脂肪酸代谢紊乱通过上调ZNF143促进胰腺癌进展的机制研究

doi:10.3969/j.issn.1674-8115.2024.10.007

摘要/Abstract

摘要：

目的·通过肿瘤转录组学筛选，明确在胰腺癌中因脂肪酸代谢紊乱引起上调的关键基因，并探究其中的锌指蛋白143（zinc finger protein 143，ZNF143）在胰腺癌中的表达及其对胰腺癌细胞迁移、侵袭能力的影响。方法·运用R语言整合Gene Expression Omnibus（GEO）数据库中GSE164760数据集、癌症基因组图谱（The Cancer Genome Atlas，TCGA）数据库中179例胰腺癌组织和4例癌旁组织及GTEx（Genotype-Tissue Expression）数据库中167例胰腺正常组织的转录组数据；筛选分析胰腺癌中脂肪酸紊乱可能会诱导表达的潜在差异基因；qRT-PCR检测使用棕榈酸（palmitic acid，PA）或油酸（oleic acid，OA）处理24 h后胰腺癌细胞中筛选基因的mRNA水平变化。按照筛选基因ZNF143表达水平的中位数，将TCGA数据库中胰腺癌患者分为高低表达2组，并将2组分析所得的差异基因进行京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）通路和基因本体论（Gene Ontology，GO）富集分析；通过细胞划痕实验、侵袭实验检测使用siRNA敲低ZNF143对胰腺癌细胞的迁移、侵袭能力的影响；使用蛋白质印迹法（Western blotting）检测敲低ZNF143对上皮-间质转化（epithelial-mesenchymal transition，EMT）相关蛋白及Wnt/β-catenin通路的影响。结果·分析了胰腺癌和肝癌中脂质代谢紊乱上调的关键基因，其中ZNF143是胰腺癌脂肪酸蓄积诱导的潜在基因之一；体外实验验证棕榈酸或油酸处理胰腺癌细胞后，ZNF143的mRNA水平均明显上调；KEGG和GO富集分析均显示ZNF143相关差异基因主要富集于细胞黏附相关通路；功能实验表明，转染ZNF143 siRNA的胰腺癌细胞迁移与侵袭能力均被下调，且EMT相关蛋白的表达也降低，可能与Wnt/β-catenin通路激活有关。结论·脂肪酸蓄积上调ZNF143在胰腺癌细胞中的mRNA表达含量，且ZNF143可能通过激活Wnt/β-catenin通路介导EMT，进而增强胰腺癌细胞的迁移和侵袭能力。

关键词: 胰腺癌, 锌指蛋白143, 脂肪酸蓄积, 生物信息学分析, 上皮间质-转化

Abstract:

Objective ·To identify key genes that may be regulated by fatty acid alteration in pancreatic cancer through tumor transcriptome screening, and to explore the expression of zinc finger protein 143 (ZNF143) in pancreatic cancer and its effect on the migration and invasion of pancreatic cancer cells. Methods ·The R language was utilized to integrate transcriptome data, including the GSE164760 dataset from the Gene Expression Omnibus (GEO) database, 179 pancreatic cancer tissue samples and 4 adjacent non-cancerous tissue samples from The Cancer Genome Atlas (TCGA) database, as well as 167 normal pancreatic tissue samples from the Genotype-Tissue Expression (GTEx) database. We conducted screening and analysis of potential differential genes that may be induced by dysregulation of fatty acid metabolism in pancreatic cancer. After treating pancreatic cancer cells with palmitic acid (PA) and oleic acid (OA) for 24 hours, the mRNA levels of candidate genes were detected by qRT-PCR. According to the median expression level of the screened gene, pancreatic cancer patients in the TCGA database were divided into two groups with high and low expression of ZNF143. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) enrichment analyses were performed for the differential genes of the two groups. siRNA was used to knock down the expression of ZNF143 in pancreatic cancer cells, and the effects on cell migration and invasion were examined by wound healing assay and invasion assay. Western blotting was used to explore the impact of ZNF143 on epithelial mesenchymal transition (EMT)-related proteins and the Wnt/β-catenin pathway. Results ·The bioinformatics database was processed to analyze key genes associated with the up-regulation of genes in lipid metabolism disorders in pancreatic cancer and liver cancer. Among them, ZNF143 was a potential gene associated with fatty acid accumulation in pancreatic cancer. In vitro experiments confirmed that the mRNA level of ZNF143 was significantly up-regulated after treating pancreatic cancer cells with palmitic acid or oleic acid. Both KEGG and GO enrichment analyses demonstrated that the differentially expressed genes associated with ZNF143 were predominantly enriched in adhesion pathways. In functional experiments, the migration and invasion abilities of pancreatic cancer cells transfected with ZNF143 siRNA were reduced, and the expression of EMT-related proteins was also decreased, potentially related to the activation of the Wnt/β- catenin pathway. Conclusion ·Fatty acid accumulation up-regulates the mRNA expression of ZNF143 in pancreatic cancer cells, and ZNF143 may enhance the migration and invasion of these cells by facilitating EMT through activation of the Wnt/β-catenin pathway.

