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Journal of Southern Medical University logoLink to Journal of Southern Medical University
. 2023 Jul 20;43(7):1116–1126. [Article in Chinese] doi: 10.12122/j.issn.1673-4254.2023.07.08

猪重组NK-lysin抑制肝细胞性肝癌的转移：基于下调FKBP3表达、抑制氧化磷酸化和糖酵解

Procine recombinant NK-lysin inhibits hepatocellular carcinoma metastasis by downregulating FKBP3 and inhibiting oxidative phosphorylation and glycolysis: a proteomic analysis

Yifan FAN 1,2, Zhiwei FENG 1,2, Kuohai FAN 1,2, wei YIN 1,2, Na SUN 1,2, Panpan SUN 1,2, Yaogui SUN 1,2, Hongquan LI 1,*
PMCID: PMC10366521  PMID: 37488794

Abstract

Objective

To investigate the potential mechanisms that mediate the inhibitory effect of porcine recombinant NKlysin (prNK-lysin) against liver cancer cell metastasis.

Methods

HPLC-tandem mass spectrometry was used to identify the differentially expressed proteins in prNK-lysin-treated hepatocellular carcinoma SMMOL/LC-7721 cells in comparison with the control and PBS-treated cells. GO functional annotation and KEGG pathway analysis of the differentially expressed proteins were performed using GO and KEGG databases. RT-qPCR was used to determine the mRNA expression levels of polypeptide-N-acetylgalactosaminotransferase 13 (GALNT13), transmembrane protein 51 (TMEM51) and FKBP prolyl isomerase 3 (FKBP3) in the cells, and the protein expression of FKBP3 was verified using Western blotting.

Results

Proteomic analysis identified 1989 differentially expressed proteins in prNK-lysin-treated cells compared with the control cells, and 2753 compared with PBS-treated cells. Fifteen proteins were differentially expressed between PBS-treated and the control cells, and 1909 were differentially expressed in prNK- lysin group compared with both PBS and control groups. These differentially expressed proteins were involved mainly in the viral process, translational initiation and RNA binding and were enriched mainly in ribosome, protein process in endoplasmic reticulum, and RNA transport pathways. RT-qPCR showed that compared with the control group, prNK-lysin treatment significantly increased the mRNA expressions of GALNT13 (P < 0.05) and TMEM51 (P < 0.01) and lowered FKBP3 mRNA expression (P < 0.05). Western blotting also showed a significantly decreased expression of FKBP3 protein in prNK-lysin-treated cells (P < 0.001).

Conclusion

Treatment with prNK-lysin causes significant changes in protein expression profile of SMMOL/LC-7721 cells and inhibits hepatocellular carcinoma metastasis by downregulating FKBP3 protein and affecting the cellular oxidative phosphorylation and glycolysis pathways.

Keywords: hepatocellular carcinoma, proteomics, porcine recombinant NK-lysin, differentially expressed proteins

原发性肝癌是2020年全球第6大最常见的癌症，也是癌症死亡的第3大原因[1]，其中肝细胞癌（HCC）是最常见的肝癌类型，占原发性肝癌的75%~85%[2]，病程快、易转移和高复发是HCC的主要特点[3, 4]。目前，HCC仍主要通过手术切除、肝移植和全身治疗来治疗，临床上主要应用Sorafenib[5]，Regorafenib[6]、Lenvatinib[7]等药物治疗晚期肝癌。但这些方法仍有一定的局限性和不良反应[8]，因此仍需通过研究寻找治疗肝癌的方法和药物。

目前，治疗癌症药物研究的主要有分子靶向药物[9]，纳米技术药物[10]，抗菌肽[11]等。其中研究表明阳离子抗菌肽（CAPs）具有抗癌活性，带正电荷的CAPs和带负电荷的癌细胞膜之间的静电相互作用能够导致细胞溶解并选择性的杀死癌细胞[12]。还有部分抗菌肽在选择性的进入癌细胞后可以破坏线粒体膜，从而诱导癌细胞凋亡[13]。NK-lysin是一种由细胞毒性T淋巴细胞和NK细胞产生的阳离子抗菌肽[14]，对多种细菌和真菌有抑制作用[15, 16]，且能够抑制癌细胞的增殖[17]。本课题组前期研究发现猪重组NK-lysin（prNK-lysin）可以显著抑制肝癌细胞系（SMMOL/LC-7721、MHCC97H、HepG2）的增殖、迁移、侵袭和黏附[18]。但prNK-lysin抑制肝癌细胞转移的机制尚不清楚，仍需进一步研究。

蛋白质组学是以蛋白质组为研究对象，大规模、有系统地研究蛋白质的特征及结构[19]。利用蛋白质组学能够发现新的与癌症转移相关的蛋白[20]，新的癌症治疗靶点[21]以及癌症的耐药靶点[22]。因此，本研究应用蛋白组学分析prNK-lysin作用于肝癌细胞后蛋白组的变化，通过比较差异表达蛋白预测prNK-lysin抑制肝癌细胞转移可能的机制和靶点，为后续研究提供思路和基础。

