miR-1229-3p抑制结直肠癌疾病进展及作为潜在生物标志物的研究

doi:10.16150/j.1671-2870.2023.05.003

摘要/Abstract

摘要：

目的：探讨微小RNA（microRNA, miRNA）在结直肠癌（colorectal cancer, CRC）疾病进展中的作用及临床应用价值。方法：基于TCGA数据库，筛选分析人类结肠癌及癌旁组织的转录组差异表达miRNA，以miRNA-1229-3p作为研究对象，在GEO数据库与Starbase数据库中验证其表达水平。采用生物信息学方法对miR-1229-3p的生物功能特征进行分析，构建miR-1229-3p过表达细胞系及miR-1229-3p敲降细胞系，通过CCK-8实验、克隆形成实验、EdU实验、Transwell实验、流式细胞术实验在体外探究miR-1229-3p对CRC细胞增殖、转移、凋亡的影响，并通过裸鼠移植瘤试验探究miR-1229-3p对CRC细胞体内增殖的影响。通过DIANA 、TargetScan、miRDB数据库筛选miR-1229-3p的靶基因，并通过KEGG富集分析、GO分析靶基因的生物学功能。检测60例CRC患者及60例健康体检者血浆外泌体中的miR-1229-3p含量，采用受试者工作特征曲线（receiver operating characteristic curve, ROC）分析其对CRC患者的诊断效能。结果：数据分析显示，miR-1229-3p在CRC细胞和组织中表达下调。生物信息学方法预测结果显示，miR-1229-3p与肿瘤的进展途径（如上皮间质转化、血管形成等）呈显著负相关（R<0，P<0.05）。体内、外研究表明，过表达miR-1229-3p后，CRC细胞的增殖、迁移和侵袭能力受到抑制，且其潜在靶基因SETD7在细胞中的表达下调；而敲低miR-1229-3p后可抑制CRC细胞的凋亡。CRC患者血浆外泌体中的miR-1229-3p含量较健康体检者降低，ROC曲线分析显示，miR-1229-3p诊断CRC的约登指数最大值为0.586 8，曲线下面积为0.863 2（P<0.01）。结论：miR-1229-3p在CRC中表达下调，SETD7是其潜在的靶基因，miR-1229-3p可抑制CRC细胞增殖、迁移与侵袭，促进CRC细胞凋亡，有望作为诊断CRC的潜在生物标志物。

关键词: 结直肠癌, miR-1229-3p, SETD7, 外泌体, 生物标志物

Abstract:

Objective: To investigate the role and clinical application of microRNA in the malignant progression of colorectal cancer (CRC). Methods: The transcriptome data of human colorectal cancer and adjacent tissues from TCGA database were analyzed to screen out the differentially expressed microRNAs (miRNAs), among which miR-1229-3p with the most significant difference was selected as the target miRNA, and furthermore the expression of miR-1229-3p was verified through GEO and Starbase databases. The biological functional characteristics of miR-1229-3p was analyzed through Bioinformatics. The miR-1229-3p overexpression cell line and miR-1229-3p knockdown cell line were constructed. The effect of miR-1229-3p on the proliferation, metastasis, and apoptosis of CRC cells was explored in vitro by CCK-8 experiment, cloning formation experiment, EdU experiment, transwell, and flow cytometry experiment. The effect of miR-1229-3p on the prolife-rative capacity of CRC in vivo was explored by nude mouse transplantation tumor experiment. The target genes of miR-1229-3p were screened out by DIANA, TargetScan, and miRDB databases. KEGG and GO were used to analyze the biological function of target genes. The expression of miR-1229-3p in plasma exosomes of 60 CRC patients and 60 healthy examiners obtained from Nanjing First Hospital were analyzed, and receiver operating characteristic curve (ROC) was used to distinguish its diagnostic efficacy for CRC patients. Results: The expression of miR-1229-3p was downregulated in CRC cells and tissues. Bioinformatics predicted a significant negative correlation between miR-1229-3p and tumor progression pathways, such as epithelial-mesenchymal transition and angiogenesis (R<0, P<0.05). In vitro and in vivo studies showed that after the overexpression of miR-1229-3p, the proliferation, migration and invasion of CRC cells were inhibited, and the potential target gene SETD7 was downregulated, while the apoptosis of CRC cells could be inhibited after downregulating miR-1229-3p. miR-1229-3p from plasma exosomes of CRC was downregulated, which could distinguish CRC patients from normal people. ROC curve showed that the optimal critical value of miR-1229-3p for diagnosing CRC was 0.586 8, and the area under the curve was 0.863 2 (P<0.01). Conclusions: The expression of miR-1229-3p is downregulated in CRC, and SETD7 is a potential target gene for miR-1229-3p. miR-1229-3p can inhibit the proliferation, migration and invasion of CRC cells, and promote apoptosis of CRC cells. miR-1229-3p can be served as a potential biomarker for CRC diagnosis.

