Targeting the MIR34C-5p-ATG4B-autophagy axis enhances the sensitivity of cervical cancer cells to pirarubicin

Abstract

Pirarubicin (THP) is a newer generation anthracycline anticancer drug. In the clinic, THP and THP-based combination therapies have been demonstrated to be effective against various tumors without severe side effects. However, previous clinical studies have shown that most patients with cervical cancer are not sensitive to THP treatment, and the associated mechanisms are not clear. Consistent with the clinical study, we confirmed that cervical cancer cells were resistant to THP in vitro and in vivo. Our data demonstrated that THP induced a protective macroautophagy/autophagy response in cervical cancer cells, and suppression of this autophagy dramatically enhanced the cytotoxicity of THP. By scanning the mRNA level change of autophagy-related genes, we found that the upregulation of ATG4B (autophagy-related 4B cysteine peptidase) plays an important role in THP-induced autophagy. Moreover, THP increased the mRNA level of ATG4B in cervical cancer cells by promoting mRNA stability without influencing its transcription. Furthermore, THP triggered a downregulation of MIR34C-5p, which was associated with the upregulation of ATG4B and autophagy induction. Overexpression of MIR34C-5p significantly decreased the level of ATG4B and attenuated autophagy, accompanied by enhanced cell death and apoptosis in THP-treated cervical cancer cells. These results for the first time reveal the presence of a MIR34C-5p-ATG4B-autophagy signaling axis in THP-treated cervical cancer cells in vitro and in vivo, and the axis, at least partially, accounts for the THP nonsensitivity in cervical cancer patients. This study may provide a new insight for improving the chemotherapeutic effect of THP, which may be beneficial to the further clinical application of THP in cervical cancer treatment.

Keywords: ATG4B; MIR34C-5p; autophagy; cervical cancer; pirarubicin.

Figure 1.

Cervical cancer cells resist THP in vitro. (A) Cervical cancer cells including C33A, SiHa and HeLa were treated with THP at the indicated dose for 24 h, and then imaged under a phase-contrast microscope at 200× magnification. (B) The cells were seeded in 96-well plates and treated in triplicate with different doses of THP for 24 h, and then the CCK8 assay was performed to evaluate the cytotoxicity of THP. Scale bar: 100 µm. (C) A total of 1 × 10⁴ cells were seeded in a 12-well plate overnight, and then treated with different doses of THP for 24 h. Subsequently, the cells were trypsinized and counted after trypan blue staining. Data are presented as the percentage of dead cells. Results are mean ± SD from 3 independent experiments.

Figure 2.

THP induces apoptosis in cervical cancer cells. (A and B) After treatment with THP at the indicated dose for 24 h, SiHa and HeLa cells were harvested and stained with ANXA5-APC and PI. Then apoptosis was assayed using a flow cytometer (A), and the percentage of apoptotic cells was calculated as the percentage of PI and ANXA5-APC double-positive cells (B). Data are mean ± SD from 3 independent experiments. *, P < 0.05; **, P < 0.01. ((C)and D) After treatment as in (A), the cells were stained with Hoechst 33258 and observed under a fluorescence microscope (C), and the activation of PARP was evaluated using western blot with TUBA as a loading control (D). Scale bars: 20 µm.

Figure 3.

THP induces cytoprotective autophagy in cervical cancer cells. (A) The cells expressing GFP-MAP1LC3 were treated with 200 ng/ml of THP or vehicle control (PBS) for 24 h, and then observed under a fluorescence microscope. White arrows indicate the characteristic puncta of GFP-MAP1LC3. DAPI was used to stain the nucleus. Bar: 5 µm. (B) Quantification of the data from (A), which are expressed as the percentage of cells containing 5 or more GFP-MAP1LC3 puncta. (C) SiHa and HeLa cells were treated with different doses of THP in the presence or absence of CQ (20 μM) for 24 h. Then the cells were harvested and the protein levels of MAP1LC3 and SQSTM1 were detected using western blot. (D-F) SiHa and HeLa cells were treated with THP (200 ng/ml) or vehicle control in the presence or absence of CQ (20 μM) or bafilomycin A₁ (Baf, 0.2 μM) for 24 h. Then the cell viability was examined using a CCK-8 kit (D), the total cell death was calculated using trypan blue staining (E) and the apoptotic cells were analyzed using flow cytometry (F). Data are mean ± SD from 3 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 4.

The upregulation of ATG4B plays a key role in THP-induced autophagy. (A) HeLa cells were treated with THP at the indicated dose for 24 h, and then the mRNA levels of 9 autophagy-related genes were evaluated using qRT-PCR. The data are expressed as the fold change over the control. (B) HeLa cells were treated with THP at the indicated dose for 24 h. Then the protein levels of ATG3, ATG4B, ATG5, BECN1, ATG7, ATG10, ATG12, PIK3C3 and ATG16L1 were detected using western blot. (Cand D) After transiently transfecting with ATG4B shRNA or control shRNA for 24 h, the HeLa cells were treated with THP at the indicated dose for 24 h. Then ATG4B and MAP1LC3 proteins were analyzed by western blot (C), and cell viability was detected using the CCK-8 kit (D). Data are mean ± SD from 3 independent experiments. *, P < 0.05; **, P < 0.01.