1. 材料和方法

1.1. 实验材料

肝癌细胞SMMOL/LC-7721购自赛百慷（上海）生物技术股份有限公司；prNK-lysin根据本课题组前期试验方法进行制备[23]；RPMI 1640培养基（GIBCO）；胎牛血清（BI）；细胞培养皿/瓶（NEST）；TMTpro 16标记试剂盒、BCA试剂盒（ThermoSicientific）。

1.2. 药物作用

根据实验室前期研究结果[18]，本实验选用90.8 μg/mL为处理组浓度，作用时间为6 h。复苏SMMOL/LC-7721，用10%FBS RPMI 1640培养基培养，培养至细胞呈对数生长期时进行后续试验。待培养皿中细胞生长至80%~ 90%时，弃原有培养基，加入含有不同浓度prNK-lysin的2%FBS RPMI 1640培养基。实验分为空白对照组、PBS处理组和prNK-lysin处理组，每组作用总体积均为10 mL。空白对照组仅加入2%FBS RPMI 1640培养基；prNK-lysin处理组作用浓度为90.8 μg/mL，加入相应量的prNK-lysin，其余为2%FBS RPMI 1640培养基；PBS处理组中PBS和2%FBS RPMI 1640培养基的量与prNK-lysin处理组相同。加药后置于5% CO2、37 ℃饱和湿润培养箱内继续培养6 h。

1.3. 蛋白组学分析

1.3.1. 质谱检测

将药物作用6 h后的SMMOL/LC-7721细胞取出，提取细胞总蛋白，并用BCA法测定蛋白浓度。每个样品取50 μg蛋白用裂解液稀释至相同浓度和体积，经处理后用胰蛋白酶酶解。按照TMTpro16标记试剂盒说明书步骤标记样品。标记好的样品用高pH反相高效液相色谱分离，流速为300 μL/min；检测波长为紫外210 nm；流动相A相为ACN-H2O（2:98，V/V），B相为ACN-H2O（90:10，V/V），并用氨水将两种流动相的pH调至10，对样品进行梯度洗脱。收集8~60 min的反相色谱分离样品进行色谱串联质谱检测。

1.3.2. 质谱数据分析

将得到的质谱数据用Proteome Discover 2.4（Thermofisher）分析。具体搜索库参数设置如表 1。对检索到的原始数据按照Score Sequest HT > 0且unique peptide≥1，并去除空白值的标准筛选可信蛋白。在可信蛋白的基础上，按Foldchange=1.2倍且P < 0.05筛选出差异表达蛋白（DEPs）。差异表达蛋白的富集分析及蛋白互作分析使用GO/KEGG数据库和STRING(https://string-db.org/)数据库完成。

表 1.

质谱检索参数

Spectral search parameters

Items Settings
Static modification TMT (N-tem, K); Carbamidomethyl (C)
Dynamic modification Oxidation (M), Acetyl (N-term)
Digestion Trypsin
Instrument Orbitrap Fusion
MS1 tolerance 10ppm
MS2 tolerance 0.02Da
Missed Cleavages 2
Database Nuiprot-reviewed_yes+taxonomy_9606.fasta
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1.4. RT-qPCR检测TMEM51、FKBP3、GALNT13 mRNA的相对表达量

将SMMOL/LC-7721细胞以1×105/mL接种到六孔细胞培养板中，培养至贴壁后弃掉上清，分别加入含有22.7、45.4、90.8、181.6 μg/mL prNK-lysin的2%1640培养基，继续作用6 h，收集细胞。用Trizol试剂从细胞中提取总RNA。采用核酸浓度检测仪检测总RNA浓度。按照TaKaRa反转录试剂盒说明书进行反转录。RT-qPCR用ABI 7500 Real-Time PCR系统进行检测。TMEM51的引物序列：F 5'-AGCTACGAGGAAGTG ATGAACA-3'；R 5'-GTGACTCATAGGACGGGAGA G-3'；FKBP3的引物序列：F 5'-CAGGAACACGGTTC AGATTCG-3'；R 5'-TATTTTGGTGGACCCTCATCCA-3'；GALNT13的引物序列：F 5'-GCCACCATATGA GAGGAAATCAGTTA-3'；R 5'-GGTTCATCGAGAC ATTGGTTACTGTTA-3'；β-actin的引物序列F 5'-CAG AGCCTCGCCTTTGCC-3'，R 5'-ACCATCACGCCCT GGTGC-3'。反应结束后，将得出的CT值采用相对定量法进行分析，按照2-ΔΔCt法计算出目的基因mRNA的相对表达量。

1.5. Western blotting检测FKBP3的蛋白表达水平

用1.4相同的处理方法处理细胞后，细胞刮板收集细胞，用含有1 mmol/L的蛋白酶抑制剂、1 mmol/L磷酸酶抑制剂的RIPA裂解液裂解细胞，提取细胞总蛋白，按照BCA蛋白检测试剂盒测定蛋白浓度。将蛋白样品用10%的SDS-聚丙烯酰胺凝胶（SDS-PAGE）电泳后转移到PVDF膜上，用5%脱脂奶粉室温封闭2 h后，4 ℃下过夜孵育一抗：β-actin多克隆兔抗（1∶20 000），FKBP3多克隆抗体（1∶1000）。然后用TBST洗涤PVDF膜3次，用山羊抗兔二抗（1∶20 000）在室温下孵育1 h。最后用化学发光成像系统检测目标蛋白，用Image J软件进行灰度值分析。