Key words: Colorectal cancer, miR-1229-3p, SETD7, Exosomes, Biomarker

中图分类号:

R735.3

秦晓丹, 孙慧玲, 潘蓓, 潘玉琴, 王书奎. miR-1229-3p抑制结直肠癌疾病进展及作为潜在生物标志物的研究[J]. 诊断学理论与实践, 2023, 22(05): 429-440.

QIN Xiaodan, SUN Huiling, PAN Bei, PAN Yuqin, WANG Shukui. miR-1229-3p inhibits the malignant progression of colorectal cancer and serves as a potential biomarker[J]. Journal of Diagnostics Concepts & Practice, 2023, 22(05): 429-440.

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参考文献 36

[1]	SIEGEL R L, MILLER K D, WAGLE N S, et al. Cancer statistics, 2023[J]. CA Cancer J Clin, 2023, 73(1):17-48. doi: 10.3322/caac.v73.1 URL
[2]	中华人民共和国国家卫生健康委员会. 中国结直肠癌诊疗规范(2023版)[J]. 中华消化外科杂志, 2023, 22(6):667-698.
	National Health Commission of the People′s Republic of China. Chinese protocol of diagnosis and treatment of colorectal cancer (2023 edition)[J]. Chin J Dig Surg, 2023, 22(6):667-698.
[3]	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. doi: 10.3322/caac.v71.3 URL
[4]	ALLEMANI C, MATSUDA T, DI CARLO V, et al. Global surveillance of trends in cancer survival 2000-14 (CONCORD-3): analysis of individual records for 37 513 025 patients diagnosed with one of 18 cancers from 322 population-based registries in 71 countries[J]. Lancet, 2018, 391(10125):1023-1075. doi: S0140-6736(17)33326-3 pmid: 29395269
[5]	张忠涛. 中国结直肠外科20年回顾与展望[J]. 中华消化外科杂志, 2022, 21(01):53-56.
	ZHANG Z T. Two-decade retrospect and prospect of colorectal surgery in China[J]. Chin J Dig Surg, 2022, 21(1):53-56.
[6]	杨盈, 孟文建, 王自强. 结直肠癌的综合治疗[J]. 中华消化外科杂志, 2022, 21(06):753-765.
	YANG Ying, MENG Wenjian, WANG Ziqiang. Comprehensive treatment of colorectal cancer[J]. Chin J Dig Surg, 2022, 21(06):753-765.
[7]	HARRIS T J, MCCORMICK F. The molecular pathology of cancer[J]. Nat Rev Clin Oncol, 2010, 7(5):251-265. doi: 10.1038/nrclinonc.2010.41 pmid: 20351699
[8]	AMBROS V, BARTEL B, BARTEL D P, et al. A uniform system for microRNA annotation[J]. RNA, 2003, 9(3):277-279. pmid: 12592000
[9]	POY M N, ELIASSON L, KRUTZFELDT J, et al. A pancreatic islet-specific microRNA regulates insulin secretion[J]. Nature, 2004, 432(7014):226-230. doi: 10.1038/nature03076
[10]	CALAME K. MicroRNA-155 function in B Cells[J]. Immunity, 2007, 27(6):825-827. doi: 10.1016/j.immuni.2007.11.010 pmid: 18093533
[11]	GRECO S J, RAMESHWAR P. MicroRNAs regulate synthesis of the neurotransmitter substance P in human me-senchymal stem cell-derived neuronal cells[J]. Proc Natl Acad Sci U S A, 2007, 104(39):15484-15489. doi: 10.1073/pnas.0703037104 URL
[12]	STEPONAITIENE R, KUPCINSKAS J, LANGNER C, et al. Epigenetic silencing of miR-137 is a frequent event in gastric carcinogenesis[J]. Mol Carcinog, 2016, 55(4):376-386. doi: 10.1002/mc.22287 URL
