MiR-26a和异黏蛋白的关系及在裸鼠体内荷瘤实验的验证

doi:10.16098/j.issn.0529-1356.2022.01.011

摘要/Abstract

摘要：

目的探讨miR-26a和异黏蛋白（MTDH）的关系。方法采用免疫组织化学SP染色法检测86例食管癌病理组织芯片中MTDH的表达，采用原位杂交检测miR-26a的表达，分析两者表达的相关性。通过生物信息学预测软件Targetscan Human7.2预测MTDH在miR-26a序列上的结合片段。采用荧光素酶报告实验验证MTDH与miR-26a的靶向调控关系。用慢病毒干扰和过表达的食管癌细胞系进行裸鼠皮下注射，观察瘤块的形成，采用免疫组织化学染色法和原位杂交法检测各组瘤体中MTDH和miR-26a的表达，分析其关系。结果 MiR-26a在食管癌组织中的表达明显低于配对癌旁正常食管组织，而MTDH在食管癌组织中的表达明显高于配对癌旁正常食管组织。MiR-26a的表达与患者病理分级（P<0.05）、N分期（P<0.05)、肿瘤体积（P<0.01）有关。MTDH在食管癌中的表达与N分期（P<0.05）、分化程度（P<0.01）有关。采用Targetscan Human7.2生物信息学软件预测发现，MTDH基因中含有1个hsa-miR-26a的靶序列。荧光素酶报告基因实验也证实了miR-26a与MTDH的靶向调控关系。MiR-26a在KYSE-450细胞中表达最高，在Eca109细胞中表达量最低，本研究采用KYSE-450细胞系进行miR-26a干扰实验的裸鼠实验，采用Eca109细胞系进行过表达实验。过表达miR-26a慢病毒转染的细胞注射裸鼠皮下，成瘤后miR26a的表达升高，MTDH的表达降低。干扰miR-26a慢病毒转染的细胞注射裸鼠皮下，成瘤后瘤块中miR-26a的表达降低，MTDH表达升高。结论 MiR-26a能抑制食管癌细胞MTDH表达，在体外和体内实验，均可验证miR-26a与MTDH的靶向调控关系，miR-26a可能通过MTDH通路在食管癌的发生和发展中扮演着癌基因的角色。

关键词: 微小RNA-26a, 异黏蛋白, 移植瘤, 食管癌, 免疫组织化学SP染色法, 原位杂交, 荧光素酶报告实验, 裸鼠

Abstract:

Objective To explore the relationship between miR-26a and metadherin (MTDH), and to verify the relationship between miR-26a and MTDH in vivo in nude mice. Methods Immunohistochemical SP staining method was used to detect the expression of MTDH and in situ hybridization was used to detect the expression of miR-26a in 86 cases of esophageal cancer, and the correlation between the expressions was analyzed. The bioinformatics prediction Targetscan Human 7.2 software could predicte the binding fragment of MTDH on the miR-26a sequence, and the luciferase report experiment was used to verify the targeted regulatory relationship between MTDH and miR-26a. Nude mice were injected esophageal cancer cell lines subcutaneously which were lentiviral interferenced and overexpressed miR-26a to observe the formation of tumors. The tumors from nude mice were made into paraffin. and each was detected. The expression of MTDH in miR-26a in the tumor groups was detected by immunohistochemical staining and in situ hybridization and the relationship was analyzed. Results The expression of miR-26a in esophageal cancer tissues was significantly lower than that in paired normal esophageal tissues, and the expression of MTDH in esophageal cancer tissues was significantly higher than that in paired normal esophageal tissues. The expression of miR-26a was related to the patient’s pathological grade (P<0.05), N stage (P<0.05), and tumor volume (P<0.01). The expression of MTDH in esophageal cancer was related to the N stage (P<0.05) and the degree of differentiation (P<0.01). Targetscan Human7.2 bioinformatics software predicted that the MTDH gene contained a target sequence of hsa-miR-26a.The luciferase reporter gene experiment also verified the targeted regulation relationship between miR-26a and MTDH. The expression of miR-26a was the highest in KYSE-450 cells and the lowest in Eca109 cells. The mRNA expression of MTDH in the lv-miR-26a group was significantly lower than that in the lv-NC group, and the mRNA expression in the lv-miR-26a-inhibitor group was significantly higher than that in the lv-NC group. After tumor formation, miR-26a expression increased and MTDH expression decreased in miR-26a group. After tumor formation, the expression of miR-26a decreased and the expression of MTDH increased in miR-26a inhibitor group. Conclusion MiR-26a can inhibit the expression of MTDH in esophageal cancer cells. Both in vitro and in vivo experiments can verify the targeted regulatory relationship between miR-26a and MTDH. MiR-26a may play a role in the occurrence and development of esophageal cancer through the MTDH.