1.6. 统计学分析

采用GraphPad Prism 8.0（GraphPad Software, Inc. California, USA）统计软件分析数据，所有数据均采用均数±标准差进行统计描述；采用t检验将各组与空白对照组相比，P < 0.05时认为差异具有统计学意义。所有实验均独立重复3次。

2. 结果

2.1. prNK-lysin作用于肝癌细胞SMMOL/LC-7721的差异表达蛋白分析

将空白对照组，PBS处理组和prNK-lysin处理组两两相比，在可信蛋白的基础上一共鉴定到4757个DEPs，prNK-lysin处理组与空白对照组相比，共鉴定到1989个DEPs，上调805个DEPs，下调1184个DEPs；prNK-lysin处理组与PBS处理组相比，共鉴定到2753个DEPs，上调1281个DEPs，下调1472个DEPs；PBS处理组和空白对照组相比共有15个DEPs，上调11个DEPs，下调4个DEPs。相对于PBS处理组和空白对照组，prNK-lysin处理组共发现1909个相同的DEPs，其中上调752个DEPs，下调1157个DEPs（图 1）。表 2、表 3分别是1909个差异表达蛋白中上调和下调差异倍数较大的前10个差异表达蛋白。

图 1.

图 1

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差异表达蛋白火山图分布

Volcano plots showing distribution of the differentially expressed proteins. A: prNK-lysin group vs control group. B: prNK-lysin group vs PBS group. C: Control group vs PBS group.

表 2.

差异倍数较大的10种呈上调趋势的差异蛋白

Top 10 up-regulated proteins in prNK-lysin-treated SMMOL/LC-7721 cells

Accession Gene Name Foldchange
All P < 0.05.GALNT13: Polypeptide N-acetylgalactosaminyltransferase 13; H2AW: Histone H2A type 3; APOH: β-2-glycoprotein 1; TMEM51: Transmembrane Protein 51; CCDC82: Coiled-coil domain-containing protein 82; CYP51A1: Lanosterol 14-α demethylase; UBIAD1: UbiA prenyltransferase domain-containing protein 1; DKK1: Dickkopfrelated protein 1; CD99L2: CD99 antigen-like protein 2; TEX264: Testis-expressed protein 264.
Q8IUC8 GALNT13 2.025
Q7L7L0 H2AW 1.933
P02749 APOH 1.876
Q9NW97 TMEM51 1.837
Q8N4S0 CCDC82 1.705
Q16850 CYP51A1 1.697
Q9Y5Z9 UBIAD1 1.622
O94907 DKK1 1.589
Q8TCZ2 CD99L2 1.582
Q9Y619 TEX264 1.567
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表 3.

差异倍数较大的10种呈下调趋势的差异蛋白

Top 10 downregulated proteins in prNK-lysin-treated SMMOL/LC-7721 cells

Accession Gene Name Foldchange
All P < 0.05.PMFBP1: Polyamine-modulated factor 1-binding protein 1; CERT1: Ceramide transfer protein; FKBP3: FKBP prolyl isomerase 3; NACA: Nascent polypeptide-associated complex subunit alpha; CEP164: Centrosomal protein of 164; MOB2: MOB kinase activator 2; GGA3: ADP-ribosylation factor-binding protein GGA3; TPM2: Tropomyosin beta chain; CKS1B: Cyclin-dependent kinases regulatory subunit 1; CEP295: Centrosomal protein of 295.
Q8TBY8 PMFBP1 0.496
Q9Y5P4 CERT1 0.496
Q00688 FKBP3 0.505
Q13765 NACA 0.508
Q9UPV0 CEP164 0.513
Q70IA6 MOB2 0.513
Q9NZ52 GGA3 0.522
P07951 TPM2 0.526
P61024 CKS1B 0.526
Q9C0D2 CEP295 0.535
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2.2. 差异表达蛋白的GO分析

根据GO蛋白数量富集分析，prNK-lysin处理组与空白对照组(图 2A)的差异表达蛋白主要分布于cytosol、cytoplasm、extracellular exosome等细胞组分（CC）中；主要参与neutrophil degranulation、viral process、translational initiation等生物过程（BP）；其发挥的分子功能（MF）主要包括RNA binding、identical protein binding、ATP binding等。prNK-lysin处理组与PBS处理组（图 2B）的差异表达蛋白主要分布于cytosol、cytoplasm、membrane等细胞组分中；主要参与viral process、translation、translational initiation等生物过程；其发挥的分子功能主要包括RNA binding、identical protein binding、ATP binding等。根据GO蛋白富集分值分析的前15的GO条目（图 3），对prNK-lysin处理组与空白对照组和prNK-lysin处理组与PBS处理组共同的差异表达蛋白进行GO分析，这些蛋白主要集中于细胞组分方面，在CC中大部分差异蛋白位于Cytosol中，且差异极显著；差异表达蛋白发挥的分子功能主要包括protein binding、RNA binding、ATP binding等；主要参与translation、protein folding、intracellular protein transport等的生物过程。

图 2.