[13]	WANG F, YING H Q, HE B S, et al. Upregulated lncRNA-UCA1 contributes to progression of hepatocellular carcinoma through inhibition of miR-216b and activation of FGFR1/ERK signaling pathway[J]. Oncotarget, 2015, 6(10):7899-7917. doi: 10.18632/oncotarget.3219 pmid: 25760077
[14]	LIU R, GU J, JIANG P, et al. DNMT1-microRNA126 epigenetic circuit contributes to esophageal squamous cell carcinoma growth via ADAM9-EGFR-AKT signaling[J]. Clin Cancer Res, 2015, 21(4):854-863. doi: 10.1158/1078-0432.CCR-14-1740 pmid: 25512445
[15]	LEE Y S, DUTTA A. MicroRNAs in cancer[J]. Annu Rev Pathol, 2009, 4:199-227. doi: 10.1146/annurev.pathol.4.110807.092222 pmid: 18817506
[16]	GILLIES J K, LORIMER I A. Regulation of p27Kip1 by miRNA 221/222 in glioblastoma[J]. Cell Cycle, 2007, 6(16):2005-2009. doi: 10.4161/cc.6.16.4526 pmid: 17721077
[17]	JOHNSON C D, ESQUELA-KERSCHER A, STEFANI G, et al. The let-7 microRNA represses cell proliferation pathways in human cells[J]. Cancer Res, 2007, 67(16):7713-7722. doi: 10.1158/0008-5472.CAN-07-1083 pmid: 17699775
[18]	MOTT J L, KOBAYASHI S, BRONK S F, et al. mir-29 regulates Mcl-1 protein expression and apoptosis[J]. Oncogene, 2007, 26(42):6133-6140. pmid: 17404574
[19]	BOMMER G T, GERIN I, FENG Y, et al. p53-mediated activation of miRNA34 candidate tumor-suppressor genes[J]. Curr Biol, 2007, 17(15):1298-1307. doi: 10.1016/j.cub.2007.06.068 pmid: 17656095
[20]	SCOTT G K, GOGA A, BHAUMIK D, et al. Coordinate suppression of ERBB2 and ERBB3 by enforced expression of micro-RNA miR-125a or miR-125b[J]. J Biol Chem, 2007, 282(2):1479-1486. doi: 10.1074/jbc.M609383200 pmid: 17110380
[21]	KUMAR M S, LU J, MERCER K L, et al. Impaired microRNA processing enhances cellular transformation and tumorigenesis[J]. Nat Genet, 2007, 39(5):673-677. doi: 10.1038/ng2003 pmid: 17401365
[22]	JUNG G, HERNÁNDEZ-ILLÁN E, MOREIRA L, et al. Epigenetics of colorectal cancer: biomarker and therapeutic potential[J]. Nat Rev Gastroenterol Hepatol, 2020, 17(2):111-130. doi: 10.1038/s41575-019-0230-y pmid: 31900466
[23]	FARHAN M, WANG H, GAUR U, et al. FOXO signaling pathways as therapeutic targets in cancer[J]. Int J Biol Sci, 2017, 13(7):815-827. doi: 10.7150/ijbs.20052 pmid: 28808415
[24]	OUDHOFF M J, BRAAM M J S, FREEMAN S A, et al. SETD7 controls intestinal regeneration and tumorigene-sis by regulating Wnt/β-Catenin and Hippo/YAP signa-ling[J]. Dev Cell, 2016, 37(1):47-57. doi: 10.1016/j.devcel.2016.03.002 URL
