Nlrp6过表达通过调控AMPK-Srebp1c轴抑制脂质合成抑制肝癌细胞的增殖

doi:10.12122/j.issn.1673-4254.2024.10.09

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (10): 1910-1917.doi: 10.12122/j.issn.1673-4254.2024.10.09

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Nlrp6过表达通过调控AMPK-Srebp1c轴抑制脂质合成抑制肝癌细胞的增殖

黄萃园(), 孙运平, 李文强, 刘丽, 王伟, 张静()

三峡大学第一临床医学院（宜昌市中心人民医院中心实验室）//缺血性心血管病湖北省重点实验室//湖北省缺血性心血管疾病临床医学研究中心，湖北宜昌 443003

收稿日期:2024-06-28 出版日期:2024-10-20 发布日期:2024-10-31
通讯作者: 张静 E-mail:284559087@qq.com;zhangjing@ctgu.edu.cn
作者简介:黄萃园，博士，主管药师，E-mail: 284559087@qq.com
基金资助:
国家自然科学基金(82300969);湖北省自然科学基金青年项目(2022CFB633);湖北省教育厅科中青年人才项目(Q20221211);湖北省重点研发计划(2022BCE001);湖北省卫生健康委面上项目(WJ2023M151);宜昌市医疗卫生研究项目(A24-2-018)

Nlrp6 overexpression inhibits lipid synthesis to suppress proliferation of hepatocellular carcinoma cells by regulating the AMPK-Srebp1c axis

Cuiyuan HUANG(), Yunping SUN, Wenqiang LI, Li LIU, Wei WANG, Jing ZHANG()

Central Laboratory, First College of Clinical Medical Science of China Three Gorges University (Yichang Central People's Hospital)//Hubei Key Laboratory of Ischemic Cardiovascular Disease//Hubei Provincial Clinical Research Center for Ischemic Cardiovascular Disease, Yichang 443003, China

Received:2024-06-28 Online:2024-10-20 Published:2024-10-31
Contact: Jing ZHANG E-mail:284559087@qq.com;zhangjing@ctgu.edu.cn
Supported by:
National Natural Science Foundation of China(82300969)

摘要/Abstract

摘要：

目的探讨Nlrp6通过调控脂质合成抑制肝细胞癌（HCC）进展的作用及其机制。方法通过分析癌症基因组图谱（TCGA）数据库中的RNA-seq数据和临床信息，评估Nlrp6在不同病理分级的HCC组织中的表达变化，并进行Kaplan-Meier生存分析及相关性分析。将腺病毒转染HepG2细胞过表达或敲低Nlrp6，使用棕榈酸（PA）构建肝脂肪变性模型。油红O染色评估Nlrp6对肝癌细胞HepG2脂质沉积的影响，CCK-8、Edu染色及克隆形成实验探讨Nlrp6对HepG2细胞增殖的影响，实时荧光定量PCR检测脂质合成相关基因的表达，Western blotting检测AMPK-Srebp1c轴相关蛋白的表达水平。使用肝脏特异性敲除Nlrp6小鼠，进行高脂饮食喂养24周，构建动物肝脂肪变性模型。取小鼠肝脏进行组织学染色，检测纤维化及肝癌标志物相关基因的表达，同时验证AMPK-Srebp1c信号通路的变化。结果生信分析结果显示在HCC患者肝脏组织中，Nlrp6的表达显著降低，且随着病理分级增加而进一步降低，同时Nlrp6的表达与患者生存期显著相关（P<0.0001）。细胞实验结果显示，Nlrp6过表达抑制了HepG2细胞的脂质沉积和细胞增殖，而Nlrp6敲低则产生相反效果（P<0.05）。q-PCR结果显示，Nlrp6可抑制脂质合成相关基因的表达（P<0.05），Western blotting结果表明过表达Nlrp6可促进AMPK的磷酸化，抑制调控脂质合成的转录因子Srebp1c的表达（P<0.05）。而Nlrp6敲低则抑制AMPK的磷酸化，促进Srebp1c的活化（P<0.05）。动物组织病理学染色结果表明，肝脏特异性敲除Nlrp6会促进肝脂肪变性及胶原沉积，q-PCR检测纤维化基因与染色结果一致（P<0.05），而肝癌标志物AFP的表达水平明显上升（P=0.06）。肝脏组织样本Western blotting结果证实Nlrp6的缺失会抑制AMPK的磷酸化，上调Srebp1c的表达（P<0.05）。结论 Nlrp6通过AMPK-Srebp1c轴抑制脂质合成，其可能是抑制肝癌细胞增殖的重要途径之一，进而改善HCC的进展。

关键词: Nlrp6, 肝癌细胞, 增殖, 脂质合成, 肝脂肪变性

Abstract:

