微小RNA-140-3p靶向细胞分裂周期相关蛋白8抑制肺腺癌细胞侵袭转移的生物信息学分析及验证

doi:10.16098/j.issn.0529-1356.2021.04.014

摘要/Abstract

摘要：

目的在生物信息学基础上探讨微小RNA（miR)-140-3p靶向细胞分裂周期相关蛋白8（CDCA8）抑制肺腺癌细胞的侵袭和转移。方法通过GEO数据库中的GEO2R分析肺腺癌芯片数据中差异表达的miRNA。TargetScanHuman7.2和miRWalk数据库查找miR-140-3p的靶基因。Cytoscape3.7.2软件筛选出Hub基因。GEPIA数据库查询靶基因在肺腺癌组织和正常肺组织中的表达水平，在肺腺癌不同分期中的表达水平以及靶基因的表达水平与肺腺癌患者总体生存率的关系。StarBase数据库查找miR-140-3p在肺腺癌中的生存分析及与CDCA8的表达水平在肺腺癌中的相关性。Real-time PCR检测miR-140-3p在正常肺上皮细胞BEAS-2B和肺腺癌细胞A549的表达水平以及感染效率。Real-time PCR和Western blotting实验检测过表达miR-140-3p后CDCA8 mRNA和蛋白的表达水平。双荧光素酶报告基因实验验证miR-140-3p是否与CDCA8直接结合。Transwell侵袭实验检测过表达miR-140-3p和CDCA8对肺腺癌细胞侵袭力的影响。结果通过GEO等数据库的分析结果显示，miR-140-3p在正常肺组织中的表达水平明显高于在肺腺癌中的表达水平，其预测靶基因CDCA8在肺腺癌中的表达水平明显高于在正常肺组织中的表达水平，且CDCA8与miR-140-3p的表达水平在肺腺癌中成负相关。实验结果显示，miR-140-3p在A549细胞中的表达明显低于在BEAS-2B细胞中的表达 (P＜0.05)；用过表达慢病毒感染细胞后miR-140-3p的表达水平明显升高 (P＜0.05)；过表达miR-140-3p后CDCA8的mRNA和蛋白表达水平明显下调 (P＜0.05)；双荧光素酶报告基因实验结果显示，miR-140-3p能与CDCA8直接结合 (P＜0.05)；与对照组相比，过表达miR-140-3p可抑制肺腺癌细胞A549的侵袭转移，而CDCA8可逆转miR-140-3p对肺腺癌细胞A549侵袭力的抑制作用 (P＜0.05)。结论 MiR-140-3p靶向CDCA8抑制肺腺癌细胞的侵袭转移。

关键词: 肺腺癌, 微小RNA-140-3p, 细胞分裂周期相关蛋白8, 侵袭, 转移, 免疫印迹法

Abstract:

Objective To investigate the effect of microRNA(miR)-140-3p targeting cell division cycle associated 8(CDCA8) on invasion and metastasis of lung adenocarcinoma cells. Methods The differentially expressed miRNAs were analyzed by GEO2R in GEO database. The target genes of miR-140-3p were searched by TargetScan human7.2 and miRWalk databases. The hub gene was screened by Cytoscape 3.7.2 software. GEPIA database was used to query the expression levels of target gene in lung adenocarcinoma tissues and normal lung tissues, the expression levels in different stages of lung adenocarcinoma, and the relationship between the expression levels of target gene and the overall survival rate of lung adenocarcinoma patients. The survival analysis of mi-140-3p in lung adenocarcinoma and the correlation between miR-140-3p and CDCA8 expression levels were searched in starBase database. Real-time PCR was used to detect the expression levels of miR-140-3p in normal lung epithelial cells BEAS-2B and lung adenocarcinoma cells A549, as well as the efficiency of infection. Expression levels of CDCA8 mRNA and protein were detected by Real-time PCR and Western blotting experiments after overexpresion of miR-140-3p. Dual-luciferase reporter assay verified whether miR-140-3p directly binds to CDCA8. Transwell invasion assay detected the effect of overexpression of miR-140-3p and CDCA8 on the invasiveness of lung adenocarcinoma cells. Results Analysis result from GEO and other databases showed that the expression level of miR-140-3p in normal lung tissues was significantly higher than that in lung adenocarcinoma, and its predicted target gene CDCA8 expression level in lung adenocarcinoma was significantly higher than that in normal lung tissues, and CDCA8 was negatively correlated with the expression level of miR-140-3p in lung adenocarcinoma. The experimental result showed that the expression of miR-140-3p in A549 cells was significantly lower than that in BEAS-2B cells (P＜0.05). The expression level of miR-140-3p increased significantly after lentiviral infection (P＜0.05). CDCA8 mRNA and protein expression levels were significantly down-regulated after overexpression of miR-140-3p (P＜0.05). Dual-luciferase reporter assay result showed that miR-140-3p could directly bind to CDCA8 (P＜0.05). Compared with the control group, overexpression of miR-140-3p inhibited the invasion and metastasis of lung adenocarcinoma A549, while CDCA8 revrsed the inhibition of miR-140-3p on the invasion and metastasis of lung adenocarcinoma A549 (P＜0.05). Conclusion MiR-140-3p targeting CDCA8 inhibits the invasion and metastasis of lung adenocarcinoma cells.

