中国医科大学学报  2019, Vol. 48 Issue (5): 393-397

文章信息

张新生, 郭琳琳, 刘超群, 许何丽, 窦文静, 刘梦瑶, 张越
ZHANG Xinsheng, GUO Linlin, LIU Chaoqun, XU Heli, DOU Wenjing, LIU Mengyao, ZHANG Yue
hsa-miR-367-3p靶基因预测及功能分析
Prediction and Functional Analysis of hsa-miR-367-3p Target Genes
中国医科大学学报, 2019, 48(5): 393-397
Journal of China Medical University, 2019, 48(5): 393-397

文章历史

收稿日期:2018-04-19
网络出版时间:2019-05-13 11:30
hsa-miR-367-3p靶基因预测及功能分析
沈阳医学院公共卫生学院职业卫生教研室, 沈阳 110034
摘要目的 通过生物信息学方法预测hsa-miR-367-3p的靶基因及其靶基因的功能。方法 运用miRbase在线数据库、miRGator v3.0在线数据库TargetScan、miRDB在线数据库和DAVID在线数据库进行预测和分析。结果 hsa-miR-367-3p在多个物种之间具有高度保守性;在干细胞中表达丰富度较高;功能富集分析发现,hsa-miR-367-3p的靶基因涉及心室发育、转录调节等38个生物过程(P < 0.05),富集在神经元等21个细胞组分(P < 0.05)。结论 hsa-miR-367-3p个别预测出来的靶基因与通路已经被证实,更多的预测结果需要进一步的研究加以验证。本研究为将来验证hsa-miR-367-3p的靶基因和可能调控的通路指明了方向。
关键词hsa-miR-367-3p    靶基因    功能富集分析    
Prediction and Functional Analysis of hsa-miR-367-3p Target Genes
Department of Occupational Health and Medicine, School of Public Health, Shenyang Medical College, Shenyang 110034, China
Abstract: Objective To predict target genes of hsa-miR-367-3p using bioinformatics tools and to determine pathways that hsa-miR-367- 3p might regulate. Methods Online databases like, miRGator v3.0, TargetScan, miRDB, and DAVID were used to predict and analyze the functions of hsa-miR-367-3p. Results hsa-miR-367-3p was found to be highly conserved among multiple species and was enriched in stem cells; functional enrichment analysis revealed the target genes of hsa-miR-367-3p. Target genes of hsa-miR-367-3p were found to be involved in 38 biological processes, such as ventricular development and regulation of transcription(P < 0.05), and were enriched in 21 cellular components such as the neurons(P < 0.05). Conclusion Some predicted target genes and pathways of hsa-miR-367-3p have been experimentally verified; however, some others require experimental validation. Our analysis paves way for future experiments, which might help in verifying target genes and pathways of hsa-miR-367-3p.

微小RNA(microRNA,miRNA)是大约22个ntRNA的内源性基因,通过降解下游mRNA或抑制蛋白质表达在动物和植物的发育中起重要的调节作用[1-2]。miRNA在调节细胞增殖分化和凋亡等各种生命过程和疾病发生中扮演重要角色[3]。据报道[4],异常的miRNA与人类中的多种疾病(心肌疾病和癌症等)有关。对miRNA进行生物信息学预测可以为今后其他研究提供参考和依据,具有重要意义。

1 材料与方法 1.1 hsa-miR-367-3p的保守性分析以及在不同物种间的基因序列进化分析

用miRbase在线数据库对hsa-miR-367-3p进行物种保守性分析。用Clustal Omega(https://www.ebi.ac.uk/Tools/msa/clustalo/)软件对mir-367-3p在物种中的基因序列进行系统进化树分析,步骤1选择RNA,步骤3选择CLUSTALW。

1.2 hsa-miR-367-3p的表达情况、靶基因预测、功能富集分析和转导通路富集分析

用mirGator v3.0数据库对hsa-miR-367-3p在组织器官中的表达情况,用TargetScan和miRanda 2种数据库来预测hsa-miR-367-3p的靶基因。用DAVID软件进行功能富集分析,选择本物种所有的基因作为背景基因。采用DAVID在线数据库进行信号转导通路富集分析。

2 结果 2.1 hsa-miR-367-3p的保守性分析

通过miRbase在线数据库对hsa-miR-367-3p进行保守性分析,发现此miRNA在人、小鼠、玻璃海鞘、家兔等多个物种之间具有高度保守性,见表 1

表 1 不同物种hsa-miR-367-3p成熟序列 Tab.1 Sequence of mature hsa-miR-367-3p in different species
ID miRNA Conserved sequence
MIMAT0000719 hsa-miR-367-3p AAUUGCACUUUAGCAAUGGUGA
MIMAT0003181 mmu-miR-367-3p AAUUGCACUUUAGCAAUGGUGA
MIMAT0016416 cin-miR-367-3p UAUUGCACAUUGGAAUGGUA
MIMAT0036325 ocu-miR-367-3p AAUUGCACUUUAGCAAUGGUGA
MIMAT0037872 cpi-miR-367-3p AAUUGCACUUUAGCAAUGGUG
MIMAT0038274 ami-miR-367-3p AAUUGCACUUUAGCAAUGGUG
MIMAT0038627 cli-miR-367-3p AAUUGCACUUUAGCAAUGGUGA
MIMAT0047198 cpo-miR-367-3p AAUUGCACUUUAGCAAUGGUGA
MIMAT0047839 dno-miR-367-3p AAUUGCACUUUAGCAAUGGUGA