Key words: pancreatic cancer, zinc finger protein 143 (ZNF143), fatty acid accumulation, bioinformatics analysis, epithelial mesenchymal transition (EMT)

中图分类号:

R735.9

俞思薇, 徐梓淇, 陶梦玉, 范广建. 脂肪酸代谢紊乱通过上调ZNF143促进胰腺癌进展的机制研究[J]. 上海交通大学学报（医学版）, 2024, 44(10): 1255-1265.

YU Siwei, XU Ziqi, TAO Mengyu, FAN Guangjian. Mechanistic study on the promotion of pancreatic cancer progression through upregulation of ZNF143 by dysregulated fatty acid metabolism[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(10): 1255-1265.

图/表 6

表1 siRNA及qRT-PCR引物序列表

Tab 1 Sequences of siRNA and qRT-PCR Primer

Target gene		Forward (5′→3′)	Reverse (5′→3′)
siRNA	Control siRNA	UUCUCCGAACGUGUCACGUTT	ACGUGACACGUUCGGAGAATT
	ZNF143 siRNA#1	GGACAUGCUACAAGAGUAATT	UUACUCUUGUAGCAUGUCCTT
	ZNF143 siRNA#2	GGAAGAAGCCAUCAGAAUATT	UAUUCUGAUGGCUUCUUCCTT
qPCR Primer	FAS	TCTGGTTCTTACGTCTGTTGC	CTGTGCAGTCCCTAGCTTTCC
	ZNF143	AAGTCCCGCAGTCTGACAC	CCTGCTACACTTTCACTTCCATC
	HNRNPA1L2	TCTAAGTCAGCGTCTCCAAAAGA	TGGCGTTGTATTCATAGCTGC

图1 筛选脂肪酸紊乱在胰腺癌中影响的相关基因Note： A. Comparison of the differential genes among the four groups in the GSE164760 dataset. B. Venn diagram of selected target genes in three differential gene sets. C. The expression levels of 28 candidate genes in Healthy, NASH (non-alcoholic steatohepatitis), NASH-PNT (non-alcoholic steatohepatitis-related paracancerous normal tissue) and NASH-T (non-alcoholic steatohepatitis-related hepatocellular carcinoma) by box plot. D. Expression of 28 candidate genes in pancreatic cancer in TCGA database. E—G. Effect of expression levels of FAS (E), ZNF143 (F), and HNRNPA1L2 (G) on the prognosis of patients with pancreatic cancer. ①P<0.01, ②P<0.05, ③P<0.001, ④P=0.014, ⑤P=0.063, ⑥P=0.289.

Fig 1 Screening for genes in pancreatic cancer that are affected by fatty acid disorders

图2 脂肪酸蓄积上调胰腺癌中 ZNF143 的表达Note： A. Correlation analysis of the expression of FAS, ZNF143, and HNRNPA1L2 with fatty acid degradation or fatty acid biosynthesis-related genes in pancreatic cancer. B?D. mRNA expression levels of FAS (B), ZNF143 (C), and HNRNPA1L2 (D) in AsPC-1 and Capan2 cells treated with BSA control or BSA-bound FFAs for 24 h following a 12 h?16 h starvation period. ①P=0.018, ②P=0.003, ③P<0.001, ④P=0.002.

Fig 2 Fatty acid accumulation up-regulates the expression of ZNF143 in pancreatic cancer

图3 TCGA数据库中 ZNF143 相关信号通路和功能富集分析Note： A. Differentially expressed genes associated with ZNF143 in pancreatic cancer. B. GO enrichment analysis. C. KEGG pathway enrichment analysis.