Key words: MmicroRNA-26a, Metadherin, Transplanted tumor, Esophageal cancer, Immunohistochemistry SP staining, In situ hybridization, Luciferase reporter assay, Nude mouse

中图分类号:

R735.1

杨晨晨范宇琴. MiR-26a和异黏蛋白的关系及在裸鼠体内荷瘤实验的验证[J]. 解剖学报, 2022, 53(1): 82-91.

YANG Chen-chen FAN Yu-qin . Relationship between miR-26a and metadherin and the verification of tumor-bearing experiment in nude mice[J]. Acta Anatomica Sinica, 2022, 53(1): 82-91.

参考文献

［1］ Bautista-Sánchez D,Arriaga-Canon C,Pedroza-Torres A,et al. The promising role of miR-21 as a cancer biomarker and its importance in RNA-based therapeutics［J］.Mol Ther Nucleic Acids, 2020, 20:409-420.

［2］ Rouleau S,Glouzon JS,Brumwell A,et al. 3’UTR G-quadruplexes regulate miRNA Binding ［J］.RNA,2017,23(8):1172-1179.

［3］ Shao ZhX, Xie JJ, Xu ZhH, et al.Transfection of microRNA-21 mediating the biological behavior andcellular radiosensitivity of esophageal squamous carcinoma cells［J］. Acta Anatomica Sinica, 2020,51(3):385-391. (in Chinese)

邵志雄,谢俊杰,徐振华,等.微小RNA-21转染介导食管鳞癌细胞生物学行为及细胞放射敏感性的变化［J］.解剖学报,2020,51(3):385-391.

［4］ Wang Y,Lu J,Chen L,et al. Tumor-derived EV-encapsulated miR-181b-5p induces angiogenesis to foster tumorigenesis and metastasis of ESCC［J］. Mol Ther Nucleic Acids,2020, 20:421-437.

［5］ He JY, Xu Q, Yang ChCh. Influence of micro RNA-26a on the proliferation， migration， apoptosis and cell cycle of esophageal cancer cells Eca109［J］.Journal of Xinxiang Medical University,2019,36(2):131-135.(in Chinese)

贺家勇,徐茜,杨晨晨.微小RNA-26a对食管癌Eca109细胞增殖、迁移、凋亡及细胞周期的影响［J］.新乡医学院学报,2019,36(2):131-135.

［6］ He A, He S, Huang C,et al. MTDH promotes metastasis of clear cell renal cell carcinoma by activating SND1-mediated ERK signaling and epithelial-mesenchymal transition［J］.Aging (Albany NY), 2020,12(2):1465-1487.

［7］ Xu ST, Ma YC,Wang CH,et al. Prognostic and clinicopathologic significance of AEG-1/MTDH and E-cadherin expression in human gallbladder carcinoma［J］.Int J Clin Exp Pathol, 2018,11(12):6025-6031.

［8］ Guo R, Hu T, Liu Y,et al. Long non-coding RNA PRNCR1 modulates non-small cell lung cancer cells proliferation, apoptosis, migration, invasion and EMT through PRNCR1/miR-126-5p/MTDH axis［J］.Biosci Rep,2020,40(7):BSR20193153.

［9］ Bray F, Ferlay J, Soerjomataram Ⅰ, et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries［J］.CA Cancer J Clin,2018, 68(6):394-424.