2.2 miR-367-3p在不同物种间的基因序列进化分析

选用预测的成熟序列进行进化树分析,在miR-367-3p家族中,人的hsa-miR-367-3p和小鼠的mmu-miR-367-3p属于同一支;九带犰狳的dno-miR-367-3p和玻璃海鞘的cin-miR-367-3p属于同一支,体现了miR-367-3p在不同物种间的保守性;但是人的hsa-miR-367-3p和美国短吻鳄的ami-miR-367-3p比较有很大差异,体现了mir-367-3p在哺乳动物和爬行动物之间的差异性。见图 1

图 1 基于各物种的基因序列分析的系统进化树 Fig.1 Phylogenetic tree showing relationships between various miRs based on the gene sequence analysis of miRs from various species

2.3 hsa-miR-367-3p的表达情况分析

hsa-miR-367-3p在各个器官的干细胞中表达丰富度较高,见图 2

图 2 hsa-miR -367-3p在各个组织器官中的表达情况 Fig.2 Expression profiles of hsa-miR -367-3p in various organs

2.4 hsa-miR-367-3p的靶基因预测

TargetScan预测的hsa-miR-367-3p靶基因数量为396个,miRDB预测的靶基因个数为495个,2个数据库预测的靶基因取交集共筛选出152个靶基因,其中包括与cAMP信号通路、PI3K-AKT信号通路以及癌症信号通路相关的基因。

2.5 hsa-miR-367-3p靶基因的功能富集分析

将2个数据库都预测到基因交集的152个hsa-miR-367-3p的靶基因用DAVID数据库作功能富集分析,发现hsa-miR-367-3p的靶基因涉及心室发育、RNA聚合酶Ⅱ启动子的转录负调节、RNA聚合酶Ⅱ启动子的转录等38个生物过程(P < 0.05),富集在神经元、突触、高尔基体等21个细胞组分(P < 0.05),参与蛋白质结合和磷脂酰肌醇结合、转录调节和磷脂酰胆碱转运蛋白活性等13个分子功能(P < 0.05),见表 2

表 2 hsa-miR-367-3p靶基因功能富集分析部分结果 Tab.2 Functional enrichment analysis of target genes of hsa-miR-367-3p
Gategory GO number Term Count P
GOTERM_BP_DIRECT 0045944 Positive regulation of transcription from RNA polymeraseⅡpromoter 16 0.009
GOTERM_BP_DIRECT 0000122 Negative regulation of transcription from RNA polymeraseⅡpromoter 14 0.004
GOTERM_BP_DIRECT 0003211 Cardiac ventricle formation 2 0.038
GOTERM_BP_DIRECT 0006366 Transcription from RNA polymeraseⅡpromoter 12 0.002
GOTERM_CC_FAT 0043005 Neuron projection 10 0.001
GOTERM_CC_FAT 0097458 Intracellular membrane-bound organelle 13 0.001
GOTERM_CC_FAT 0045202 Synapse 7 0.003
GOTERM_CC_FAT 0005794 Golgi apparatus 18 0.001
GOTERM_MF_DIRECT 0005515 Protein binding 92 0.001
GOTERM_MF_DIRECT 0003730 mRNA 3’-UTR binding 4 0.007
GOTERM_MF_DIRECT 0035091 Phosphatidylinositol binding 4 0.030
GOTERM_MF_DIRECT 0001077 Transcriptional activator activity, RNA polymeraseⅡcore promoter proximal region sequence-specific binding 10 0.001
GOTERM_MF_DIRECT 0008526 Phosphatidylinositol transporter activity 2 0.046

2.6 hsa-miR-367-3p靶基因的信号转导通路分析

利用DAVID数据库对取交集的152个候选靶基因进行生物通路富集分析。预测结果为16个通路,其中,预测靶基因集合显著富集于PI3K-AKT信号通路与癌症通路(P < 0.05),见表 3图 3