Fig 3 KEGG analysis and GO analysis of ZNF143 in TCGA database

图4 敲低 ZNF143 抑制AsPC-1和Capan2细胞迁移、侵袭能力Note： A. Detection of the expression level of ZNF143 in different pancreatic cancer cells by Western blotting. B. ZNF143 expression levels in AsPC-1 and Capan2 cells transfected with Control siRNA or ZNF143 siRNA were measured by Western blotting. C/D. Effects of ZNF143 siRNA on the migration (C) and invasion (D) ability of AsPC-1 and Capan2 cells. ①P=0.003, ②P=0.007, ③P=0.005, ④P=0.029, ⑤P<0.001, ⑥P=0.002.

Fig 4 Knockdown of ZNF143 inhibited the migration and invasion ability of AsPC-1 and Capan2 cells

图5 敲低 ZNF143 下调AsPC-1和Capan2细胞中EMT、Wnt/β-catenin通路相关蛋白的表达Note： A. Correlation analysis of ZNF143 expression and EMT-related genes in pancreatic cancer. B/C. Detection of EMT and Wnt/β-catenin pathway-related proteins in AsPC-1 (B) and Capan2 (C) after ZNF143 silencing by western blotting.

Fig 5 Knockdown of ZNF143 down-regulated the expression of proteins related to EMT and Wnt/β-catenin pathway in AsPC-1 and Capan2 cells