图 2

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差异表达蛋白GO分析图

GO analysis of the differentially expressed proteins in prNK-lysin-treated SMMOL/ LC-7721 cells.A: prNK-lysin group vs control group. B: prNK-lysin group vs PBS group.

图 3.

图 3

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prNK-lysin处理组与空白对照组和prNK-lysin处理组与PBS处理组共同差异表达蛋白GO分析富集气泡图

GO analysis enrichment bubble chart of the differentially expressed proteins in prNK-lysin group compared with the control group and PBS group.

2.3. 差异表达蛋白的KEGG分析

图 4为prNK-lysin处理组与空白对照组（图 4A），prNK-lysin处理组与PBS处理组（图 4B）的差异表达蛋白KEGG富集分析的前20个通路气泡图。prNK-lysin处理组和空白对照组（图 5A），prNK-lysin处理组与PBS处理组（图 5B）的差异表达蛋白在KEGG Level 3水平下分析发现，差异表达蛋白涉及到的通路主要富集在Genetic Information Processing、Human Disease、Metabolism、Organismal Systems，其中大多数差异表达蛋白主要富集于Genetic Information Processing、Human Disease，在Genetic Information Processing中主要涉及Ribosome、Protein process in endoplasmic reticulum、RNA transport等通路。

图 4.

图 4

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差异表达蛋白KEGG富集分析通路气泡图

Bubble diagram of KEGG pathway enrichment analysis of the differentially expressed proteins. A: prNKlysin group vs control group. B: prNK-lysin group vs PBS group.

图 5.

图 5

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差异表达蛋白KEGG Level 3水平通路图

Pathway map of the differentially expressed protein KEGG Level 3. A: prNK-lysin group vs control group. B: prNK-lysin group vs PBS group.

将上调差异表达蛋白和下调差异表达蛋白在KEGG Level 2水平进行分析，与KEGG Level 3水平分析相比，Level 2水平分析多了Cellular Processes、Environment Information Processing这两方面的通路（图 6）。在Cellular Processes中差异蛋白主要富集于Cell growth and death、Cell motility、eukaryotes、Transport and catabolism通路；在Environment Information Processing中差异蛋白主要富集于Membrane transport、Signal transduction、Signaling molecules and interaction通路；在Genetic Information Processing中差异蛋白主要富集于Folding，sorting and degradation、Replication and repair、Transcription、Translation通路；在Human Diseases中差异蛋白主要富集于Cancer：overview、Infectious disease：bacterial、Neurodegenerative disease等通路；在Metabolism中差异蛋白主要富集于Amino acid metabolism、Carbohydrate metabolism、Energy metabolism等通路；在Organismal Systems中差异蛋白主要富集于Endocrine system、Environmental system、Immol/Lune system等通路。

图 6.

图 6

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差异表达蛋白KEGG Level 2水平通路图

Pathway map of the differentially expressed protein KEGG Level 2. A: prNK-lysin group vs control group. B: prNK-lysin group vs PBS group.

将prNK-lysin处理组与空白对照组和prNK-lysin处理组与PBS处理组共同的差异表达蛋白进行KEGG通路分析，选取差异显著的前20条通路进行作图。差异蛋白主要富集于Metabolic pathways中，Ribosome通路差异极显著（图 7）。

图 7.

图 7

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prNK-lysin处理组与空白对照组和prNK-lysin处理组与PBS处理组共同差异表达蛋白KEGG通路分析气泡图

Bubble plot of KEGG pathway analysis of the differentially expressed proteins in prNK-lysin-treated SMMOL/LC-7721 cells compared with the control group and PBS group.

2.4. TMEM51、FKBP3、GALNT13 mRNA相对表达量的测定结果

TMEM51、FKBP3、GALNT13 mRNA的相对表达量检测结果如图 8所示，GALNT13 mRNA相对表达量与空白对照组相比，90.8、181.6 μg/mL prNK-lysin处理组显著升高（1.54 ± 0.06 vs 1.02 ± 0.17；3.65 ± 0.14 vs 1.02±0.17；P < 0.05，图 8A）。与空白对照组相比，45.4、90.8、181.6 μg/mL prNK-lysin处理组显著升高TMEM51 mRNA相对表达量（1.27±0.07 vs 1.00±0.04；1.47 ± 0.07 vs 1.00 ± 0.04；1.76 ± 0.11 vs 1.00 ± 0.04；P < 0.01，图 8B）。22.7、90.8、181.6 μg/mL prNK-lysin处理组与空白对照组相比显著降低FKBP3 mRNA相对表达量（0.36±0.04 vs 1.02±0.24；0.43±0.06 vs 1.02±0.24；0.44±0.09 vs 1.02±0.24；P < 0.05，图 8C）。这一结果与蛋白组学测序结果基本一致。

图 8.

