Effect and mechanism of acetyl-CoA carboxylase 1 in regulating the proliferation of hepatocellular carcinoma cells
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摘要:
为了探究巨噬细胞如何促进肝癌细胞的脂滴囤积，并深入研究脂滴的关键代谢酶在肝癌细胞恶性生物学中的作用，利用肿瘤相关巨噬细胞（tumor-associated macrophages, TAMs）上清液诱导Hepa1-6脂滴（lipid droplets, LDs）生成，发现TAMs中的白细胞介素10（interleukin-10, IL-10）能够促进脂滴的积累。乙酰辅酶 A 羧化酶（acetyl-CoA carboxylase alpha, ACC1）是脂肪酸合成的关键酶之一，影响着肝癌的发生发展。本研究发现，ACC1在LDhigh Hepa1-6中高表达，之后通过ACC1的小分子抑制剂、siRNA干扰和CRISPR-cas9敲除等手段阻断ACC1的活性，发现Hepa1-6脂滴积累降低，同时减少了Hepa1-6增殖的恶性生物学行为，促进了Hepa1-6发生凋亡事件。综上所述，TAMs释放的IL-10促进了肝癌细胞的脂滴形成，导致肝癌细胞的恶性增殖与凋亡。ACC1在TAMs促进肝癌细胞脂滴积累过程中发挥关键作用，并且可能受TAMs释放的IL-10的调控，这些发现可为肝癌治疗提供新的靶点。
Abstract:In order to investigate how macrophages promote lipid droplet hoarding in hepatocellular carcinoma cells and to delve into the roles of key metabolic enzymes of lipid droplets in the malignant biology of hepatocellular carcinoma cells, the present study was conducted to induce the generation of Hepa1-6 lipid droplets (LDs) using supernatants from tumor-associated macrophages (TAMs), and found that interleukin-10 (IL-10) in TAMs was found to promote the accumulation of lipid droplets. Acetyl-CoA carboxylase alpha (ACC1) is one of the key enzymes for fatty acid synthesis and influences the development of hepatocellular carcinoma. In this study, ACC1 was found to be highly expressed in LDhigh Hepa1-6, and subsequent blockade of ACC1 activity by means of a small molecule inhibitor of ACC1, siRNA interference, and CRISPR-cas9 knockdown was found to reduce the accumulation of Hepa1-6 lipid droplets, as well as to reduce the malignant biological behavior of Hepa1-6 proliferation, and to promote the occurrence of apoptotic events. In summary, IL-10 released by TAMs promoted lipid droplet formation in hepatocellular carcinoma cells, leading to malignant proliferation and apoptosis of hepatocellular carcinoma cells. ACC1 plays a key role in the promotion of lipid droplet accumulation in hepatocellular carcinoma cells by TAMs and may be regulated by IL-10 released by TAMs, and these findings may provide a new target for hepatocellular carcinoma treatment.
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Figure 1. tumor-associated macrophage (TAMs) model establishment
A: q-PCR for CD206, ARG1, VEGFα, IL-10, inducible nitric oxide synthase (iNOS), interleukin-12 subunit alpha (IL12A), TNFα mRNA expression levels; B: flow cytometry was used to detect the expression of PD-L1 in TAMs*P < 0. 05,**P < 0. 01,***P < 0. 001
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Figure 2. LDhigh Hepa1-6 cell model constructs and TAMs infiltration induced accumulation of LDs in hepatocellular carcinoma cells
A and B: Hepa1-6 was stimulated with different concentrations of TAMs supernatants (10%, 20%, 30%, 40%), stained with BODIPY 493/503, and LDs accumulation in Hepa1-6 was detected by flow cytometry; C and D: 30% TAMs supernatants were stimulated for 1, 2 and 3 days, stained with BODIPY 493/503 and flow cytometry was used to detect the accumulation of LDs in Hepa1-6; E: Oil Red O labelled LDs; F: q-PCR for Plin 1, Plin 2, Plin 3, Plin 4, Plin5 mRNA expression levels*P < 0. 05, **P < 0. 01,***P < 0. 001 vs control group
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Figure 3. IL-10 secretion by TAMs induces accumulation of LDs in hepatocellular carcinoma cells A: q-PCR for IL-10, TGFβ, IL-12, TNFα mRNA expression levels; B and C: Stimulation with different concentrations of IL-10, BODIPY 493/503 staining, and flow cytometric detection of LDs accumulation in Hepa1-6, MFI: Mean fluorescence intensity
*P < 0. 05,**P < 0. 01,***P < 0. 001
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Figure 4. IL-10 deletion affects lipid droplet formation, cell proliferation and apoptosis