Key words: Lung adenocarcinoma, MicroRNA-140-3p, Cell division cycle associated 8, Invasion, Metastasis, Western blotting

中图分类号:

R734.2

郑荃胡雅琼白俊尹崇高李洪利刘雨清. 微小RNA-140-3p靶向细胞分裂周期相关蛋白8抑制肺腺癌细胞侵袭转移的生物信息学分析及验证[J]. 解剖学报, 2021, 52(4): 589-600.

ZHENG Quan HU Ya-qiong BAI Jun YIN Chong-gao LI Hong-li LIU Yu-qing. Bioinformatics analysis and experimental validation of microRNA-140-3p targeting cell division cycle associated 8 inhibiting invasion and metastasis of lung adenocarcinoma cells[J]. Acta Anatomica Sinica, 2021, 52(4): 589-600.

参考文献

［1］ Seijo LM, Peled N, Ajona D, et al. Biomarkers in lung cancer screening: achievements, promises, and challenges［J］. J Thorac Oncol,2019,14(3):343-357.

［2］ Wang J, Jia Y, Zhao S, et al. BIN1 reverses PD-L1-mediated immune escape by inactivating the c-MYC and EGFR/MAPK signaling pathways in nonsmall cell lung cancer［J］. Oncogene,2017,36(45):6235-6243.

［3］ Garzon R, Calin GA, Croce CM. MicroRNAs in cancer［J］. Annu Rev Med,2009,60(1):167-179.

［4］ Fan BL，Ji XH，Zhu WL. Effects of miR-100 on mitotic arrest and Polo-like kinase 1 protein expression in hepatocellular carcinoma cells［J］. Acta Acatomica Sinica,2014,45(1):70-73 (in Chinese)

范秉琳，嵇晓辉，朱武凌. MiR-100对肝癌细胞有丝分裂阻滞及Polo样激酶1蛋白表达的作用［J］. 解剖学报,2014,45(1):70-73.

［5］ Raponi M, Dossey L, Jatkoe T, et al. MicroRNA classifiers for predicting prognosis of squamous cell lung cancer［J］. Cancer Res,2009,69(14):5776-5783.

［6］ Kumar MS, Erkeland S , Pester RE, et al. Suppression of non-small cell lung tumor development by the let-7 microRNA family［J］. Proc Natl Acad Sci USA,2008,105(10):3903-3908.

［7］ Phan NN, Wang CY, Li KL, et al. Distinct expression of CDCA3, CDCA5, and CDCA8 leads to shorter relapse free survival in breast cancer patient［J］. Oncotarget,2018,9(6):6977-6992.

［8］ Chang JL, Chen TH, Wang CF, et al. Borealin/Dasra B is a cell cycle-regulated chromosomal passenger protein and its nuclear accumulation is linked to poor prognosis for human gastric cancer［J］. Exp Cell Res,2006,312(7):962-973.

［9］ Hayama S, Daigo Y, Yamabuki T, et al. Phosphorylation and activation of cell division cycle associated 8 by aurora kinase B plays a significant role in human lung carcinogenesis［J］. Cancer Res,2007,67(9):4113-4122.

［10］ Garber ME, Troyanskaya OG, Schluens K, et al. Diversity of gene expression in adenocarcinoma of the lung［J］. Proc Natl Acad Sci USA,2001,98(24):13784-13789.

［11］ Landi MT, Dracheva T, Rotunno M, et al. Gene expression signature of cigarette smoking and its role in lung adenocarcinoma development and survival［J］. PLoS One,2008,3(2):e1651.