图 8

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prNK-lysin对SMMOL/LC-7721中GALNT13、TMEM51、FKBP3 mRNA相对表达量的影响

Effect of prNK-lysin on expression levels of GALNT13 (A), TMEM51 (B) and FKBP3 (C) mRNA in hepatocellular carcinoma cells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs control group.

2.5. prNK-lysin对FKBP3蛋白表达的抑制作用

用不同浓度的prNK-lysin处理细胞6 h后，用Western blotting检测其对FKBP3蛋白表达量的影响。与空白对照组相比，不同浓度的prNK-lysin（22.7、45.4、90.8、181.6 μg/mL）均能显著抑制FKBP3蛋白的表达（0.68±0.02 vs 1.02±0.03；0.63±0.01 vs 1.02±0.03；0.38± 0.01 vs 1.02±0.03；0.31±0.01 vs 1.02±0.03，P < 0.001，图 9）。

图 9.

图 9

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prNK-lysin抑制SMMOL/LC-7721中FKBP3的蛋白表达

prNK-lysin inhibits FKBP3 protein expression in SMMOL/LC-7721 cells. **P < 0.01, ***P < 0.001, ****P < 0.0001 vs control group.

3. 讨论

本研究应用蛋白组学发现，与空白对照组和PBS处理组相比，prNK-lysin处理组中差异倍数较大且与癌症相关的蛋白有GALNT13、TMEM51、FKBP3（FKBP Prolyl Isomerase 3）等。

GALNT13是GALNT(多肽-N-乙酰半乳糖胺基转移酶)家族成员。GALNTs主要通过将N-乙酰半乳糖胺（GalNAc）从UDP-GalNAc转移到受体蛋白的丝氨酸/ 苏氨酸残基上来启动O-糖基化（或O-GALNA酰化）[24]，这一过程在多种生物过程中发挥重要作用。GALNTs在不同癌症中发挥不同的作用。GALNT12通过影响PI3K/Akt/mTOR通路促进多形性胶质母细胞瘤的发展[25]。研究发现GALNT8通过抑制上皮间充质转化过程来抑制乳腺癌的转移[26]。有研究报道GALNT3通过降低β- catenin水平抑制肺癌发展[27]。在本研究中，prNK-lysin作用后，SMMOL/LC-7721中的GALNT13表达量显著增加，说明GALNT13可能是prNK-lysin抑制肝癌细胞转移和侵袭的靶点之一。

TMEMs是一种跨越脂质双分子层宽度并永久锚定的蛋白质，能允许特定物质通过生物膜[28]。研究发现抑制TMEM106C在HCC中的表达会显著抑制HCC的增殖和转移[29]；通过对食管癌的研究发现，TMEM176A可以抑制食管癌细胞的侵袭和迁移，诱导癌细胞发生凋亡[30]。此外，TMEM还是抑癌靶点。TMEM97是miR- 152抑制前列腺癌细胞活力和侵袭潜能的作用靶点[31]；miR-381可通过负向调控TMEM16A抑制胃癌细胞的增殖和侵袭，并促进其凋亡[32]。在本研究中，prNK-lysin处理后，肝癌细胞中TMEM51的表达上调，表明prNKlysin可能通过TMEM51来发挥其抑制肝癌细胞转移和侵袭的作用。

FKBP3是第一个被确认主要位于细胞核中的FKBP家族成员，普遍存在于多种组织中[33]。FKBPs属于亲免疫家族，除参与蛋白质折叠、蛋白质稳定性、激酶活性等细胞功能外[34]，还可与参与肿瘤进展和化疗耐药等过程[35]。研究发现通过敲低鼻咽癌细胞中FKBP3的表达能抑制癌细胞生长、侵袭和迁移[36]。下调原代结直肠癌细胞中FKBP3的表达后，增强了原代结直肠癌细胞对奥沙利铂的敏感度[35]。本研究发现prNK-lysin作用后肝癌细胞FKBP3的表达量显著下降，表明prNKlysin作用后可能通过抑制FKBP3的表达来抑制肝癌细胞增殖。

对prNK-lysin处理组与空白对照组和prNK-lysin处理组与PBS处理组共同差异表达蛋白的KEGG通路分析表明，这些差异蛋白主要参与人类疾病、氨基酸和蛋白质合成、代谢等通路，其中糖酵解、氧化磷酸化、活性氧等通路值得深入探究。