［10］ Fang WT. Interpretation of 2017 National Comprehensive CancerNetwork (NCCN) guidelines for the diagnosis and treatment of esophageal squamous cell carcinoma throughthe new TNM staging of esophageal carcinoma (eighth edition) by the Union for International Cancer Control(UICC) and the American Cancer Commission (AJCC)［J］. Chinese Journal of Gastrointestinal Surgery, 2017,20(10):1122-1126. (in Chinese)

方文涛.通过食管癌TNM新分期(第八版)解读2017年NCCN食管鳞癌诊疗指南［J］.中华胃肠外科杂志,2017,20(10):1122-1126.

［11］ Zhang F, ZhangY, Sun LX, et al. Expression of CPA4 in lung cancer tissues and the corresponding metastatic lymph node tissue and its functional roles［J］.Chinese Journal of Cancer Prevention and Treatment,2019,26(6):366-372.(in Chinese)

张峰,张媛,孙立新,等.H226细胞和肺癌及其淋巴结转移组织CPA4表达以及生物学功能研究［J］.中华肿瘤防治杂志,2019,26(6):366-372.

［12］ Chang WH. Role of miR-377 in the proliferation or apoptosis of pancreatic cancer and the regulation mechanisms［D］.Chongqing: Third Military Medical University, 2016.(in Chinese)

常伟华. MiR-377在胰腺癌增殖、凋亡中的作用及机制研究［D］.重庆：第三军医大学,2016.

［13］ Huang W. MicroRNAs: biomarkers, diagnostics, and therapeutics ［J］.Methods Mol Biol, 2017,1617:57-67.

［14］ Wang H, Guo Y, Mi N, et al. miR-101-3p and miR-199b-5p promote cell apoptosis in oral cancer by targeting BICC1［J］. Mol Cell Probes,2020,52:101567.

［15］ Yang X, Zhu X,Yan Z, et al. miR-489-3p/SIX1 axis regulates melanoma proliferation and glycolytic potential［J］.Mol Ther Oncolytics, 2019, 16:30-40.

［16］ Zeng R, Huang J, Sun Y, et al. Cell proliferation is induced in renal cell carcinoma through miR-92a-3p upregulation by targeting FBXW7［J］.Oncol Lett, 2020,19(4):3258-3268.

［17］ Huang J,Lu D,Xiang T,et al.MicroRNA-132-3p regulates cell proliferation, apoptosis, migration and invasion of liver cancer by targeting Sox4［J］. Oncol Lett, 2020, 19(4):3173-3180.

［18］ Li J, Liang Y,Lv H,et al.miR-26a and miR-26b inhibit esophageal squamous cancer cell proliferation through suppression of c-MYC pathway［J］.Gene,2017,625:1-9.

［19］ Wei Z,Chang K,Fan C,et al.MiR-26a/miR-26b represses tongue squamous cell carcinoma progression by targeting PAK1［J］.Cancer Cell Int，2020，20:82.

［20］ Li HH,Wang JD,Wang W,et al. Effect of miR-26a-5p on gastric cancer cell proliferation,migration and invasion by targeting COL10A1［J］. Eur Rev Med Pharmacol Sci, 2020,24(3):1186-1194.

［21］ Rong C,Shi Y,Huang J,et al.The effect of metadherin on NF-κB activation and downstream genes in ovarian cancer［J］.Cell Transplant,2020,29:963689720905506.

［22］ Han L,Cao X,Chen Z,et al. Overcoming cisplatin resistance by targeting the MTDH-PTEN interaction in ovarian cancer with sera derived from rats exposed to GuizhiFuling wan extract［J］.BMC Complement Med Ther, 2020,20(1):57.

［23］ Jiang X, Xiao TW, Lu HL, et al.MiR-506-3p suppresses metastasis of colorectal cancer by targeting UHRF1［J］. Journal of Tongji University (Medical Science),2019,40(3):298-304.(in Chinese)

蒋逊,肖天卫,鲁贺磊,等.微小RNA-506-3p调控UHRF1抑制大肠癌细胞转移的机制［J］.同济大学学报(医学版),2019,40(3):298-304.

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