［12］ Hou J, Aerts J, den Hamer B, et al. Gene expression-based classification of non-small cell lung carcinomas and survival prediction［J］. PLoS One,2010,5(4): e10312.

［13］ Selamat SA, Chung BS, Girard L, et al. Genome-scale analysis of DNA methylation in lung adenocarcinoma and integration with mRNA expression［J］. Genome Res,2012,22(7):1197-1211.

［14］ Okayama H, Kohno T, Ishii Y, et al. Identification of genes upregulated in ALK-positive and EGFR/KRAS/ALK-negative lung adenocarcinomas［J］. Cancer Res,2012,72(1):100-111.

［15］ Zhang GX，Xu XW，Meng B，et al. MiR-186-5p inhibits epithelial mesenchymal transition in lung adenocarcinoma cells through modulating PTTG1［J］. Chinese Journal of Biochemistry and Molecular Biology, 2017, 33(4): 380-385 (in Chinese)

张国新，徐新伟，孟斌，等. MiR-186-5p通过靶向调控PTTG1抑制肺腺癌细胞的上皮-间质转化［J］. 中国生物化学与分子生物学报,2017,33(4):380-385.

［16］ Zhang B, Li H, Yin C, et al. Dock1 promotes the mesenchymal transition of glioma and is modulated by MiR-31［J］. Neuropathol Appl Neurobiol,2017,43(5):419-432.

［17］ Yin C, Mou Q, Pan X, et al. MiR-577 suppresses epithelial-mesenchymal transition and metastasis of breast cancer by targeting Rab25［J］. Thorac Cancer,2018,9(4):472-479.

［18］ Vasudevan S, Tong Y, Steitz JA. Switching from repression to activation: microRNAs can up-regulate translation［J］. Science,2007,318(5858):1931-1934.

［19］ Kai Y, Peng W, Ling W, et al. Reciprocal effects between microRNA-140-5p and ADAM10 suppress migration and invasion of human tongue cancer cells［J］. Biochem Biophys Res Commun,2014,448(3):308-314.

［20］ Yuan Y, Shen Y, Xue L, et al. miR-140 suppresses tumor growth and metastasis of non-small cell lung cancer by targeting insulin-like growth factor 1 receptor［J］. PLoS one,2013,8(9):e73604.

［21］ Ma J, Zhang F, Sun P. miR-140-3p impedes the proliferation of human cervical cancer cells by targeting RRM2 to induce cell-cycle arrest and early apoptosis［J］. Bioorg Med Chem,2020,28(3):115283.

［22］ Zhou Y, Wang B, Wang Y, et al. miR-140-3p inhibits breast cancer proliferation and migration by directly regulating the expression of tripartite motif 28［J］. Oncol Lett,2019,17(4):3835-3841.

［23］ Dong W, Yao C, Teng X, et al. MiR-140-3p suppressed cell growth and invasion by downregulating the expression of ATP8A1 in non-small cell lung cancer［J］. Tumour Biol,2016,37(3):2973-2985.

［24］ Liu ZhJ, Dou LT, Zhuang Zh, et al. Screening and survival analysis of differentially expressed genes in papillary thyroid carcinoma［J］. Acta Anatomica Sinica, 2020,51(1),51-57. (in Chinese)

刘中娇，窦龙涛，庄振，等. 乳头状甲状腺癌差异表达基因的筛选及生存分析［J］. 解剖学报,2020,51(1):51-57.

［25］ Kong X, Zhang G, Huo Y, et al. MicroRNA-140-3p inhibits proliferation, migration and invasion of lung cancer cells by targeting ATP6AP2［J］. Int J Clin Exp Pathol,2015,8(10):12845.

［26］ Ci C, Tang B, Lyu D, et al. Overexpression of CDCA8 promotes the malignant progression of cutaneous melanoma and leads to poor prognosis［J］. Int Jo Mol Med,2019,43(1):404-412.

［27］ Wang Y, Zhao Z, Bao X, et al. Borealin/Dasra B is overexpressed in colorectal cancers and contributes to proliferation of cancer cells［J］. Med Oncol,2014,31(11):248.

［28］ Bi Y, Chen S, Jiang J, et al. CDCA8 expression and its clinical relevance in patients with bladder cancer［J］. Medicine,2018,97(34): e11899.

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