在肿瘤结节或肿块的中心区域，癌细胞处于缺氧条件时，会产生“Warburg effect”[37]，即通过重新编程其代谢以利用糖酵解来产生ATP，同时这也是癌细胞适应高浓度ROS的策略[38]。细胞内的ROS主要由电子传递链产生[39, 40]，在基础水平上，ROS作为信号分子调节部分对维持生理功能至关重要的生物学过程，包括细胞增殖和分化、组织维持和衰老[38, 41]，但当ROS超过细胞可承受范围时，ROS诱导氧化应激，导致某些生物分子（核酸、蛋白质和脂肪）的损伤，最终导致细胞凋亡或衰老[38, 42, 43]。放疗和各种形式常规化疗都是通过促进细胞中ROS的积累，引起细胞毒性氧化应激来发挥其抗癌作用[42]。在多种肿瘤细胞系中的研究表明，Napabucasin(油菜素)的生物激活导致以Nqo1依赖方式产生细胞毒性水平的ROS，引起DNA损伤和细胞死亡，并影响细胞信号通路[44]。但最近研究表明，不是所有的肿瘤都有“Warburg effect”，部分具有高转移和致瘤潜能的癌症干细胞更依赖于氧化磷酸化。通过对2000例乳腺癌患者的基因表达数据分析，发现氧化磷酸化的表达显著上调，提示氧化磷酸化可能是乳腺癌的一个靶点[45]，并且研究发现抑制氧化磷酸化过程能够提高靶向治疗三阴性乳腺癌的疗效[46]。Ras驱动的胰腺导管腺癌干细胞样细胞的转录组学和代谢分析显示，其糖酵解降低，细胞能量主要依赖于氧化磷酸化[47]。二甲双胍已被证明可以抑制氧化磷酸化中的复合物Ⅰ，引起线粒体功能障碍和细胞内ROS增加，从而在癌细胞系[48, 49]中产生抗增殖或抗凋亡的作用。本研究发现差异表达蛋白富集于活性氧和氧化磷酸化通路，表明prNK-lysin可能通过影响肝癌细胞的线粒体代谢导致细胞死亡。

本研究通过GO富集分析表明差异表达蛋白主要分布于细胞质中，参与翻译、RNA结合及蛋白质结合等过程。KEGG通路分析显示，差异表达蛋白涉及的通路主要有核糖体、氧化磷酸化和活性氧通路等，表明prNK-lysin可能通过影响线粒体代谢过程发挥对肝癌细胞的抑制作用，但具体作用机制仍需进一步研究。

Biography

范艺凡，硕士，E-mail: fanyfc@163.com

Funding Statement

中央引导地方科技发展资金项目(2020-2021)

Contributor Information

范 艺凡 (Yifan FAN), Email: fanyfc@163.com.

李 宏全 (Hongquan LI), Email: livets@163.com.

