Mechanism Study of SIRT3 Enhancing Chemosensitivity of Rectal Cancer Cells to 5-Fluorouracil via FoxO3a/BNIP3-Mediated Mitophagy
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摘要:
目的 探讨沉默信息调节因子3（Sirtuin 3，SIRT3）在结直肠癌细胞5-氟尿嘧啶（5-FU）耐药中的作用，并阐明其是否通过调控叉头框蛋白O3a（Forkhead box O3a，FoxO3a）/Bcl-2相互作用蛋白3（BCL2 interacting protein 3，BNIP3）介导的线粒体自噬来增强肿瘤细胞对5-FU的敏感性。 方法 建立5-FU耐药CRC细胞系；构建SIRT3稳定过表达细胞系；CCK-8检测IC50值；流式检测细胞凋亡；Western Blot检测SIRT3、FoxO3a、BNIP3及线粒体自噬相关蛋白微管相关蛋白1轻链3（microtubule-associated protein 1 light chain 3，LC3）、死骨片1（sequestosome 1，p62）、PTEN诱导假定激酶1（PTEN-induced putative kinase 1，PINK1）、Parkin RBR E3泛素蛋白连接酶（Parkin RBR E3 ubiquitin-protein ligase，Parkin）；免疫荧光观察线粒体与自噬体共定位；免疫共沉淀检测FoxO3a乙酰化水平；构建裸鼠皮下异种移植瘤模型，评估SIRT3过表达体内抗肿瘤效果。 结果 5-FU耐药CRC细胞中SIRT3、FoxO3a及BNIP3蛋白表达显著下调（P < 0.05），线粒体与自噬体共定位减少，线粒体自噬水平受抑制。过表达SIRT3后，5-FU耐药细胞IC50值显著降低，凋亡率升高（P < 0.01），LC3-II/I比值升高，p62表达下降，PINK1和Parkin表达上调（P < 0.05），降低FoxO3a乙酰化水平，上调BNIP3表达。SIRT3过表达联合5-FU组可显著抑制肿瘤生长（P < 0.01），且肿瘤组织中自噬相关蛋白表达变化与体外一致，自噬小体增加。 结论 SIRT3通过去乙酰化激活FoxO3a/BNIP3通路诱导线粒体自噬，逆转CRC 5-FU耐药，联合治疗具协同抗瘤作用。 Abstract:Objective To investigate the role of Sirtuin 3 （SIRT3） in 5-fluorouracil （5-FU） resistance of colorectal cancer （CRC） cells and to elucidate whether it enhances tumor cell sensitivity to 5-FU by regulating Forkhead box O3a （FoxO3a）/BCL2 interacting protein 3 （BNIP3）-mediated mitophagy. Methods A 5-FU-resistant CRC cell line was established, and a SIRT3 stably overexpressing cell line was constructed via lentiviral transfection. The half-maximal inhibitory concentration （IC50） was measured by CCK-8 assay, and apoptosis was detected by flow cytometry. Western blot was used to detect the expression of SIRT3, FoxO3a, BNIP3, and mitophagy-associated proteins including microtubule-associated protein 1 light chain 3 （LC3）, sequestosome 1 （p62）, PTEN-induced putative kinase 1 （PINK1）, and Parkin RBR E3 ubiquitin-protein ligase （Parkin）. Co-localization of mitochondria and autophagosomes was observed by immunofluorescence staining. FoxO3a acetylation level was assessed by co-immunoprecipitation. A subcutaneous xenograft tumor model in nude mice was established to evaluate the anti-tumor effect of SIRT3 overexpression in vivo. Results The protein expression of SIRT3, FoxO3a, and BNIP3 was significantly downregulated in 5-FU-resistant CRC cells compared with parental cells （P < 0.05）, accompanied by decreased co-localization of mitochondria and autophagosomes. Overexpression of SIRT3 markedly reduced the IC50 value of 5-FU-resistant cells, increased the apoptosis rate （P < 0.01）, elevated the LC3-II/I ratio, decreased p62 expression, and upregulated PINK1 and Parkin expression （P < 0.05）. Furthermore, SIRT3 overexpression decreased FoxO3a acetylation and increased BNIP3 expression. The combination of SIRT3 overexpression and 5-FU significantly inhibited tumor growth in vivo （P < 0.01）, and the changes in autophagy-associated protein expression in tumor tissues were consistent with the in vitro results, with an increase in autophagosomes. Conclusion SIRT3 reverses 5-FU resistance in CRC cells by deacetylating and activating the FoxO3a/BNIP3 pathway to induce mitophagy, and combination therapy exerts a synergistic anti-tumor effect. -
Key words:
- Colorectal cancer /
- SIRT3 /
- 5-Fluorouracil /
- Chemoresistance /
- Mitophagy /
- FoxO3a /
- BNIP3
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图 1 5-FU耐药的CRC细胞中SIRT3表达下调且线粒体自噬受损（$ \bar x \pm s $，n = 3）
A： CCK-8法检测细胞增殖；B： 细胞中蛋白表达水平；C.：Mito-Tracker和LC3荧光染色；*P < 0.05；**P < 0.01；***P < 0.001。
Figure 1. SIRT3 expression is downregulated and mitophagy is impaired in 5-FU-resistant CRC cells （$ \bar x \pm s $，n = 3）
图 2 SIRT3过表达可逆转CRC细胞的5-FU耐药性（$ \bar x \pm s $，n = 3）
A： HCT116-R和HT-29-R细胞中稳定过表达SIRT3；B： CCK-8检测细胞增殖；C： 流式细胞术检测细胞凋亡；*P < 0.05；**P < 0.01；***P < 0.001。
Figure 2. SIRT3 overexpression reverses 5-FU resistance in CRC cells （$ \bar x \pm s $，n = 3）
图 3 SIRT3通过激活线粒体自噬来增强5-FU的化疗敏感性（$ \bar x \pm s $，n = 3）
A：过表达SIRT3后自噬相关蛋白表达；B：使用自噬抑制剂Mdivi-1后自噬相关蛋白表达；C：CCK-8检测细胞增殖；D：流式细胞术检测细胞凋亡；*P < 0.05；**P < 0.01；***P < 0.001。
Figure 3. SIRT3 enhanceed chemosensitivity of5-FU by activating mitophagy （$ \bar x \pm s $，n = 3）
图 4 SIRT3通过FoxO3a/BNIP3信号通路诱导线粒体自噬（$ \bar x \pm s $，n = 3）
A：乙酰化检测；B. BNIP3的蛋白表达水平；C：自噬相关蛋白表达；*P < 0.05；**P < 0.01。
Figure 4. SIRT3 induces mitophagy via the FoxO3a/BNIP3 signaling pathway （$ \bar x \pm s $，n = 3）
图 5 SIRT3激动剂联合5-FU在体内协同抑制CRC肿瘤生长（$ \bar x \pm s $，n = 5）
A：肿瘤示意图；B：肿瘤生长曲线；C：肿瘤质量；D：肿瘤细胞增殖；E：肿瘤细胞凋亡；F：肿瘤细胞凋亡相关蛋白表达；*P < 0.05；**P < 0.01；***P < 0.001。
Figure 5. SIRT3 agonist combined with 5-FU synergistically inhibits CRC tumor growth in vivo （$ \bar x \pm s $，n = 5）
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