MiR-199a-3p enhances cisplatin sensitivity of cholangiocarcinoma cells by inhibiting mTOR signaling pathway and expression of MDR1

Abstract

Several studies have reported reduced miRNA-199a-3p (miR-199a-3p) in different human malignancies, however, little is known about miR-199a-3p in cholangiocarcinoma cells. In this study, we demonstrate the essential role and mechanism of miR-199a-3p in regulating cisplatin sensitivity in cholangiocarcinoma cell lines. Using a CCK-8 cell counting assay we found that expression of miR-199a-3p was positively correlated with cisplatin sensitivity in cholangiocarcinoma cell lines. MiR-199a-3p overexpression could decrease the proliferation rate and increase apoptosis of cholangiocarcinoma cells in the presence of cisplatin, while miR-199a-3p inhibition had the opposite effect. Further study demonstrated that mTOR was the target gene of miR-199a-3p, and that miR-199a-3p mimics could inhibit expression of mTOR, which consequently reduced the phosphorylation of its downstream proteins 4EBP1 and p70s6k. Rescue experiments proved that miR-199a-3p could increase the cisplatin sensitivity of cholangiocarcinoma cell lines by regulating mTOR expression. Moreover, we also found that miR-199a-3p overexpression could reduce cisplatin induced MDR1 expression by decreasing the synthesis and increasing the degradation of MDR1, thus enhancing the effectiveness of cisplatin in cholangiocarcinoma. In conclusion, miR-199a-3p could increase cisplatin sensitivity of cholangiocarcinoma cell lines by inhibiting the activity of the mTOR signaling pathway and decreasing the expression of MDR1.

Keywords: MDR1; MiR-199a-3p; chemosensitivity; cholangiocarcinoma; mTOR.

Figure 1. Cell viability under cisplatin and miR-199a-3p expression of cholangiocarcinoma cells

(A) Cell viability of GBC-SD and RBE cell lines under different concentration of cisplatin by CCK-8 assay. (B) Expression of miR-199a-3p in GBC-SD and RBE cell lines examined by qPCR. U6 was used as the internal reference. **P < 0.01 vs. GBC-SD.

Figure 2. MiR-199a-3p enhanced cisplatin sensitivity of cholangiocarcinoma cells

(A–B) Cell viability under different concentrations of cisplatin of GBC-SD and RBE cell lines treated with miR-199a-3p mimics, inhibitor and negative control, examined by CCK-8 assay. (C–D) Cell proliferation rate under different concentrations of cisplatin of GBC-SD and RBE cell lines treated with miR-199a-3p mimics, inhibitor and negative control, examined by EdU assay. The number of EdU positive cells was counted. (E) Apoptosis incidence under certain concentrations of cisplatin in GBC-SD and RBE cell lines treated with miR-199a-3p mimics, inhibitor and negative control, examined by flow cytometry. The cell numbers in quadrants Q2 and Q4 were defined as apoptotic cells. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3. mTOR expression in cholangiocarcinoma cells and the TargetScan result

(A) TargetScan predicted mTOR was the target gene of miR-199a-3p: miR-199a-3p could bind the 129–135 positions of the mTOR 3′-UTR. (B) Western blotting detected the expression of mTOR and p-mTOR (sites 2481 and 2448) in GBC-SD and RBE cell lines; the results were quantified using ImageJ software (**P < 0.01, ***P < 0.001).

Figure 4. MiR-199a-3p could inhibit the mTOR signaling pathway

(A) Expression of mTOR, p-mTOR (sites 2481 and 2448), p-4EBP1, p-p70s6k, 4EBP1 and P70s6K were detected by Western blotting in GBC-SD and RBE cell lines under different treatments with miR-199a-3p. (B) The efficiency of miR-199a-3p mimics and inhibitor were confirmed by qPCR analysis (**P < 0.01, ***P < 0.001).

Figure 5. MiR-199a-3p enhanced cisplatin sensitivity of cholangiocarcinoma cells via the mTOR signal pathway

(A–B) Cell viability of GBC-SD and RBE cell lines was detected by CCK-8 assay under different concentrations of cisplatin. Cells were treated by mTOR siRNA alone, negative control in combination with miR-199a-3p mimics or inhibitor. (C) Western blotting was used to confirm the efficiency of mTOR siRNA (***P < 0.001).

Figure 6. MiR-199a-3p inhibited cisplatin induced MDR1 expression

(A) Protein level of MDR1 was detected in GBC-SD and RBE cell lines under treatment with miR-199a-3p mimics or cisplatin or both. The results were quantified using ImageJ software. (B) Protein expression of MDR1 was detected under treatment with MG-132 or miR-199a-3p mimics or both (upper panel), and the same with CHX (lower panel). The results were quantified using ImageJ (***P < 0.001).