参考文献 35

1	HALBROOK C J, LYSSIOTIS C A, PASCA DI MAGLIANO M, et al. Pancreatic cancer: advances and challenges[J]. Cell, 2023, 186(8): 1729-1754.
2	QIAN Y Z, GONG Y T, FAN Z Y, et al. Molecular alterations and targeted therapy in pancreatic ductal adenocarcinoma[J]. J Hematol Oncol, 2020, 13(1): 130.
3	SINN M, BAHRA M, LIERSCH T, et al. CONKO-005: adjuvant chemotherapy with gemcitabine plus erlotinib versus gemcitabine alone in patients after R0 resection of pancreatic cancer: a multicenter randomized phase III trial[J]. J Clin Oncol, 2017, 35(29): 3330-3337.
4	PERKHOFER L, GOUT J, ROGER E, et al. DNA damage repair as a target in pancreatic cancer: state-of-the-art and future perspectives[J]. Gut, 2021, 70(3): 606-617.
5	ENCARNACIÓN-ROSADO J, KIMMELMAN A C. Harnessing metabolic dependencies in pancreatic cancers[J]. Nat Rev Gastroenterol Hepatol, 2021, 18(7): 482-492.
6	HALBROOK C J, THURSTON G, BOYER S, et al. Differential integrated stress response and asparagine production drive symbiosis and therapy resistance of pancreatic adenocarcinoma cells[J]. Nat Cancer, 2022, 3(11): 1386-1403.
7	YIN X P, XU R Y, SONG J L, et al. Lipid metabolism in pancreatic cancer: emerging roles and potential targets[J]. Cancer Commun, 2022, 42(12): 1234-1256.
8	MENENDEZ J A, LUPU R. Fatty acid synthase and the lipogenic phenotype in cancer pathogenesis[J]. Nat Rev Cancer, 2007, 7(10): 763-777.
9	AUCIELLO F R, BULUSU V, OON C, et al. A stromal lysolipid-autotaxin signaling axis promotes pancreatic tumor progression[J]. Cancer Discov, 2019, 9(5): 617-627.
10	BIAN X L, LIU R, MENG Y, et al. Lipid metabolism and cancer[J]. J Exp Med, 2021, 218(1): e20201606.
11	JEONG D W, PARK J W, KIM K S, et al. Palmitoylation-driven PHF2 ubiquitination remodels lipid metabolism through the SREBP1c axis in hepatocellular carcinoma[J]. Nat Commun, 2023, 14(1): 6370.
12	CHEN X M, LI L Z, LIU X H, et al. Oleic acid protects saturated fatty acid mediated lipotoxicity in hepatocytes and rat of non-alcoholic steatohepatitis[J]. Life Sci, 2018, 203: 291-304.
13	SHEN C J, CHAN R H, LIN B W, et al. Oleic acid-induced metastasis of KRAS/p53-mutant colorectal cancer relies on concurrent KRAS activation and IL-8 expression bypassing EGFR activation[J]. Theranostics, 2023, 13(13): 4650-4666.
14	KUBO M, GOTOH K, EGUCHI H, et al. Impact of CD36 on chemoresistance in pancreatic ductal adenocarcinoma[J]. Ann Surg Oncol, 2020, 27(2): 610-619.
15	YE B Y, YANG G G, LI Y M, et al. ZNF143 in chromatin looping and gene regulation[J]. Front Genet, 2020, 11: 338.
16	YE B Y, SHEN W L, ZHANG C Y, et al. The role of ZNF143 overexpression in rat liver cell proliferation[J]. BMC Genomics, 2022, 23(1): 483.
17	CHEN X, FANG F, LIOU Y C, et al. Zfp143 regulates Nanog through modulation of Oct4 binding[J]. Stem Cells, 2008, 26(11): 2759-2767.
18	MYSLINSKI E, GÉRARD M A, KROL A, et al. A genome scale location analysis of human Staf/ZNF143-binding sites suggests a widespread role for human Staf/ZNF143 in mammalian promoters[J]. J Biol Chem, 2006, 281(52): 39953-39962.
19	NGONDO R P, CARBON P. ZNF143 is regulated through alternative 3'UTR isoforms[J]. Biochimie, 2014, 104: 137-146.
20	IZUMI H, WAKASUGI T, SHIMAJIRI S, et al. Role of ZNF143 in tumor growth through transcriptional regulation of DNA replication and cell-cycle-associated genes[J]. Cancer Sci, 2010, 101(12): 2538-2545.
21	KAWATSU Y, KITADA S, URAMOTO H, et al. The combination of strong expression of ZNF143 and high MIB-1 labelling index independently predicts shorter disease-specific survival in lung adenocarcinoma[J]. Br J Cancer, 2014, 110(10): 2583-2592.
22	PAEK A R, MUN J Y, JO M J, et al. The role of ZNF143 in breast cancer cell survival through the NAD(P)H quinone dehydrogenase 1-p53-Beclin1 axis under metabolic stress[J]. Cells, 2019, 8(4): 296.
23	VERMA V, PAEK A R, CHOI B K, et al. Loss of zinc-finger protein 143 contributes to tumour progression by interleukin-8-CXCR axis in colon cancer[J]. J Cell Mol Med, 2019, 23(6): 4043-4053.
24	CARRER A, TREFELY S, ZHAO S, et al. Acetyl-CoA metabolism supports multistep pancreatic tumorigenesis[J]. Cancer Discov, 2019, 9(3): 416-435.
25	TADROS S, SHUKLA S K, KING R J, et al. De novo lipid synthesis facilitates gemcitabine resistance through endoplasmic reticulum stress in pancreatic cancer[J]. Cancer Res, 2017, 77(20): 5503-5517.
26	LI J, GU D, LEE S S, et al. Abrogating cholesterol esterification suppresses growth and metastasis of pancreatic cancer[J]. Oncogene, 2016, 35(50): 6378-6388.
27	NATH A, LI I, ROBERTS L R, et al. Elevated free fatty acid uptake via CD36 promotes epithelial-mesenchymal transition in hepatocellular carcinoma[J]. Sci Rep, 2015, 5: 14752.
28	PAN J M, FAN Z Y, WANG Z Q, et al. CD36 mediates palmitate acid-induced metastasis of gastric cancer via AKT/GSK-3β/β- catenin pathway[J]. J Exp Clin Cancer Res, 2019, 38(1): 52.
29	MARTIN-MORENO J M, WILLETT W C, GORGOJO L, et al. Dietary fat, olive oil intake and breast cancer risk[J]. Int J Cancer, 1994, 58(6): 774-780.
30	YANG P, SU C X, LUO X, et al. Dietary oleic acid-induced CD36 promotes cervical cancer cell growth and metastasis via up-regulation Src/ERK pathway[J]. Cancer Lett, 2018, 438: 76-85.
31	NEUSCHWANDER-TETRI B A. Hepatic lipotoxicity and the pathogenesis of nonalcoholic steatohepatitis: the central role of nontriglyceride fatty acid metabolites[J]. Hepatology, 2010, 52(2): 774-788.
32	HUNING L, KUNKEL G R. Two paralogous znf143 genes in zebrafish encode transcriptional activator proteins with similar functions but expressed at different levels during early development[J]. BMC Mol Cell Biol, 2020, 21(1): 3.
33	PAEK A R, LEE C H, YOU H J. A role of zinc-finger protein 143 for cancer cell migration and invasion through ZEB1 and E-cadherin in colon cancer cells[J]. Mol Carcinog, 2014, 53(Suppl 1): E161-E168.
34	FENG Y L, CHEN D Q, VAZIRI N D, et al. Small molecule inhibitors of epithelial-mesenchymal transition for the treatment of cancer and fibrosis[J]. Med Res Rev, 2020, 40(1): 54-78.
35	WEN Z, HUANG Z T, ZHANG R, et al. ZNF143 is a regulator of chromatin loop[J]. Cell Biol Toxicol, 2018, 34(6): 471-478.

