[1]付琳琳,马保东,张书娟,等.三重基序蛋白28对乳腺癌细胞增殖能力和肿瘤免疫微环境的影响[J].新乡医学院学报,2024,(4):301-308.[doi:10.7683/xxyxyxb.2024.04.001]
　FU Linlin,MA Baodong,ZHANG Shujuan,et al.Effect of tripartite motif-containing 28 on the proliferation and tumor immune microenvironment in breast cancer[J].Journal of Xinxiang Medical University,2024,(4):301-308.[doi:10.7683/xxyxyxb.2024.04.001]
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三重基序蛋白28对乳腺癌细胞增殖能力和肿瘤免疫微环境的影响
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《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
期数:
2024年4
页码:
301-308
栏目:
基础研究
出版日期:
2024-04-05

文章信息/Info

Title:
Effect of tripartite motif-containing 28 on the proliferation and tumor immune microenvironment in breast cancer
作者:
付琳琳1; 马保东2; 张书娟3; 霍彦平1
(1.郑州大学附属郑州中心医院乳腺外科，河南　郑州　450001；2.郑州大学附属郑州中心医院干细胞再生医学转化中心，河南　郑州　450001；3.郑州大学附属郑州中心医院检验科，河南　郑州　450001)
Author(s):
FU Linlin1; MA Baodong2; ZHANG Shujuan3; HUO Yanping1
(1.Department of Breast Surgery,Zhengzhou Central Hospital Affiliated to Zhengzhou University,Zhengzhou 450001,Henan Province,China;2.Center of Stem Cell and Regenerative Medicine,Zhengzhou Central Hospital Affiliated to Zhengzhou University,Zhengzhou 450001,Henan Province,China;3.Department of Laboratory Medicine,Zhengzhou Central Hospital Affiliated to Zhengzhou University,Zhengzhou 450001,Henan Province,China)
关键词:
乳腺癌; 三重基序蛋白28; 免疫微环境; 生物信息学
Keywords:
breast cancer; tripartite motif-containing 28; immune microenvironment; bioinformatics
分类号:
R737.9
DOI:
10.7683/xxyxyxb.2024.04.001
文献标志码:
A
摘要:
目的　探讨三重基序蛋白28(TRIM28)对乳腺癌细胞增殖和集落形成能力的影响及其与肿瘤浸润免疫细胞之间的关系。
方法　选择2020年9月至2022年1月在郑州大学附属郑州中心医院进行诊治的39例乳腺癌患者的石蜡包埋肿瘤组织标本及配对的癌旁邻近组织，采用免疫组织化学染色法检测乳腺癌组织及癌旁组织中TRIM28蛋白的表达；将对数生长期MCF7细胞随机分为si-control组和si-TRIM28组，si-control组细胞转染空载质粒，si-TRIM28组细胞转染敲低TRIM28的质粒；采用Western blot法检测2组MCF7细胞中TRIM28蛋白表达水平，细胞计数盒-8实验检测2组细胞增殖能力，平板克隆形成实验检测2组细胞的集落形成能力；CIBERSORT算法分析美国癌症肿瘤基因图谱（TCGA）数据库中乳腺癌组织中肿瘤浸润免疫细胞的差异及其与TRIM28蛋白的相关性；基因组富集分析(GSEA)软件分析TRIM28基因表达水平差异之间的信号通路；TISIDB数据库获取TRIM28基因相关的免疫调节因子，对其进行京都基因和基因组百科全书(KEGG)通路富集分析。
