1. Volume 143, Issue 1, Pages 177-187.e8 (July 2012)
miR-375 Inhibits Autophagy and Reduces Viability of Hepatocellular Carcinoma Cells Under Hypoxic Conditions 
Ying Chang, Wei Yan, Xingxing He, Lemeng Zhang, Chuanjiang Li, Hai Huang, Gary Nace, David A. Geller, Jusheng Lin, Allan Tsung 
Gastroenterology 
Volume 143, Issue 1, Pages e8 (July 2012)
DOI: /j.gastro
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2. Figure 1
Autophagy was activated as a protective mechanism against hypoxic stress in HCC. (A) Hepatoma cell lines, Huh7 and Hep3B, were exposed to physiological hypoxia (1% oxygen). LC3I/II and p62 were detected by Western blotting over time. (B) ULK1 and ATG7 were detected by Western blotting after 72 h transfection with each siRNA. Scrambled siRNAs were served as control. MTT assays were performed at each time point after hypoxic exposure. Data are shown as mean ± standard deviation from 3 independent experiments. *P < .05 vs siNC.
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3. Figure 2
miR-375 is down-regulated in human HCC and modulates the hypoxia-induced autophagic response. (A) After 48 h hypoxia, LC3I/II protein was detected by Western blot in Huh7 cells transfected with indicated miRNA precursors or inhibitors. The ratio of LC3II to LC3I/β-actin was calculated by densitometric analysis. (B) Huh7 and Hep3B cells were transfected with indicated doses of pre–miR-375. LC3I/II protein was detected by Western blotting. Blots were representative of 3 experiments with similar results. (C) LC3B mRNA was detected by real-time PCR in Huh7 cells transfected with pre–miR-375 after 48 h hypoxia and was calculated by 2–ΔΔCt method. Glyceraldehyde-3-phosphate dehydrogenase was used as endogenous control. (D, E) TaqMan quantitative reverse transcription PCR was performed to analyze the miR-375 expression in primary normal hepatocytes isolated from human, rat, and mouse (n = 3 each) and in tissues of 20 pairs of human HCC and their background livers (BL). Each dot indicates the expression level of an individual case calculated by 2–ΔΔCt method.
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4. Figure 3
miR-375 inhibits hypoxia-induced autophagosome formation and autophagic flux in HCC cells. (A) Huh7 cells were transfected with pre–miR-375 or pre–miR-NC. After 18 h transfection, cells were retransfected with pGFP-LC3 plasmid and exposed to hypoxia for another 24 h before observation for GFP-LC3 puncta under fluorescence microscope. Blue indicated the nucleus stained by 4′,6-diamidino-2-phenylindole. Green indicated GFP-LC3. The percentage of cells with accumulation of GFP-LC3 in puncta was calculated in 3 random fields. (B) After 24 h transfection, cells were treated with Bafilomycin A1 and exposed to hypoxia for 10 h. LC3I/II was detected by Western blot. (C) Transmission electron microscopy showed autophagosomes (white arrow, a) and autophagosome-fused lysosomes (white arrow b, c) in Huh7 cells after 48 h hypoxia in the pre–miR-NC group, whereas broken and swollen mitochondria were observed in the pre–miR-375 group without autophagic signs. (D) In the rescue study, miR-375 precursor alone or with miR-375 inhibitor (anti–miR-375) was transfected into Huh7 cells. LC3I/II was detected by Western blotting after 48 h hypoxia. (E) Anti–miR-375 or indicated controls were transfected into human normal primary hepatocytes. miR-375 expression was detected by TaqMan real-time PCR after 48 h transfection and calculated by 2–ΔΔCt method. Data are shown as mean ± standard deviation from 3 independent experiments. *P < .05.
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5. Figure 4
miR-375 regulates autophagy independent of the PDK1/AKT/mTOR pathway. (A) The indicated proteins were detected by Western blotting in transfected Huh7 cells after hypoxia at each time point. (B) After 18 h transfection with pre–miR-375 or pre–miR-NC, cells were retransfected with siRNAs for mTOR or treated with rapamycin (50 nM) for another 48 h. LC3I/II and total mTOR were detected by Western blotting. The ratio of LC3II to LC3I was calculated by densitometric analysis. The ratios in mock groups were set as 1.
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6. Figure 5
miR-375 suppresses autophagy by targeting Atg7. (A) mRNA expression of 8 indicated autophagy-related genes was measured by real-time PCR and calculated by 2–ΔΔCt method in transfected Huh7 cells after 48 h hypoxia. (B) Atg7 protein from above cells was detected by Western blotting. Two unrelated miRNAs, miR-885-5p and miR-194, were used as controls. Blots were representative of 3 experiments with similar results. (C) Base pairing complement suggested the putative miR-375 binding position at 3′ UTR of Atg7. The luciferase activity was standardized to β-gal control. (D) Western blotting and densitometric analysis were performed to show the Atg7 expression in the human HCC (T) and their background livers (B). In each pair of tissues, photodensity in background livers was set as 1. Quantitative data are shown as mean ± standard deviation from 3 independent experiments (*P < .05).