A: Oil Red O labelled LDs; B: q-PCR for ACC1, Plin4, Plin5 mRNA expression levels; C: CFSE staining labelling to detect the effect of IL-10 deletion on the proliferation of Hepa1-6 cells; D and E: Effects of IL-10 deletion on apoptosis in Hepa1-6 cells*P < 0. 05 ,**P < 0. 01, ***P < 0. 001 vs control group
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Figure 5. High expression of ACC1 in LDhigh Hepa1-6 cells and implications for cancer
A: q-PCR for ACC1, FASN, ACSL, DGAT1, DGAT2 mRNA expression levels; B: Western blotting for ACC1 protein levels; C: ACC1 expression levels in clinical samples of hepatocellular carcinoma compared with normal tissues; D: ACC1 expression levels in different hepatocellular carcinoma stages (stage1, stage2, stage3, stage4); E: ACC1 expression levels in different hepatocellular carcinoma grades (grade1, grade2, grade3, grade4); F: Survival curves of patients with ACC1 high expression and F: Survival curves of patients with high ACC1 expression and low ACC1 expression; G: Survival curves of patients with high ACC1 expression and low ACC1 expression in different liver cancer grades (grade1, grade2, grade3, grade4) (*P < 0. 05,**P < 0. 01,***P < 0. 001)
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Figure 6. ACC1 mediates the accumulation of LDs in LDhigh Hepa1-6 cells
A: Western blotting to detect ACC1 protein level; B and C: BODIPY 493/503 staining and flow cytometry to detect the accumulation of LDs in TOFA and ND-630-treated Hepa1-6 cells, MFI: Mean fluorescence intensity; D: Oil Red O labelling of LDs; E: TG content of the cells; F: q-PCR to detect the expression level of ACC1 mRNA and assess the effect of ACC1-siRNA interference; G: Western blotting to detect ACC1 expression level and assess the effect of ACC1-siRNA interference; H and I: BODIPY 493/503 staining and flow cytometry to detect the effect of siRNA interference on the accumulation of LDs in Hepa1-6, MFI: Mean fluorescence intensity (*P < 0. 05,**P < 0. 01,***P < 0. 001 vs control group)
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Figure 7. ACC1 knockdown inhibits the accumulation of LDs in LDhigh Hepa1-6 cells
A: Western blotting to detect the expression level of ACC1 and evaluate the effect of ACC1 knockdown; B and C: BODIPY 493/503 staining and flow cytometry to detect the effect of ACC1 knockdown on the accumulation of LDs in Hepa1-6; D: triglyceride (TG) content of the cells, MFI: Mean fluorescence intensity*P < 0. 05,**P < 0. 01,***P < 0. 001
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Figure 8. ACC1 affects Hepa1-6 cell viability
A: Detection of LDH release to assess the toxicity of TOFA and ND-630 on Hepa1-6 cells; B: CCK-8 to detect the effect of TOFA and ND-630 on the viability of Hepa1-6 cells; C: CCK-8 to detect the effect of ACC1 knockdown on the viability of Hepa1-6 cells***P < 0. 001, **P < 0. 01, *P < 0. 05
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Figure 9. ACC1 regulates Hepa1-6 cell proliferation
A: CFSE staining labeling to detect the effect of TOFA and ND-630 on the proliferation of Hepa1-6 cells; B: CFSE staining labeling to detect the effect of ACC1 knockdown on the proliferation of Hepa1-6 cells; C and D: Effect of TOFA and ND-630 on the cell cycle of Hepa1-6 cells; E and F: The effect of ACC1 knockdown on the cell cycle of Hepa1-6 cells
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Figure 10. ACC1 regulates apoptosis in Hepa1-6 cells
A and B: Effects of TOFA and ND-630 on apoptosis in Hepa1-6 cells; C and D: Effects of ACC1 knockdown on apoptosis in Hepa1-6 cells*P < 0. 05,**P < 0. 01,***P < 0. 001
Table 1 Sequence for guide RNA (gRNA) about acetyl-CoA carboxylase alpha (ACC1)
gRNA Forward primer Reverse primer gRNA-1 CACCGAATGCATGCGATCTATCCGT AAACACGGATAGATCGCATGCATTC gRNA-2 CACCGAAGTGTATCTGAGCTGACGG AAACCCGTCAGCTCAGATACACTTC gRNA-3 CACCGCAAACGTGAATGCTTGACCA AAACTGGTCAAGCATTCACGTTTGC 
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