结果　乳腺癌组织中TRIM28蛋白阳性染色定位于细胞质中，呈棕黄色细颗粒状。TRIM28蛋白在癌旁组织中呈阴性或弱阳性表达，而在乳腺癌组织中呈强阳性表达；肿瘤组织中TRIM28蛋白表达水平显著高于癌旁组织（t=2.258，P<0.05）。si-TRIM28组MCF7细胞中TRIM28蛋白相对表达量显著低于si-control组（t=61.654,P<0.05）。培养第0天，2组MCF7细胞存活率比较差异无统计学意义（t=-0.547，P>0.05）。培养第4天，si-TRIM28 组MCF7细胞的存活率显著低于si-control组（t=13.920，P<0.05）。si-TRIM28组MCF7细胞克隆形成数量显著少于si-control组（t=11.528,P<0.05）。乳腺癌组织和癌旁组织中巨噬细胞亚群、B细胞亚群、T细胞亚群、活化的自然杀伤细胞、单核细胞和嗜酸性粒细胞计数比较差异均有统计学意义（P<0.05）；乳腺癌组织中TRIM28蛋白表达水平与浸润的单核细胞和调节性T细胞呈正相关（r=0.150、0.100，P<0.05），与活化的树突状细胞、活化的记忆CD4 T细胞、静息的记忆CD4 T细胞、γδ T细胞呈负相关（r=-0.100、-0.160、-0.099、-0.190，P<0.05）；GSEA分析结果表明，TRIM28基因参与了多种免疫/癌症相关的信号通路，包括癌症通路、T细胞受体信号通路、Notch信号通路和Wnt信号通路。从TISIDB数据库中获取了TRIM28基因相关的58个免疫调节因子；对这58个免疫调节因子进行KEGG信号通路富集分析结果显示，免疫调节因子CSF1R、CXCL12、CD27、CD40和CD40LG共同参与了T细胞受体信号通路、自然杀伤细胞介导的细胞毒性、核因子-κB信号通路等信号通路。
结论　TRIM28蛋白在乳腺癌中高度表达，敲低TRIM28基因可抑制乳腺癌细胞的增殖能力和集落形成能力，TRIM28蛋白可能通过多种免疫/癌症相关信号通路参与调节乳腺癌免疫微环境。
Abstract:
Objective　To explore the effect of tripartite motif-containing 28 (TRIM28) on the proliferation and colony-forming ability of breast cancer cells and the relationship with tumor-infiltrating immune cells.
Methods　A total of 39 patients with breast cancer undergoing treatment in Zhengzhou Central Hospital Affiliated to Zhengzhou University from September 2020 to January 2022 were selected as the research subjects.Paraffin-embedded tumor tissue specimens and paired paracancerous tissues were collected.The expression of TRIM28 protein in these tissues was determined by immunohistochemical staining.The MCF7 cells in the logarithmic phase were divided into the si-control group and the si-TRIM28 group.The cells in the si-control group were transfected with empty plasmids,and the cells in the si-TRIM28 group were transfected with si-TRIM28 plasmids,which specifically knocked down TRIM28.The expression level of TRIM28 protein in the two groups of MCF7 cells was detected by Western blot,the cell proliferation in the two groups was detected by cell counting kit-8 assay,and the colony formation of the MCF7 cells in the two groups was detected by colony formation assay.Use CIBERSORT algorithm to analyze the difference of tumor infiltrating immune cells in breast cancer sample tissues in The Cancer Genome Atlas（TCGA） database and its correlation with TRIM28 protein.The signaling pathways between the differences in TRIM28 gene expression levels were analyzed by gene set enrichment analysis (GSEA).TRIM28-related immunomodulatory factors were obtained from the TISIDB database and analyzed by the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis.