[25]	DEWS M, HOMAYOUNI A, YU D, et al. Augmentation of tumor angiogenesis by a Myc-activated microRNA cluster[J]. Nat Genet, 2006, 38(9):1060-1065. doi: 10.1038/ng1855 pmid: 16878133
[26]	MERTENS-TALCOTT S U, CHINTHARLAPALLI S, LI X, et al. The oncogenic microRNA-27a targets genes that regulate specificity protein transcription factors and the G2-M checkpoint in MDA-MB-231 breast cancer cells[J]. Cancer Res, 2007, 67(22):11001-110011. doi: 10.1158/0008-5472.CAN-07-2416 URL
[27]	HOU Y, ZHANG Q, PANG W, et al. YTHDC1-mediated augmentation of miR-30d in repressing pancreatic tumorigenesis via attenuation of RUNX1-induced transcriptional activation of Warburg effect[J]. Cell Death Differ, 2021, 28(11):3105-3124. doi: 10.1038/s41418-021-00804-0 pmid: 34021267
[28]	KONOSHENKO M Y, BRYZGUNOVA O E, LAKTIONOV P P. miRNAs and androgen deprivation therapy for prostate cancer[J]. Biochim Biophys Acta Rev Cancer, 2021, 1876(2):188625. doi: 10.1016/j.bbcan.2021.188625 URL
[29]	HU X, CHEN Q, GUO H, et al. Identification of target PTEN-based miR-425 and miR-576 as potential diagnostic and immunotherapeutic biomarkers of colorectal cancer with liver metastasis[J]. Front Oncol, 2021, 11:657984. doi: 10.3389/fonc.2021.657984 URL
[30]	LONE W, BOUSKA A, SHARMA S, et al. Genome-wide miRNA expression profiling of molecular subgroups of peripheral T-cell lymphoma[J]. Clin Cancer Res, 2021, 27(21):6039-6053. doi: 10.1158/1078-0432.CCR-21-0573 URL
[31]	KONISHI H, SATO H, TAKAHASHI K, et al. Tumor-progressive mechanisms mediating miRNA-protein interaction[J]. Int J Mol Sci, 2021, 22(22):12303. doi: 10.3390/ijms222212303 URL
[32]	BUTKYTĖ S, ČIUPAS L, JAKUBAUSKIENĖ E, et al. Splicing-dependent expression of microRNAs of mirtron origin in human digestive and excretory system cancer cells[J]. Clin Epigenetics, 2016, 8:33. doi: 10.1186/s13148-016-0200-y pmid: 27019673
[33]	NISHIBEPPU K, KOMATSU S, IMAMURA T, et al. Plasma microRNA profiles: identification of miR-1229-3p as a novel chemoresistant and prognostic biomarker in gastric cancer[J]. Sci Rep, 2020, 10(1):3161. doi: 10.1038/s41598-020-59939-8 pmid: 32081926
[34]	ZHAO X, CUI L. A robust six-miRNA prognostic signature for head and neck squamous cell carcinoma[J]. J Cell Physiol, 2020, 235(11):8799-8811. doi: 10.1002/jcp.29723 pmid: 32342519
[35]	ZHANG C, ZHANG Q, LI H, et al. miR-1229-3p as a prognostic predictor facilitates cell viability, migration, and invasion of hepatocellular carcinoma[J]. Horm Metab Res, 2021, 53(11):759-766. doi: 10.1055/a-1646-8415 pmid: 34740278
[36]	CAO L, WANG M, XU K. Research progress of role and mechanism of SETD7 in tumor occurrence and progression[J]. Zhongguo Fei Ai Za Zhi, 2023, 26(1):38-45.