, t test.
Fig 3. PKM2 is a direct target of miR-122.
(A) Putative miR-122 binding sequence in the 3′-UTR of PKM2 mRNA. (B) Huh7/R cells were co-transfected with luciferase reporter plasmids, pRL-TK vector and miR-122 (or NCO) using lipofectamine 2000 reagent. After transfection for 48 h, luciferase activities were measured by a dual luciferase reporter assay. The pRL-TK vector was used as an internal control. The results were determined as relative luciferase activity (Firefly LUC/Renilla LUC). *p<0.05, t test.
Fig 4. PKM2 is overexpressed in doxorubicin-resistant Huh7 cells.
(A) The expression of PKM2 was detected by qPCR and western blot analysis in Huh7/R cell line and its parental Huh7 cell line. *p<0.05, t test. (B) The Huh7 and Huh7/R cells were transfected with PKM2 recombinant vector (2 μg/ml) for 24 h. The Huh7 and Huh7/R cells were then treated with 1.5 μg/ml DOX and 15 μg/ml DOX for another 48 h, respectively. Cell viability was measured by MTT assay. *p<0.05, t test. (C) Transfection of miR-122 decreased the PKM2 level in Huh7/R cells. *p<0.05, t test. (D) Transfection of miR-122 didn’t influence the doxorubicin accumulation in Huh7/R cells. (E) Western blot analysis was performed to evaluate the expression of Bcl-2, Bcl-xl, Bcl-w, Bax, and Bad in Huh7/R cells treated with miR-122 or doxorubicin.
Fig 5. Huh7/R cells exhibit increased glucose metabolism via miR-122-PKM2 pathway.
(A) Glucose uptake of Huh7/R cells was increased. *p<0.05, t test. (B) Lactate production in Huh7/R cells was increased. *p<0.05, t test. (C) ATP production in Huh7/R cells was decreased. *p<0.05, t test. (D) MiR-122 but not doxorubicin significantly decreased the glucose uptake in Huh7/R cells. *p<0.05, t test. (E) MiR-122 but not doxorubicin significantly decreased the lactate production in Huh7/R cells. *p<0.05, t test. (F) MiR-122 but not doxorubicin further reduced the ATP production in Huh7/R cells. *p<0.05, t test. (G) Overexpression of miR-122 inhibited the proliferation of Huh7/R cells, and significantly enhanced the anti-proliferation effect of DOX on Huh7/R cells. *p<0.05, t test.
Fig 6. MiR-122 sensitized Huh7/R cells to doxorubicin inducing cell death via miR-122-PKM2 pathway.
(A) Huh7/R cells were transfected with miR-122 mimics as well as PKM2 recombinant vector for 24 h, and then the cells were treated with doxorubicin (10 μg/ml) for another 48 h. Cell viability was measured by MTT assay. *p<0.05, t test. (B) Huh7/R cells were transfected with miR-122 mimics as well as PKM2 recombinant vector for 24 h, and then the cells were treated with doxorubicin (10 μg/ml) for another 24 h. Cell apoptosis was measured using Annexin V/PI staining. (C) Cleavage of caspase-3 and PARP was evaluated by western blot analysis.
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