Results　The TRIM28 protein in breast cancer tissues was positively stained and localized in the cytoplasm,presenting as brownish-yellow fine granules.TRIM28 in paracancerous tissues was expressed either negatively or weakly positive,while the expression of TRIM28 protein in breast cancer tissues was observed with a robust positive signal.The expression level of TRIM28 protein in tumor tissues was significantly higher than that in paracancerous tissues (t=2.258,P<0.05).The relative expression level of TRIM28 protein of MCF7 cells in the si-TRIM28 group was significantly lower than that in the si-control group (t=61.654,P<0.005).On day 0 of cultivation,there was no significant difference in MCF7 cell survival rate between the two groups (t=-0.547,P>0.05).On day 4 of cultivation,the cell survival rate of MCF7 cells in the si-TRIM28 group was significantly lower than that in the si-control group (t=13.920,P<0.05).The number of colony formation in MCF7 cells in the si-TRIM28 group was significantly fewer than that in the si-control group (t=11.528,P<0.05).There were significant differences in the counts of macrophage subpopulations,B-cell subpopulations,T-cell subpopulations,activated natural killer cells,monocytes,and eosinophils in breast cancer tissues and paracancerous tissues (P<0.05).The expression level of TRIM28 in breast cancer tissues was positively correlated with infiltrating monocytes and regulatory T cells (r=0.150,0.100;P<0.05) and negatively correlated with activated dendritic cells,activated CD4 memory T cells,resting CD4 memory T cells and γδ T cells (r=-0.100,-0.160,-0.099,-0.190;P<0.05).GSEA analysis showed that TRIM28 was involved in multiple immune/cancer-related signaling pathways,including cancer pathway,T-cell receptor signaling pathway,Notch signaling pathway,and Wnt signaling pathway.Fifty-eight immunomodulatory factors related to TRIM28 gene were obtained from the TISIDB database.The KEGG pathway enrichment analysis on these immunomodulatory factors showed that CSF1R,CXCL12,CD27,CD40 and CD40LG collectively participated in the T-cell receptor signaling pathway,natural killer cell-mediated cytotoxicity and nuclear factor-κB signaling pathway.
Conclusion　TRIM28 protein is highly expressed in breast cancer.The proliferative and colony-forming abilities of breast cancer cells can be inhibited by knocking down TRIM28 gene.TRIM28 protein may be involved in regulating the immune microenvironment in breast cancer through multiple immune/cancer-related signaling pathways.

参考文献/References:

［1］　SIEGEL R L,MILLER K D,JEMAL A.Cancer statistics,2020［J］.CA Cancer J Clin,2020,70(1):7-30.
［2］　SUNG H,FERLAY J,SIEGEL R L,et al.Global cancer statistics 2020:GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries［J］.CA Cancer J Clin,2021,71(3):209-249.
［3］　TRAYES K P,COKENAKES S E H.Breast cancer treatment［J］.Am Fam Physician,2021,104(2):171-178.
［4］　KERR A J,DODWELL D,MCGALE P,et al.Adjuvant and neoadjuvant breast cancer treatments:a systematic review of their effects on mortality［J］.Cancer Treat Rev,2022,105:102375.
［5］　EMENS L A.Breast cancer immunotherapy:facts and hopes［J］.Clin Cancer Res,2018,24(3):511-520.
［6］　DE MELO GAGLIATO D,BUZAID A C,PEREZ-GARCIA J,et al.Immunotherapy in breast cancer:current practice and clinical challenges［J］.BioDrugs,2020,34(5):611-623.
［7］　CZERWI〖KG-\*3〗N〖DD(-\*2〗＇SKA P,MAZUREK S,WIZNEROWICZ M.The complexity of TRIM28 contribution to cancer［J］.J Biomed Sci,2017,24(1):63.
［8］　CHIKUMA S,SUITA N,OKAZAKI I M,et al.TRIM28 prevents autoinflammatory T cell development in vivo［J］.Nat Immunol,2012,13(6):596-603.
［9］　CZERWINSKA P,JAWORSKA A M,WLODARCZYK N A,et al.Melanoma stem cell-like phenotype and significant suppression of immune response within a tumor are regulated by TRIM28 protein［J］.Cancers,2020,12(10):2998.
［10］　CZERWINSKA P,MACKIEWICZ A A.Low levels of TRIM28-interacting KRAB-ZNF genes associate with cancer stemness and predict poor prognosis of kidney renal clear cell carcinoma patients［J］.Cancers,2021,13(19):4835.
［11］　LIU J,HAN X,CHEN L,et al.TRIM28 is a distinct prognostic biomarker that worsens the tumor immune microenvironment in lung adenocarcinoma［J］.Aging,2020,12(20):20308-20331.