References

  • 1.Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CACancer J Clin. 2021;71(3):209–49. doi: 10.3322/caac.21660. [DOI] [PubMed] [Google Scholar]
  • 2.中华人民共和国国家卫生健康委员会医政医管局 原发性肝癌诊疗指南(2022年版) 中国实用外科杂志. 2022;42(3):241–73. [Google Scholar]
  • 3.Sia D, Villanueva A, Friedman SL, et al. Liver cancer cell of origin, molecular class, and effects on patient prognosis. Gastroenterology. 2017;152(4):745–61. doi: 10.1053/j.gastro.2016.11.048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.樊 宏英, 赵 瑞君. 抗菌肽抗肿瘤作用机制的研究进展. 国际医学寄生虫病杂志. 2011;38(2):96–100. [Google Scholar]
  • 5.Llovet JM, Ricci S, Mazzaferro V, et al. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med. 2008;359(4):378–90. doi: 10.1056/NEJMoa0708857. [DOI] [PubMed] [Google Scholar]
  • 6.Bruix J, Qin S, Merle P, et al. Regorafenib for patients with hepatocellular carcinoma who progressed on sorafenib treatment (RESORCE): a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet. 2017;389(10064):56–66. doi: 10.1016/S0140-6736(16)32453-9. [DOI] [PubMed] [Google Scholar]
  • 7.Kudo M, Finn RS, Qin S, et al. Lenvatinib versus sorafenib in firstline treatment of patients with unresectable hepatocellular carcinoma: a randomised phase 3 non-inferiority trial. Lancet. 2018;391(10126):1163–73. doi: 10.1016/S0140-6736(18)30207-1. [DOI] [PubMed] [Google Scholar]
  • 8.Ghaziani TT, Dhanasekaran R. Recent progress in systemic therapy for hepatocellular cancer (HCC) Curr Treat Options Gastroenterol. 2021;19(2):351–68. doi: 10.1007/s11938-021-00346-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Lee YT, Tan YJ, Oon CE. Molecular targeted therapy: Treating cancer with specificity. Eur J Pharmacol. 2018;834(5):188–96. doi: 10.1016/j.ejphar.2018.07.034. [DOI] [PubMed] [Google Scholar]
  • 10.Chaturvedi VK, Singh A, Singh VK, et al. Cancer nanotechnology: a new revolution for cancer diagnosis and therapy. Curr Drug Metab. 2019;20(6):416–29. doi: 10.2174/1389200219666180918111528. [DOI] [PubMed] [Google Scholar]
  • 11.Tornesello AL, Borrelli A, Buonaguro L, et al. Antimicrobial peptides as anticancer agents: functional properties and biological activities. Molecules. 2020;25(12):2850. doi: 10.3390/molecules25122850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Chen BD, Le WJ, Wang YL, et al. Targeting negative surface charges of cancer cells by multifunctional nanoprobes. Theranostics. 2016;6(11):1887–98. doi: 10.7150/thno.16358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Mader JS, Salsman J, Conrad DM, et al. Bovine lactoferricin selectively induces apoptosis in human leukemia and carcinoma cell lines. Mol Cancer Ther. 2005;4(4):612–24. doi: 10.1158/1535-7163.MCT-04-0077. [DOI] [PubMed] [Google Scholar]
  • 14.Dassanayake RP, Falkenberg SM, Register KB, et al. Antimicrobial activity of bovine NK-lysin-derived peptides on Mycoplasma bovis. PLoS One. 2018;13(5):e0197677. doi: 10.1371/journal.pone.0197677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Zhu R, Wu YS, Liu XX, et al. Membrane disruptive antimicrobial potential of NK-lysin from yellow catfish (Pelteobagrus fulvidraco) Fish Shellfish Immol/Lunol. 2020;97(3):571–80. doi: 10.1016/j.fsi.2019.10.046. [DOI] [PubMed] [Google Scholar]
  • 16.Liu B, Liu GD, Guo HY, et al. Functional characterization of NKlysin in golden pompano Trachinotus ovatus (Linnaeus 1758) Dev Comp Immol/Lunol. 2020;107(10):103658. doi: 10.1016/j.dci.2020.103658. [DOI] [PubMed] [Google Scholar]
  • 17.Yan J, Liang X, Bai C, et al. NK-18, a promising antimicrobial peptide: anti-multidrug resistant leukemia cells and LPS neutralizing properties. Biochimie. 2018;147(8):143–52. doi: 10.1016/j.biochi.2018.02.001. [DOI] [PubMed] [Google Scholar]
  • 18.Khan A, Fan K, Sun N, et al. Recombinant porcine NK-lysin inhibits the invasion of hepatocellular carcinoma cells in vitro. Int J Biol Macromol. 2019;140(8):1249–59. doi: 10.1016/j.ijbiomac.2019.08.212. [DOI] [PubMed] [Google Scholar]
  • 19.Aebersold R. Constellations in a cellular universe. Nature. 2003;422(6928):115–6. doi: 10.1038/422115a. [DOI] [PubMed] [Google Scholar]
  • 20.宋 琳岚, 杨 敏, 蒋 小燕, et al. 基于iTRAQ技术筛选肝癌转移相关差异蛋白. 重庆医科大学学报. 2016;41(10):983–9. [Google Scholar]
  • 21.Jiang Y, Sun AH, Zhao Y, et al. Proteomics identifies new therapeutic targets of early-stage hepatocellular carcinoma. Nature. 2019;567(7747):257–61. doi: 10.1038/s41586-019-0987-8. [DOI] [PubMed] [Google Scholar]
  • 22.Jung JH, You S, Oh JW, et al. Integrated proteomic and phosphoproteomic analyses of cisplatin-sensitive and resistant bladder cancer cells reveal CDK2 network as a key therapeutic target. Cancer Lett. 2018;28(437):1–12. doi: 10.1016/j.canlet.2018.08.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Fan KH, Li HQ, Wang ZR, et al. Expression and purification of the recombinant porcine NK-lysin inPichia pastorisand observation of anticancer activity in vitro. Prep Biochem Biotechnol. 2016;46(1):65–70. doi: 10.1080/10826068.2014.979206. [DOI] [PubMed] [Google Scholar]
  • 24.Ten Hagen KG, Fritz TA, Tabak LA. All in the family: the UDPGalNAc: polypeptide N-acetylgalactosaminyltransferases. Glycobiology. 2003;13(1):1R–16R. doi: 10.1093/glycob/cwg007. [DOI] [PubMed] [Google Scholar]
  • 25.Zheng YJ, Liang MT, Wang BW, et al. GALNT12 is associated with the malignancy of glioma and promotes glioblastoma multiforme in vitro by activating Akt signaling. Biochem Biophys Res Commol/Lun. 2022;610(7):99–106. doi: 10.1016/j.bbrc.2022.04.052. [DOI] [PubMed] [Google Scholar]
