MiR-200b regulates autophagy associated with chemoresistance in human lung adenocarcinoma

Abstract

Chemoresistance remains a major clinical problem in combating human lung adenocarcinoma (LAD), and abnormal autophagy is closely associated with this phenomenon. In the present study, an inverse correlation between miR-200b and autophagy-associated gene 12 (ATG12) expressions was observed in docetaxel-resistant (SPC-A1/DTX and H1299/DTX) and sensitive (SPC-A1 and H1299) LAD cells as well as in tissue samples. Further study showed that miR-200b directly targeted ATG12 in LAD. Moreover, miR-200b-dependent ATG12 downregulation inhibited autophagy and enhanced the chemosensitivity of SPC-A1/DTX and H1299/DTX cells both in vivo and in vitro. LAD chemoresistance is therefore closely related to downregulation of miR-200b and the corresponding upregulation of ATG12. These results provide new evidence for the mechanisms governing the microRNA (miRNA)-ATG12 network and their possible contribution to autophagy modulation and LAD chemoresistance.

Keywords: ATG12; autophagy; chemoresistance; human lung adenocarcinoma; miR-200b.

Figure 1. Forced expression of miR-200b blocks autophagy in SPC-A1/DTX cells

A. SPC-A1/DTX cells were transfected with the indicated miRNA precursors. Whole cell lysates were analyzed by western blot for LC3-II/LC3-I protein. LC3-II/LC3-I and LC3-II/GAPDH ratios were calculated by Image J densitometric analysis (three independent experiments gave similar results). B. Docetaxel-resistant SPC-A1/DTX cells transfected with PmiR-200b and parental SPC-A1 cells transfected with AmiR-200b were exposed to docetaxel (100 μg/L and 10 μg/L, respectively) and cisplatin (1.5 μg/ml and 0.5 μg/ml, respectively) for 48 h. Whole cell lysates were analyzed by western blot using LC3 and P62 antibodies (control: GAPDH). LC3-II/LC3-I and P62/GAPDH ratios were calculated using Image J densitometric analysis (three independent experiments gave similar results). C. SPC-A1/DTX cells and SPC-A1 cells were transfected with a GFP-LC3 construct and either PmiR-200b or AmiR-200b, following treatment with docetaxel (100 μg/L and 10 μg/L, respectively) or cisplatin (1.5 μg/ml and 0.5 μg/ml, respectively). GFP-LC3 dot formation was analyzed as described in the Materials and Methods section (bar: 50 μm). The results are presented as mean ± SD of values obtained in three independent experiments. *P < 0.05; **P < 0.01. D. SPC-A1 cells were transfected with AmiR-NC or AmiR-200b for 48 h. Cell samples were prepared for transmission electron microscopy analysis. Arrows indicate autophagosomes. N: nucleus. E. SPC-A1/DTX cells transfected with PmiR-200b were treated with docetaxel (100 μg/L) after incubation with Baf A1 (20 nM) for 2 h or not. SPC-A1 cells transfected with AmiR-200b were treated with Baf A1 (20 nM, 2 h) or not. Cell lysates were analyzed by western blot for LC3 and P62 (control: GAPDH). LC3-II/GAPDH and P62/GAPDH ratios were calculated using Image J densitometric analysis (three independent experiments gave similar results). F. Western blot analysis for LC3, P62 and ATG5 in SPC-A1 cells co-transfected with AmiR-200b and ATG5 siRNA. LC3-II/LC3-I and P62/GAPDH ratios were calculated using Image J densitometric analysis (three independent experiments gave similar results).

Figure 2. MiR-200b inhibits autophagy by directly targeting ATG12

A. Predicted miR-200b consensus sequences in the ATG12 3′UTR. B. Luciferase activity analysis of ATG12 3′UTR (wild type and mutant constructs) were performed after co-transfection with PmiR-200b in HEK-293 cells by using the Dual-luciferase Reporter Assay System. The data are expressed as the mean ± SD (n = 3). *P < 0.05 vs the PmiR-NC. C. Western blot and qRT-PCR was used to analyze the expression of ATG12 in both parental and docetaxel-resistant cells. ATG12/GAPDH ratios were calculated using Image J densitometric analysis (three independent experiments gave similar results). D. SPC-A1 cells and H1299 cells were transfected with AmiR-200b or AmiR-NC whereas SPC-A1/DTX cells and H1299/DTX cells were transfected with PmiR-200b or PmiR-NC. ATG12 expression was determined by western blot and qRT-PCR analysis. ATG12/GAPDH ratios were calculated using Image J densitometric analysis. Data are represented as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01. E. SPC-A1/DTX cells and H1299/DTX cells were transfected with ATG12 siRNA, followed by treatment with docetaxel (100 μg/L) or cisplatin (1.5 μg/ml). LC3, P62 and ATG12 expression levels were evaluated by western blot. LC3-II/LC3-I, P62/GAPDH and ATG12/GAPDH ratios were calculated using Image J densitometric analysis (three independent experiments gave similar results). F. Western blot for LC3, p62 and ATG12 of whole lysates of SPC-A1/DTX cells and H1299/DTX cells transfected with PmiR-200b, pDsRed1 ATG12 or both. LC3-II/LC3-I, P62/GAPDH and ATG12/GAPDH ratios were calculated using Image J densitometric analysis (three independent experiments gave similar results). G. SPC-A1/DTX cells and H1299/DTX cells were co-transfected with GFP-LC3 plasmids and PmiR-200b, pDsRed1 ATG12 or both combinations (bar: 50 μm). Data are represented as mean ± SD of three independent experiments. *P < 0.05; **P < 0.01.