[1]	杜少倩, 陶梦玉, 曹源, 王红霞, 胡孝渠, 范广建, 臧丽娟. CXCL9在乳腺癌中的表达及其与肿瘤免疫浸润特征的相关性研究[J]. 上海交通大学学报（医学版）, 2023, 43(7): 860-872.
[2]	秦雅含, 张珂, 张梦雨, 沈洁, 彭美玉. MDSC靶向免疫治疗胰腺癌的研究进展[J]. 上海交通大学学报（医学版）, 2023, 43(10): 1317-1323.
[3]	马芳芳, 秦洁洁, 任灵杰, 唐笑梅, 刘佳, 施敏敏, 蒋玲曦. 基于水凝胶微球建立胰腺癌原代细胞的3D培养模型[J]. 上海交通大学学报（医学版）, 2023, 43(1): 79-87.
[4]	许静轩, 杜少倩, 曹源, 王红霞, 黄伟翼. MMP14在胰腺癌中的表达及其与肿瘤免疫微环境特征的相关性研究[J]. 上海交通大学学报（医学版）, 2022, 42(3): 312-322.
[5]	王建茹, 彭广操, 朱明军. 基于GEO数据库和生物信息学分析筛选小鼠心肌缺血再灌注损伤相关的潜在枢纽基因[J]. 上海交通大学学报(医学版), 2022, 42(1): 51-62.
[6]	刘俐, 耿子龙, 陈嘉焕, 张沙沙, 张冰. 血管内皮生长因子A调控人脐静脉内皮细胞miRNA的全基因表达谱分析[J]. 上海交通大学学报(医学版), 2021, 41(9): 1183-1189.
[7]	李静威, 王俐文, 蒋玲曦, 詹茜, 陈皓, 沈柏用. 胰腺癌免疫抑制性肿瘤微环境研究综述[J]. 上海交通大学学报(医学版), 2021, 41(8): 1103-1108.
[8]	杨鹿笛, 王高明, 胡仁豪, 蒋小华, 崔然. 生物信息学方法筛选胰腺癌进展相关的核心基因[J]. 上海交通大学学报(医学版), 2021, 41(5): 571-578.
[9]	高境泽，吴霞. 卵巢肿瘤组织中CXCL9 mRNA表达与患者的预后、免疫微环境特征的相关性研究[J]. 上海交通大学学报（医学版）, 2020, 40(4): 457-.
[10]	张伟然1, 2，林雪峰3，李鑫2，张浩2，王猛2，孙伟2，韩兴鹏2，孙大强1, 4. 转录组分析鉴定肺腺癌潜在的生物标志物[J]. 上海交通大学学报(医学版), 2020, 40(12): 1598-1606.
[11]	梁雨，江明杰，田聆. 前列腺素 E 2重塑胰腺肿瘤微环境的作用机制研究进展[J]. 上海交通大学学报（医学版）, 2019, 39(8): 923-.
[12]	褚以忞 1，周锋利 1，徐莹 1，蒯榕 1，李吉 1，侯照远 2，杨大明 1，彭海霞 1. VTCN1调控结肠癌细胞长链非编码 RNA和 mRNA的全基因表达谱分析[J]. 上海交通大学学报（医学版）, 2019, 39(3): 270-.
[13]	马静，沈鸣，段友容，杜联芳 . 载紫杉醇纳米诊疗体的制备及初步应用[J]. 上海交通大学学报（医学版）, 2017, 37(2): 166-.
[14]	李小平，张晓伟，郭伟剑，等. CD44在胰腺癌中的表达及其临床意义[J]. 上海交通大学学报（医学版）, 2015, 35(9): 1354-.
[15]	胡斌，许磊，白永瑞，等. 117例无远处转移胰腺癌的放疗效果分析[J]. 上海交通大学学报（医学版）, 2015, 35(8): 1153-.