  • 26.Huang T, Meng F, Huang H, et al. GALNT8 suppresses breast cancer cell metastasis potential by regulating EGFR O-GalNAcylation. Biochem Biophys Res Commol/Lun. 2022;23(601):16–23. doi: 10.1016/j.bbrc.2022.02.072. [DOI] [PubMed] [Google Scholar]
  • 27.Park MS, Yang AY, Lee JE, et al. GALNT3 suppresses lung cancer by inhibiting myeloid-derived suppressor cell infiltration and angiogenesis in a TNFR and c-MET pathway-dependent manner. Cancer Lett. 2021;17(521):294–307. doi: 10.1016/j.canlet.2021.08.015. [DOI] [PubMed] [Google Scholar]
  • 28.Schmit K, Michiels C. TMEM proteins in cancer: a review. Front Pharmacol. 2018;6(9):1345. doi: 10.3389/fphar.2018.01345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Duan JC, Qian YW, Fu XH, et al. TMEM106C contributes to the malignant characteristics and poor prognosis of hepatocellular carcinoma. Aging. 2021;13(4):5585–606. doi: 10.18632/aging.202487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Wang Y, Zhang Y, Herman JG, et al. Epigenetic silencing of TMEM176A promotes esophageal squamous cell cancer development. Oncotarget. 2017;8(41):70035–48. doi: 10.18632/oncotarget.19550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Ramalho-Carvalho J, Gonçalves CS, Graça I, et al. A multiplatform approach identifies miR-152-3p as a commol/Lon epigenetically regulated onco-suppressor in prostate cancer targeting TMEM97. Clin Epigenetics. 2018;10(1):1–15. doi: 10.1186/s13148-017-0434-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.柯 超, 蒋 斌, 周 红见, et al. miR-381靶向TMEM16A对胃癌细胞增殖和侵袭的影响. 安徽医科大学学报. 2020;55(12):1866–71. [Google Scholar]
  • 33.Jin YJ, Burakoff SJ. The 25-kDa FK506-binding protein is localized in the nucleus and associates with casein kinase Ⅱ and nucleolin. Proc NatlAcad Sci USA. 1993;90(16):7769–73. doi: 10.1073/pnas.90.16.7769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Solassol J, Mange A, Maudelonde T. FKBP family proteins as promising new biomarkers for cancer. Curr Opin Pharmacol. 2011;11(4):320–5. doi: 10.1016/j.coph.2011.03.012. [DOI] [PubMed] [Google Scholar]
  • 35.Tong JF, Shen Y, Chen X, et al. FKBP3 mediates oxaliplatin resistance in colorectal cancer cells by regulating HDAC2 expression. Oncol Rep. 2019;42(4):1404–12. doi: 10.3892/or.2019.7259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Qiu SJ, Dong JD, Chen JJ, et al. Knockdown of FKBP3 suppresses nasopharyngeal carcinoma cell growth, invasion and migration, deactivated NF-κB/IL-6 signaling pathway through inhibiting histone deacetylase 2 expression. Chin J Physiol. 2023;66(2):85. doi: 10.4103/cjop.CJOP-D-22-00075. [DOI] [PubMed] [Google Scholar]
  • 37.Warburg O. On the origin of cancer cells. Science. 1956;123(3191):309–14. doi: 10.1126/science.123.3191.309. [DOI] [PubMed] [Google Scholar]
  • 38.Gorrini C, Harris IS, Mak TW. Modulation of oxidative stress as an anticancer strategy. Nat Rev Drug Discov. 2013;12(12):931–47. doi: 10.1038/nrd4002. [DOI] [PubMed] [Google Scholar]
  • 39.Kumari S, Badana AK, G MMOL/L, et al. Reactive oxygen species: a key constituent in cancer survival. Biomark Insights. 2018;13(6):1177271918755391. doi: 10.1177/1177271918755391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Hawkins CL, Davies MJ. Detection, identification, and quantification of oxidative protein modifications. J Biol Chem. 2019;294(51):19683–708. doi: 10.1074/jbc.REV119.006217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Schieber M, Chandel NS. ROS function in redox signaling and oxidative stress. Curr Biol. 2014;24(10):R453–62. doi: 10.1016/j.cub.2014.03.034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Panieri E, Santoro MMOL/L ROS homeostasis and metabolism: a dangerous liason in cancer cells. Cell Death Dis. 2016;7(6):e2253. doi: 10.1038/cddis.2016.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Sosa V, Moliné T, Somoza R, et al. Oxidative stress and cancer: an overview. Ageing Res Rev. 2013;12(1):376–90. doi: 10.1016/j.arr.2012.10.004. [DOI] [PubMed] [Google Scholar]
  • 44.Froeling FEM, Swamynathan MMOL/L, Deschênes A, et al. Bioactivation of napabucasin triggers reactive oxygen speciesmediated cancer cell death. Clin Cancer Res. 2019;25(23):7162–74. doi: 10.1158/1078-0432.CCR-19-0302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Whitaker-Menezes D, Martinez-Outschoorn UE, Flomenberg N, et al. Hyperactivation of oxidative mitochondrial metabolism in epithelial cancer cells in situ: visualizing the therapeutic effects of metformin in tumor tissue. Cell Cycle. 2011;10(23):4047–64. doi: 10.4161/cc.10.23.18151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Evans KW, Yuca EK, Scott SS, et al. Oxidative phosphorylation is a metabolic vulnerability in chemotherapy-resistant triple-negative breast cancer. Cancer Res. 2021;81(21):5572–81. doi: 10.1158/0008-5472.CAN-20-3242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Viale A, Pettazzoni P, Lyssiotis CA, et al. Oncogene ablationresistant pancreatic cancer cells depend on mitochondrial function. Nature. 2014;514(7524):628–32. doi: 10.1038/nature13611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Hsieh Li SM, Liu ST, Chang YL, et al. Metformin causes cancer cell death through downregulation of p53-dependent differentiated embryo chondrocyte 1. J Biomed Sci. 2018;25(1):1–13. doi: 10.1186/s12929-018-0478-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Mogavero A, Maiorana MV, Zanutto S, et al. Metformin transiently inhibits colorectal cancer cell proliferation as a result of either AMPK activation or increased ROS production. Sci Rep. 2017;7(1):15992. doi: 10.1038/s41598-017-16149-z. [DOI] [PMC free article] [PubMed] [Google Scholar]

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