Figure 5.

THP enhances the mRNA stability of ATG4B instead of its transcriptional activity. (A) Schematic representation of the ATG4B promoter region containing the putative binding sites for several transcription factors. The region (−2888 to +155) was cloned into the pGL3-Basic reporter vector and was named pGL3-ATG4B. (B) After transfection with pGL3-ATG4B for 12 h, HeLa cells were treated with various doses of THP or vehicle control for another 24 h, and then the luciferase activity was assayed using the Dual-Luciferase Reporter System, and normalized to the control. (Cand D) HeLa cells were treated with 5 μg/ml actinomycin D (Act D) in the presence or absence of 200 ng/ml of THP for the indicated times, then the level of ATG4B mRNA was assayed using RT-PCR (C) and qRT-PCR (D). Data are represented as the mean ± SD from 3 independent experiments. n.s., no significance; **, P < 0.01; ***, P < 0.001.

Figure 6.

THP promotes ATG4B mRNA stability through decreasing the level of MIR34C-5p. (A) The predicted microRNAs that target the 3′-UTR of ATG4B mRNA using 3 different website tools. (Band C) HeLa cells were transfected with control MIR, MIR34A mimics or MIR34C-5p mimics for 24 h, then the mRNA and protein levels of ATG4B were examined by qRT-PCR (B) and western blot (C), respectively. (D) HeLa cells were treated with different doses of THP for 24 h, then the levels of MIR34A and MIR34C-5p were quantified using qRT-PCR, taking RNU6-2 as a control. (E) After transfection with control MIR or MIR34C-5p mimics for 24 h, the HeLa cells were treated with 200 ng/ml of THP or vehicle control for an additional 24 h. Then the mRNA level of ATG4B was quantified using qRT-PCR. (F) After transfection with control MIR or MIR34C-5p mimics for 24 h, the HeLa cells were treated with actinomycin D (Act D, 5 µg/ml) in the presence or absence of THP (200 ng/ml) for the indicated times. Then the mRNA level of ATG4B was quantified using qRT-PCR. (G) Schematic representation of a predicted binding site of MIR34C-5p in the 3′-UTR of human ATG4B mRNA, and the mutant ATG4B 3′-UTR (ATG4B 3′UTR-mu). (H) HeLa cells were cotransfected with the reporter plasmids (pmir-ATG4B, pmir-ATG4B-mu or empty pmir-GLO) and RNA oligonucleotides (control MIR or MIR34C-5p mimics) for 24 h. Then the luciferase activity was determined using the Dual-Luciferase Reporter System. Data are mean ± SD from 3 independent experiments. n.s., no significance; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 7.

MIR34C-5p inhibits THP-induced autophagy and enhances the cytotoxicity of THP in cervical cancer cells. (A) After transfection with the indicated RNA oligonucleotides (control MIR or MIR34C-5p mimics) for 24 h, HeLa cells were treated with THP at the indicated dose for an additional 24 h. Then the protein levels of ATG4B, MAP1LC3 and PARP were detected using western blot. (B) After cotransfecttion with MIR34C-5p mimics and pCMV5-ATG4B for 24 h, the HeLa cells were treated with THP at the indicated dose for an additional 24 h. Then the levels of MAP1LC3 and ATG4B were analyzed using western blot. (C) HeLa cells were treated as in (A), and then the cell viability was evaluated using the CCK8 assay. Data are mean ± SD from 3 independent experiments. *, P < 0.05; **, P < 0.01; ***, P <0.001.

Figure 8.

THP represses the growth of cervical cancer xenograft accompanied by the downregulation of MIR34C-5p and upregulation of ATG4B. HeLa cells (2 × 10⁶) were subcutaneously injected into the flanks on both sides of each mouse. When the tumors reached 100 mm³, the mice were randomized into 2 groups (5–8 mice/group), and then treated with THP (intravenously) at a 1 mg/kg dose or vehicle control. One wk later, the xenograft tumors were harvested (A) and the tumor size was measured (B). The xenograft tumor lysates were used for detecting ATG4B and MAP1LC3 proteins by western blot (C), and analyzing MIR34C-5p using qRT-PCR (D). Data are mean ± SD. *, P < 0.05; ***, P < 0.001.

Figure 9.

Schematic illustration for the mechanisms of action of THP in cervical cancer cells. On the one side, THP induces apoptosis, which results in cell death. On the other side, THP activates the MIR34C-5p-ATG4B-autophagy signaling axis via the sequential triggering of MIR34C-5p downregulation, ATG4B mRNA stability enhancement, and ATG4B upregulation and autophagy induction. Moreover, autophagy protects cervical cancer cells from cell death. The autophagy inhibitor CQ sensitizes cervical cancer cells to THP treatment.