MicroRNA-377-3p inhibits hepatocellular carcinoma growth and metastasis through negative regulation of CPT1C-mediated fatty acid oxidation

Figures

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  Fig. 1. MiR-377-3p targets the 3’-UTR of CPT1C and inhibits its expression in HCC. A HEK-293T cells were transfected with negative control (NC) or mimics of candidate miRNAs as indicated. The representative western blot shows CPT1C expression. β-actin was used as a loading control for western blot. B, C Western blot analysis of CPT1C expression after transfected with (B) NC or miR-377-3p mimics, or (C) scramble or miR-377-3p inhibitor in HepG2 and MHCC97H cells. Histograms show relative CPT1C expression by RT-qPCR. The values of the control groups in HepG2 and MHCC97H cells were set to 1. D Levels of CPT1C mRNA in HepG2 and MHCC97H cells transfected as in (B) and (C). The values of the control groups in HepG2 and MHCC97H cells were set to 1. E Luciferase reporter gene assay was conducted in HepG2 and MHCC97H cells to compare CPT1C 3′-UTR activity among plasmids within wild-type (WT) or mutant (Mut) CPT1C 3′-UTR luciferase reporter with overexpression of miR-377-3p. The top panel indicates WT and Mut forms of putative miR-377-3p target sequences of CPT1C 3′-UTR. Blue font indicates the putative miR-377-3p binding sites within human CPT1C 3′-UTR. Red font indicates the mutations introduced…

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  Fig. 2. MiR-377-3p regulates FAO of lipid metabolism through CPT1C. A Representative images of CY3-labeled palmitate (red) and staining of mitochondria (green) in HepG2 cells transfected with CPT1C expression plasmid or miR-377-3p mimics or miR-377-3p mimics plus CPT1C expression plasmid. The colocalization was shown as yellow. Graphs at the right panel show relative colocalization number of mitochondria and CPT1C. Results shown are mean ± SD of 3 independent experiments. Scale bar 10 μm. B FAO assay of HepG2 cells transfected as in A. Cells treated with FCCP were used as positive control, and cells treated with Eto were used as negative control. Eto, Etomoxir. Graphs at the right panel show relative FAO rate. Data shown are mean ± SD of 3 independent experiments. C HepG2 cells were transfected as in A and stained with BODIPY 493/503 and then the lipid droplet content was determined by flow cytometry. Graphs at the right panel show relative ratio of positive cells. Data shown are mean ± SD of three independent experiments. D Cells were transfected as in A and stained with Oil Red O. Graphs at the right panel show relative LD content. Data shown are mean ± SD of three independent…

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  Fig. 3. MiR-377-3p suppresses proliferation, migration and invasion through inhibition of CPT1C expression in HCC cells. A The proliferation curve of HepG2 cells transfected with CPT1C expression plasmid or miR-377-3p mimics or miR-377-3p mimics plus CPT1C expression plasmid. Western blot analysis shows CPT1C expression. RT-qPCR analysis indicates miR-377-3p expression. B Colony formation assay of HepG2 cells transfected as in A. Graphs at the right panel show relative colony number. Data shown are mean ± SD of 3 independent experiments. C Wound scratch healing assays of HepG2 cells after transfection as in A. Scale bar 50 μm. Histograms show relative cell migration. D, E Transwell assay of HepG2 cells transfected as in A without D or with E Matrigel. Graphs at the right panel show relative cell migration and invasion. The values of the control groups in HepG2 cells were set to 1. Data are mean ± SD (n = 3). **p < 0.01

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  Fig. 4. The miR-377-3p/CPT1C axis regulates the proliferation, migration, and invasion of HCC cells mainly through FAO. A HepG2 cells were transfected with CPT1C expression plasmid or empty vector and treated with indicated dose of Etomoxir. Cells were stained with BODIPY 493/503 and the lipid droplet content was determined by flow cytometry. Eto, Etomoxir. Graphs at the right panel show relative ratio of positive cells. Data shown are mean ± SD of 3 independent experiments. B The proliferation curve of HepG2 and MHCC97H cells transfected with miR-377-3p inhibitor or scramble and treated with Etomoxir (5 μM) as indicated. Western blot analysis shows CPT1C expression. RT-qPCR analysis indicates miR-377-3p expression. C, D Wound scratch healing (C) and invasion (D) assays of HepG2 and MHCC97H cells after transfection as in (B). Scale bar 50 μm. Histograms show relative cell migration and invasion. The values of the control groups in HepG2 and MHCC97H cells were set to 1. Data are mean ± SD (n = 3). **p < 0.01

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  Fig. 5. The miR-377-3p/CPT1C axis regulates HCC tumor growth and metastasis in nude mice. A HepG2 cells stably infected with lentivirus carrying CPT1C shRNA or control shRNA were treated with antagomir miR-377-3p (anti-miR-377-3p) or antagomir NC (scramble) and injected into nude mice as indicated. After 42 days, mice were euthanized to harvest tumors. Images of all xenograft tumors excised at day 42 were shown. The tumor growth curves were plotted. ATP level of tumor tissues (mouse no. V) was determined. MiR-377-3p expression in representative tumor tissues (mouse no. V) was examined by MISH and RT-qPCR, and CPT1C expression was determined by IHC and western blot, and lipid content was conducted by BODIPY 493/503, respectively. Tumor volumes are presented as means ± SD (n = 6). **p < 0.01 at day 42. Data shown are mean ± SD of quintuplicate measurements for ATP level and were repeated 3 times with similar results. **p < 0.01. B Representative lung tissues and H&E-stained sections of the lung tissues. The number of tumor nodules were shown (right panel). **p < 0.01

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  Fig. 6. The clinical significance of miR-377-3p and CPT1C in HCC patients. A Representative miRNA in situ hybridization of miR-377-3p or immunohistochemical staining of CPT1C in 90 paired HCC tumor and adjacent normal tissues. Scale bar 50 μm. H scores of miR-377-3p or CPT1C expression between tumor and normal tissues were compared by Mann-Whitney U test. B Kaplan-Meier survival curves for overall survival of 72 HCC patients according to the relative expression of miR-377-3p and CPT1C. C Representative MISH of miR-377-3p or IHC staining of CPT1C or BODIPY 493/503 staining of lipid droplets in 90 HCC tissues. Correlation was determined between miR-377-3p and CPT1C expression (left panel), between CPT1C expression and lipid droplets (middle panel) and between miR-377-3p expression and lipid droplets (right panel) in 90 HCC patients. Scale bar 50 μm. The p value was generated using Spearman’s rank correlation test