The dual-inhibitory effect of miR-338-5p on the multidrug resistance and cell growth of hepatocellular carcinoma

Abstract

Chemotherapeutic treatments against hepatocellular carcinoma (HCC) are necessary for both inoperable patients to improve prospects for survival and surgery patients to improve the outcome after surgical resection. However, multidrug resistance (MDR) is a major obstacle to obtaining desirable results. Currently, increasing the chemotherapy sensitivity of tumor cells or discovering novel tumor inhibitors is an effective therapeutic strategy to solve this issue. In the present study, we uncovered the dual-inhibitory effect of miR-338-5p: on the one hand, it could downregulate ABCB1 expression and sensitize HCC cells to doxorubicin and vinblastine by directly targeting the 3'-untranslated region (3'-UTR) of ABCB1, while, on the other hand, it could suppress the proliferation of HCC cells by directly targeting the 3'-UTR of EGFR and reducing EGFR expression. Since EGFR regulates ABCB1 levels, the indirect action of miR-338-5p in ABCB1 modulation was revealed, in which miR-338-5p inhibits ABCB1 expression by targeting the EGFR/ERK1/2 signaling pathway. These data indicate that the miR-338-5p/EGFR/ABCB1 regulatory loop plays a critical role in HCC, and a negative correlation between miR-338-5p and EGFR or ABCB1 was also detected in HCC clinical samples. In conclusion, these findings reveal a critical role for miR-338-5p in the regulation of MDR and proliferation of HCC, suggesting the potential therapeutic implications of miR-338-5p in HCC treatment.

Fig. 1. ABCB1 is a direct target of miR-338-5p.

ABCB1 was detected by qRT–PCR in Hep3B and Huh7 cells treated with miR-338-5p mimics a or inhibitors b. ABCB1 mRNA levels were normalized to GAPDH mRNA levels. P-gp expression in hepatoma cells transfected with miR-338-5p mimics c or inhibitors d was analyzed by western blotting. Hep3B e and Huh7 f cells were treated with the ABCB1 vector (ABCB1, hereafter) or empty plasmid (Vector, hereafter), and then, the levels of P-gp were measured. g Left: alignment of miR-338-5p with ABCB1 3′-UTR. Right: luciferase assay for the direct targeting of the 3′-UTR of ABCB1 by miR-338-5p. The wide-type or mutant of ABCB1 3′-UTR plasmid was co-transfected with miR-338-5p mimics or NC in Hep3B cells, and subsequently, luciferase activity was detected. The data in all experiments are presented as the means ± SD of three independent experiments. **P < 0.01, ***P < 0.001 vs. NC or Vector

Fig. 2. miR-338-5p regulates the sensitivity of HCC cells to DOX and VBL by targeting ABCB1.

The ectopic expression of miR-338-5p enhanced the sensitivity to chemotherapeutics and increased the intracellular concentration of DOX in HCC cells. Cells were treated with medium containing DOX or VBL for 48 h, and then, cell viability was detected. Overexpression of miR-338-5p in Hep3B a or Huh7 b cells increased the sensitivity to DOX or VBL. Knockdown of miR-338-5p reduced the sensitivity to DOX or VBL of Hep3B c and Huh7 d cells. Intracellular DOX accumulation was measured by flow cytometry in cells transfected with miR-338-5p mimics e or inhibitors f. e–f Left: the output results of the intracellular DOX fluorescence intensity measured by flow cytometry. Right: the statistical results. (g–h) The sensitivity of cells to DOX or VBL was detected by CCK-8 assay. (a–d) and (g–h) Left: The sensitivity of cells to DOX. Right: The sensitivity of cells to VBL. Two-way analysis of variance was employed to analyze the drug sensitivity data. The data in all experiments are presented as the means ± SD of three independent experiments. ***P < 0.001 vs. NC

Fig. 3. miR-338-5p suppresses proliferation of Hep3B and Huh7 cells.

a–b CCK-8 assays were used to evaluate the effect of upregulation of miR-338-5p on Hep3B or Huh7 cell proliferation. c–d Cell proliferation was promoted after transfection with miR-338-5p inhibitors. e–f Cell colony formation assays showed the effects of overexpressing or knocking down miR-338-5p on HCC cell growth. Student’s t-test (two-tailed) was employed to analyze the data. Data in all experiments are presented as the means ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. NC

Fig. 4. EGFR is another direct target of miR-338-5p.

a The target genes of miR-338-5p were predicted using TargetScan, MicroCosm, and MiRanda. b–c The mRNA levels of potential target genes of miR-338-5p were measured after cells were transfected with miR-338-5p for 72 h. d EGFR mRNA expression was detected after cells were treated with miR-338-5p inhibitor by qRT–PCR. The protein expression of EGFR in HCC cells transfected with miR-338-5p mimics e or inhibitors f was analyzed by western blotting. g Left: sequence complementarity between the 3′-UTR of EGFR mRNA and the seed region of miR-338-5p. Right: luciferase activity in Hep3B cells transfected with miR-338-5p and reporter plasmids containing wt (wild-type) or mt (mutant) EGFR 3′-UTR was analyzed. h Cells were treated with the EGFR vector (EGFR, hereafter), and then, the protein levels of EGFR were measured by western blotting. The proliferation i–j and colony formation k of Hep3B and Huh7 cells were remarkably promoted after transfection with the EGFR vector compared with the control vector. Student’s t-test (two-tailed) or one-way or two-way analysis of variance was employed to analyze the data. Data in all experiments are presented as the means ± SD of three independent experiments. **P < 0.01, ***P < 0.001 vs. NC

Fig. 5. miR-338-5p inhibits ABCB1 expression by targeting the EGFR/ERK1/2 signaling pathway.

a The expression levels of P-gp were determined by western blotting after transfection with EGFR siRNA. b P-gp was increased in cells treated with the EGFR vector. c–d The P-gp levels in cells after co-transfection with miR-338-5p and EGFR vector. Western blot showing expression levels of p-EGFR, ERK1/2, and p-ERK1/2 in cells transfected with miR-338-5p mimics e–f or inhibitors g–h. i–j HCC cells were treated with 15 μM U0126 (an inhibitor of ERK1/2 signaling) for 24 h, and then, the protein levels of genes were detected by western blot analysis. Student’s t-test (two-tailed) or one-way analysis of variance was employed to analyze the data. Data in all experiments are presented as the means ± SD of three independent experiments. **P < 0.01, ***P < 0.001 vs. NC, Vector or Ctrl

Fig. 6. The miR-338-5p level is negatively correlated with the ABCB1 and EGFR mRNA levels in HCC clinical samples.

a–c The expression of miR-338-5p, ABCB1, and EGFR were detected by qRT–PCR. d The expression of ABCB1 mRNA was inversely associated with the miR-338-5p expression in the HCC tissues. e The correlation between the expression of EGFR mRNA and miR-338-5p in the HCC tissues. Student’s t-test (two-tailed) or Spearman’s correlation test was used to analyze the data. Data in all experiments are presented as the means ± SD of three independent experiments. *P < 0.05, **P < 0.01 vs. non-tumor tissues

Fig. 7

Schematic model depicting the miR-338-5p/EGFR/ABCB1 regulatory loop in the drug resistance and proliferation of HCC