［12］　WEI C,CHENG J,ZHOU B,et al.Tripartite motif containing 28 (TRIM28) promotes breast cancer metastasis by stabilizing TWIST1 protein［J］.Sci Rep,2016,6:29822.
［13］　HAO L,LENG J,XIAO R,et al.Bioinformatics analysis of the prognostic value of Tripartite Motif 28 in breast cancer［J］.Oncol Lett,2017,13(4):2670-2678.
［14］　AZIZI E,CARR A J,PLITAS G,et al.Single-cell map of diverse immune phenotypes in the breast tumor microenvironment［J］.Cell,2018,174(5):1293-1308.e36.
［15］　GOFF S L,DANFORTH D N.The role of immune cells in breast tissue and immunotherapy for the treatment of breast cancer［J］.Clin Breast Cancer,2021,21(1):e63-e73.
［16］　王璐,刘敏.乳腺癌患者血清中B7-同源物2和B7-同源物3的表达及临床意义［J］.新乡医学院学报,2022,39(9):827-832,837.
WANG L,LIU M.Expression and clinical significance of B7 homolog 2 and B7 homolog 3 in serum of patients with breast cancer［J］.J Xinxiang Med Univ,2022,39(9):827-832,837.
［17］　吴文章,张凡,贾祺,等.MAGE-A4在肿瘤诊断与免疫治疗中的作用研究进展［J］.解放军医学杂志,2023,48(7):834-840.
WU W Z,ZHANG F,JIA Q,et al.Research progress on MAGE-A4 in tumor diagnosis and immunotherapy［J］.Med J Chin PLA,2023,48(7):834-840.
［18］　刘逸贤,陈嵘,程忠平.免疫治疗在晚期或复发性子宫内膜癌中的应用进展［J］.中华全科医学,2023,21(10):1753-1756,1797.
LIU Y X,CHEN R,CHENG Z P.Advances in immunotherapy in advanced or recurrent endometrial carcinoma［J］.Chin J Gen Pract,2023,21(10):1753-1756,1797.
［19］　YANG Y H,LIU J W,LU C,et al.CAR-T cell therapy for breast cancer:from basic research to clinical application［J］.Int J Biol Sci,2022,18(6):2609-2626.
［20］　ZHOU B,LIN W,LONG Y,et al.Notch signaling pathway:architecture,disease,and therapeutics［J］.Signal Transduct Target Ther,2022,7(1):95.
［21］　DE FRANCESCO E M,MAGGIOLINI M,MUSTI A M.Crosstalk between Notch,HIF-1α and GPER in breast cancer EMT［J］.Int J Mol Sci,2018,19(7):E2011.
［22］　EDWARDS A,BRENNAN K.Notch signalling in breast development and cancer［J］.Front Cell Dev Biol,2021,9:692173.
［23］　鹿涛,高志棣.骨转移瘤的信号通路及中西医治疗研究进展［J］.青岛大学学报(医学版),2022,58(4):628-632.
LU T,GAO Z D.Research advances in signaling pathways and integrated traditional Chinese and western medicine therapy for bone metastases［J］.J Qingdao Univ Med Sci,2022,58(4):628-632.
［24］　ABREU DE OLIVEIRA W A,LAITHY Y E,BRUNA A,et al.Wnt signaling in the breast:from development to disease［J］.Front Cell Dev Biol,2022,10:884467.
［25］　VAN SCHIE E H,VAN AMERONGEN R.Aberrant WNT/CTNNB1 signaling as a therapeutic target in human breast cancer:weighing the evidence［J］.Front Cell Dev Biol,2020,8:25.
［26］　AYYANAN A,CIVENNI G,CIARLONI L,et al.Increased Wnt signaling triggers oncogenic conversion of human breast epithelial cells by a Notch-dependent mechanism［J］.Proc Natl Acad Sci U S A,2006,103(10):3799-3804.
［27］　LING J,KUMAR R.Crosstalk between NFkB and glucocorticoid signaling:a potential target of breast cancer therapy［J］.Cancer Lett,2012,322(2):119-126.

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更新日期/Last Update: 2024-04-05