Figure 3. MiR-200b inhibits starvation and rapamycin-induced autophagy in SPC-A1 cells

A. SPC-A1 cells were incubated in HBSS for different time periods (0 to 8 h). Exogenous miR-200b was transfected into cells at 4 h. The relative expression levels of miR-200b mRNA and ATG12 mRNA were assessed by qRT-PCR, with expression in the control set as 1.0. B, D. SPC-A1 cells were transfected with PmiR-200b, ATG12 siRNA or NC, followed by treatment with B. HBSS for 4 h or D. rapamycin (50 nM) for 2 h. The protein levels of LC3, P62 and ATG12 were assayed by western blot. LC3-II/LC3-I, P62/GAPDH and ATG12/GAPDH ratios were calculated using Image J densitometric analysis (three independent experiments gave similar results). C, E. SPC-A1 cells were co-transfected with either PmiR-200b or ATG12 siRNA and GFP-LC3 plasmid, and then exposed to C. HBSS for 4 h or E. rapamycin (50 nM) for 2 h. Data are shown as mean ± SD of three replicates and are representative of three independent experiments. *P < 0.05; **P < 0.01. F. Western blot analysis detected the expression of LC3 and P62 in AmiR-200b-transfected SPC-A1 cells after treatment with rapamycin (50 nM) for 2 h. LC3-II/LC3-I and P62/GAPDH ratios were calculated using Image J densitometric analysis (three independent experiments gave similar results).

Figure 4. MiR-200b increases chemosensitivity of SPC-A1/DTX cells by targeting ATG12

A, B. SPC-A1/DTX cells were transfected with PmiR-200b, pDsRed1 ATG12, or both or with ATG12 siRNA, followed by exposure to indicated doses of docetaxel or cisplatin for 48 h. A. MTT assay showing cell viability; B. colony formation assay showing cell proliferation. C, D. SPC-A1 cells were transfected with AmiR-200b, ATG12 siRNA or both, and then treated with the indicated concentrations of docetaxel or cisplatin for 48 h. C. MTT assay showing cell viability; D. colony formation assay showing cell proliferation. Data are shown as mean ± SD of three replicates and are representative of three independent experiments. *P < 0.05; **P < 0.01.

Figure 5. MiR-200b increases the sensitivity of SPC-A1/DTX cells to apoptosis by targeting ATG12

A, B. SPC-A1/DTX cells were transfected with PmiR-200b, pDsRed1 ATG12, or both, or with ATG12 siRNA, followed by exposure to docetaxel (100 μg/L) or cisplatin (1.5 μg/ml) for 48 h. Apoptosis was measured by A. flow cytometry with Annexin-V staining (Data are expressed as mean ± SD of three independent experiments. *P < 0.05; **P < 0.01); and B. western blotting was performed for apoptosis markers (c-caspase-3 and c-PARP). C-caspase3/caspase3, c-PARP/PARP and ATG12/GAPDH ratios were calculated using Image J densitometric analysis (three independent experiments gave similar results). C, D. SPC-A1 cells were transfected with AmiR-200b, ATG12 siRNA or both, and then treated with docetaxel (20 μg/L) or cisplatin (0.8 μg/ml) for 48 h. Apoptosis was measured by C. flow cytometry with Annexin-V staining (Data are expressed as mean ± SD of three independent experiments. *P < 0.05; **P < 0.01.); and D. western blot for apoptosis markers (c-caspase-3 and c-PARP). C-caspase3/caspase3, c-PARP/PARP and ATG12/GAPDH ratios were calculated using Image J densitometric analysis (three independent experiments gave similar results).

Figure 6. MiR-200b enhances the antitumor efficacy of docetaxel in vivo

A. Nude mice were subcutaneously injected with 5 × 10⁶ cells stably transfected with pcDNA miR-200b or pcDNA miR-NC and treated with docetaxel (1mg/kg) beginning at day eight. Tumor size was measured every 2 days after docetaxel treatment (n = 3, *P < 0.05). B. Representative photographs of tumors formed at 16 days after subcutaneous transplantation are displayed. C. Hematoxylin and eosin (H&E)-stained, ATG12-stained, ki67-stained and TUNEL staining in paraffin sections of the tumors. D. Western blot analysis of LC3 and ATG12 in implanted tumors. Data shown are representative of three identical experiments. LC3-II/LC3-I and P62/GAPDH ratios were calculated using Image J densitometric analysis (three independent experiments gave similar results).

Figure 7. Downregulation of miR-200b expression correlates with upregulation of ATG12 expression, decreased sensitivity to docetaxel and poor prognosis in lung adenocarcinoma tissues

A. qRT-PCR was performed to analyze the relative mRNA expression of miR-200b and ATG12 in docetaxel-sensitive (n = 27) and insensitive (n = 33) LAD tissues. MiR-200b and ATG12 mRNA abundances were normalized to U6 RNA and GAPDH, respectively. B. Protein expression of ATG12 was determined by immunochemical staining (magnification 200×). C. Expression levels of miR-200b and ATG12 mRNA were inversely correlated among tissue samples as measured by linear regression analysis. D. The Kaplan–Meier survival curve shows that patients with high miR-200b expression have shorter progression-free survival (PFS) than those with low miR-200b expression (log-rank test, P = 0.021). Data are expressed as mean ± SD of three independent experiments. *P < 0.05.

Figure 8. Mechanism whereby miR-200b modulates ATG12-dependent chemoresistance

MiR-200b inhibits ATG12 expression, which leads to a decrease in autophagy that contributes to chemoresistance.