Objective To investigate the mechanism of Nlrp6 for regulating hepatocellular carcinoma (HCC) progression in light of lipid synthesis regulation. Methods Nlrp6 expression level in HCC tissues of different pathological grades was investigated using RNA-seq data from The Cancer Genome Atlas (TCGA) database, and its correlation with the patients' survival was analyzed with Kaplan-Meier survival analysis. HepG2 cells with adenovirus-mediated Nlrp6 overexpression or knockdown were treated with palmitic acid (PA), and the changes in lipid deposition and cell proliferation were evaluated using Oil Red O staining, CCK-8 assay, EdU staining, and colony formation assay. RT-qPCR and Western blotting were used to detect the changes in expression of lipid synthesis-related genes and the proteins in the AMPK-Srebp1c axis. In a mouse model of hepatic steatosis established in liver-specific Nlrp6 knockout mice by high-fat diet feeding for 24 weeks, liver fibrosis was examined with histological staining, and the changes in expressions of HCC markers and the AMPK-Srebp1c signaling pathway were detected. Results Nlrp6 expression was significantly reduced in HCC tissues with negative correlations with the pathological grades and the patients' survival (P<0.0001). In HepG2 cells, Nlrp6 overexpression significantly inhibited lipid deposition and cell proliferation, whereas Nlrp6 knockdown produced the opposite effects. Nlrp6 overexpression strongly suppressed the expression of lipid synthesis-related genes, promoted AMPK phosphorylation, and inhibited Srebp1c expression. The mice with liver-specific Nlrp6 knockout and high-fat feeding showed increased hepatic steatosis, collagen deposition, and AFP expression with reduced AMPK phosphorylation and increased Srebp1c expression. Conclusion Nlrp6 overexpression inhibits lipid synthesis in HCC cells by regulating the AMPK-Srebp1c axis, which might be a key pathway for suppressing HCC cell proliferation.

Key words: Nlrp6, hepatocellular carcinoma cells, proliferation, lipid synthesis, hepatic steatosis

黄萃园, 孙运平, 李文强, 刘丽, 王伟, 张静. Nlrp6过表达通过调控AMPK-Srebp1c轴抑制脂质合成抑制肝癌细胞的增殖[J]. 南方医科大学学报, 2024, 44(10): 1910-1917.

Cuiyuan HUANG, Yunping SUN, Wenqiang LI, Li LIU, Wei WANG, Jing ZHANG. Nlrp6 overexpression inhibits lipid synthesis to suppress proliferation of hepatocellular carcinoma cells by regulating the AMPK-Srebp1c axis[J]. Journal of Southern Medical University, 2024, 44(10): 1910-1917.

图/表 6

图1 Nlrp6在HCC患者肝脏组织中表达降低

Fig.1 Reduced Nlrp6 expression in liver tissues is associated with poor survival outcomes of hepatocellular carcinoma (HCC) patients. A: Nlrp6 expression levels in HCC patients with varying pathological grades based on RNA-seq and clinical data of patients from The Cancer Genome Atlas (TCGA) dataset. B: Kaplan-Meier survival curve analysis based on TCGA dataset (n=371). C: Spearman correlation analysis between Nlrp6 expression and tumor proliferation markers using TCGA dataset (n=371). D: Comparative analysis of Nlrp6, lipid metabolism, and proliferation-related gene expressions in HCC tissues (n=371) versus adjacent tissues (n=50) using TCGA dataset. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

图2 Nlrp6抑制HepG2细胞脂质沉积

Fig.2 Nlrp6 overexpression inhibits lipid accumulation in HepG2 cells. A: Validation of Nlrp6 overexpression and knockdown (n=3). B: Oil Red O staining after stimulation with 0.5 μmol/L palmitic acid (PA) for 24 h. *P<0.05 vs Ad-NC; #P<0.05 vs Ad-shNC.

图3 Nlrp6抑制HepG2细胞增殖

Fig.3 Nlrp6 overexpression inhibits proliferation of HepG2 cells. A: CCK-8 assay for detecting cell proliferation (n=5). B: Colony formation assay. C: EdU staining. **P<0.01 vs Ad-shNC; #P<0.05 vs Ad-NC.

图4 Nlrp6通过脂代谢调控肝癌细胞增殖

Fig.4 Nlrp6 regulates HepG2 cell proliferation by modulating lipid metabolism. A: Q-PCR assay for assessing the effect of Nlrp6 down-regulation on expressions of genes related to lipid synthesis in HepG2 cells. B: Effect of Nlrp6 overexpression on expressions of genes related to lipid synthesis in HepG2 cells (n=3). *P<0.05 vs Ad-shNC; ##P<0.01 vs Ad-NC.

图5 AMPK-Srebp1c轴介导Nlrp6抑制HCC进展

Fig. 5 The AMPK-Srebp1c axis mediates Nlrp6 overexpression-induced inhibition of HepG2 cell proliferation. A: Western blotting for assessing the effect of Nlrp6 downregulation on the AMPK-Srebp1c signaling pathway in HepG2 cells. B: Western blotting for assessing the effect of Nlrp6 upregulation on the AMPK-Srebp1c signaling pathway in HepG2 cells. *P<0.05 vs Ad-shNC; #P<0.05 vs Ad-NC.

图6 肝脏特异性敲除Nlrp6通过AMPK-Srebp1c轴促进脂代谢紊乱诱导的损伤

Fig.6 Liver-specific Nlrp6 knockout promotes lipid metabolism disorder-induced injury via the AMPK-Srebp1c axis. A: HE staining, Masson staining and Sirius scarlet staining of mouse livers after modeling with high-fat diet (HFD) in wild-type mice (Loxp) and Nlrp6 liver-specific knockout mice (LKO). B: RT-qPCR for detecting expressions of the relevant genes in the liver tissues. C: Western blotting for assessing the effect of liver-specific NLRP6 knockout on the AMPK-Srebp1c signaling pathway. *P<0.05, **P<0.01 vs Loxp-HFD.

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Nlrp6过表达通过调控AMPK-Srebp1c轴抑制脂质合成抑制肝癌细胞的增殖

Nlrp6 overexpression inhibits lipid synthesis to suppress proliferation of hepatocellular carcinoma cells by regulating the AMPK-Srebp1c axis

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