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7. Figure 6
miR-375 inhibits mitophagy and resensitizes tumor cells to hypoxic stress. (A) Huh7 and Hep3B cells were transfected with pre–miR-375 or pre–miR-NC. MTT assays were performed after hypoxia. Data are shown as mean ± standard deviation from 3 independent experiments. *P < .05 vs pre–miR-NC. (B) The ultrastructure of treated Huh7 cells was observed by electron micrography after 48 h hypoxia. In the pre–miR-NC cells, white arrows indicated the typical mitophagosomes (double-membrane enclosed damaged mitochondria) and mitophagosome-fused lysosomes. In the pre–miR-375 group, damaged mitochondria dispersed in the cytoplasma with no signs of autophagy. (C) Pre–miR-375/pre–miR-NC or siAtg7/siNC was transfected into Huh7 cells, respectively. After 18 h transfection, cells were retransfected with pGPF-LC3 plasmids and exposed to hypoxia for another 24 h. Immunofluorescence staining showed the colocalization (overlay yellow indicated mitophagy) of Tom20 (red indicated mitochondrial location) and GFP-LC3 (green indicated autophagic vacuoles). (D, E) Total protein and cytosolic protein from transfected Huh7 cells were isolated after 48 h hypoxia. The indicated proteins were detected by Western blotting. Blots shown are representative of 3 experiments with similar results.
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8. Figure 7
miR-375 inhibits autophagy and tumor growth of hepatoma xenografts in vivo. (A) Data are presented as tumor growth curves of hepatoma xenografts in nude mice with Lenti-miR-375 Huh7 transfectants and Lenti-GFP-NC transfectants, which were used as a control. *P < .05. (B) Hypoxia-inducing factor–1α was measured by Western blotting in Huh7 cells under normoxia and in 3 representative xenograft samples of each group. (C) Immunofluorescence staining was performed to show LC3B puncta in representative xenograft tissue sections. Red indicates LC3B; blue indicates nuclei. (D) LC3I/II and Atg7 in 3 representative xenograft tissues in each groups were detected by Western blotting. (E) TEM showed the ultramicrostructure of representative xenograft tissue samples. White arrows in the Lenti-GFP-NC group indicated autophagosome and autophagosome-fused lysosome. White arrows in the Lenti-miR-375 group indicated uneliminated mitochondria. (F) H&E staining for representative xenograft tissue sections. Dash line indicates necrotic areas. Area-a indicates a necrotic area; Area-b indicates tumor cells of regular morphology; black arrow indicates a new blood vessel. Imaging shown is representative of tissues.
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9. Supplementary Figure 1
Flow cytometry was performed to determine the transfection efficiency of miRNA precursors or inhibitors by using Cy3-labeled miRNA negative control.
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10. Supplementary Figure 2
MiR-192 accelerated autophagic flux and hastened the degradation of LC3II. Huh7 cells were transfected with pre–miR-192 or pre–miR-NC. (A) The indicated proteins were detected by Western blotting. (B) After 24 h transfection, cells were treated with Bafilomycin A1 and exposed to hypoxia for 3 h. LC3I/II was detected by Western blotting. (C) Transmission electron microscopy showed the ultramicrostructure of representative cell samples. White arrows in the pre–miR-NC group indicate the earlier mitophagosome in which the shape of mitochondria is faintly visible. White arrows in the pre-miR-192 group indicate the later autophagic vacuoles in which most of contents had been degraded.
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11. Supplementary Figure 3
miR-375 inhibited autophagy in Hep3B cell line. Transmission electron microscopy showed the ultramicrostructure of representative cell samples. White arrows indicate the mitophagosomes or mitophagosome-fused lysosomes.
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12. Supplementary Figure 4
miR-375 indirectly regulated p62 expression through targeting transcription factor Sp1. (A) Huh7 cells were transfected with pre–miR-375 or pre–miR-NC and then exposed to hypoxia for 48 h. Sp1 protein from above cells was detected by Western blotting. Two unrelated miRNAs, miR-885-5p and miR-194, were used as controls. Data were representative of 3 experiments with similar results. (B) Base pairing complement suggested the putative miR-375 binding position at 3’ UTR of Sp1. The empty, wild-type, mutated constructs were used to detect luciferase activity. The luciferase activity was standardized to β-gal control. Data were shown as mean ± standard deviation from 3 independent experiments. (C) Indicated proteins were detected by Western blotting in Huh7 cells transfected with siRNA of Sp1 or p-CMV-Sp1 plasmid. Scrambled siRNAs (siNC) or p-CMV was used as negative control. (D) p62 mRNA was measured by real-time PCR and calculated by 2−ΔΔCt method. (E) p62 and LC3I/II expression were detected by Western blotting after 72 h transfection. Scrambled siRNAs were used as control. MTT assays were performed at each time point after hypoxia exposure. Data are shown as mean ± standard deviation from 3 independent experiments.*P < .05 vs siNC.
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13. Supplementary Figure 5
TaqMan real-time PCR results indicated miR-375 expression in xenografts in Lenti-miR-375 and control groups. Data were shown as mean ± standard deviation. P < .05 vs Lenti-GFP-NC.
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14. Supplementary Figure 6
Transmission electron microscopy showed the ultramicrostructure of representative tissue samples from xenografts of nude mice in Lenti-GFP-NC and Lenti-miR-375 groups.
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