表 3 hsa-miR-367-3p靶基因的信号转导通路富集分析结果 Tab.3 Enrichment analysis showing the target genes of hsa-miR-367-3p involved in various signaling pathways
Term Genotype P
PI3K-Akt signaling pathway CCNE2ITGA6TSC1ITGA5ITGAVFASLGPIK3AP1PIK3R3 0.003
Pathways prevalent in cancer CCNE2ITGA6ITGAVFASLGMAPK8APPL1PIK3R3 0.020
cAMP signaling pathway HCN2ADRB1ATP2B4PPP1R12AMAPK8PIK3R3 0.001
Proteoglycans prevalent in cancer ITGA5ANK3ITGAVPPP1R12AFASLGPIK3R3ITPR1 0.002
Focal adhesion ITGA6ITGA5ITGAVPPP1R12AMAPK8PIK3R3 0.002
Regulation of actin cytoskeleton WASLITGA5ITGA6ITGAVPIK3R3PPP1R12A 0.002
Small cell lung cancer ITGA6ITGAVPIK3R3CCNE2 0.022
Progesterone-mediated oocyte maturation CPEB2CPEB3MAPK8PIK3R3 0.024
Choline metabolism in cancer WASLMAPK8TSC1PIK3R3 0.035
Hepatitis B DDX3XFASLGCCNE2MAPK8PIK3R3 0.019
Oocyte meiosis CCNE2CPEB2CPEB3ITPR1 0.044
Arrhythmogenic right ventricular cardiomyopathy DSC2ITGA5ITGA6ITGAV 0.012
Dilated cardiomyopathy ADRB1ITGA5ITGA6ITGAV 0.022

图 3 hsa-miR-367-3p的靶基因参与cAMP信号通路 Fig.3 Predicted target genes of hsa-miR-367-3p involved in the cAMP signaling pathway

3 讨论

miRbase分析发现,hsa-miR-367-3p在人、小鼠、玻璃海鞘、家兔等多个物种之间具有高度保守性。通过miRGator v3.0数据库对hsa-miR-367-3p在各个器官的表达情况进行分析,结果显示其在干细胞中表达丰富。TargetScan预测的hsa-miR-367-3p靶基因数量为395个,miRDB预测的靶基因个数为495个,2个数据库预测的靶基因取交集共筛选出152个靶基因,其中包括PI3K-AKT信号通路的基因:CCNE2ITGA6TSC1ITGA5ITGAVFASLGPIK3AP1等;癌症信号通路相关的基因:CCNE2ITGA6MAPK8PIK3R3FASLGAPPL1等。功能富集分析发现,hsa-miR-367-3p的靶基因作用于多种生物过程、细胞组分和分子功能等,包括心室发育、神经元、突触、高尔基体以及蛋白质结合等。相关研究[5]表明,hsa-miR-367-3p作用于相关癌症,包括脑胶质瘤、子宫内膜癌、肝癌。研究[6]发现,miR-367-3p的靶基因CEBPA在脑胶质瘤中发挥作用。MA等[7]研究结果表明,miR-367-3p通过介导的HMGA2基因在子宫内膜癌中发挥作用,这表明miR-367-3p介导的HMGA2靶基因可能是子宫内膜癌的生物标志物。XU等[8]研究发现,miR-367-3p可以调节pAkt和pERK信号通路抑制肝癌细胞侵袭,其中的pAkt信号通路与预测出的hsa-miR-367-3p其中靶基因的信号转导通路相吻合。但是,hsa-miR-367-3p对癌症的作用机制还有待进一步研究,本研究通过对hsa-miR-367-3p进行生物信息学预测,个别预测出来的靶基因与通路已经被证实,更多的预测结果需要进一步的研究加以验证,本研究分析结果为hsa-miR-367-3p今后的相关研究提供了参考依据,并为癌症机制的研究提供了一些新的思路和方向。

参考文献
[1]
RAJAGOPALAN R, VAUCHERET H, TREJO J, et al. A diverse and evolutio-narily fluid set of microRNAs in arabidopsis thaliana[J]. Genes Dev, 2006, 20(24): 3407-3425. DOI:10.1101/gad.1476406
[2]
ROOIJ EV, SUTHERLAND LB, LIU N, et al. Olson ENA signature patternof stress-responsive microRNAs that can evoke cardiac hypertrophy and hea-rt failure[J]. Proc Nat Acad Sci, 2006, 103(48): 18255-18260. DOI:10.1073/pnas.0608791103
[3]
MATKOVICH SJ, HU Y, ESCHENBACHER WH, et al. Direct and indirect involvement of microRNA-499 in clinical and experimental cardiomyopathy[J]. Circ Res, 2012, 111(5): 521-531. DOI:10.1161/CIRCRESAHA.112.265736
[4]
THUM T, GROSS C, FIEDLER J, et al. MicroRNA-21 contributes to myocard-ial disease by stimulating map kinase signalling in fibroblasts[J]. Nature, 2008, 456(7224): 980-984. DOI:10.1038/nature07511
[5]
BHAYANI MK, CALIN GA, LAI SY. Functional relevance of miRNA seq-uences in human disease[J]. Mutat Res, 2012, 731(1/2): 14-19. DOI:10.1016/j.mrfmmm.2011.10.014
[6]
LIU XB, ZHENG J, XUE YX, et al. Piwil3/oip5-as1/miR-367-3p/cebpa feedback loop regulates the biological behavior of glioma cells[J]. Theranostics, 2018, 8(4): 1084-1105. DOI:10.7150/thno.21740
[7]
MA J, LI D, KONG FF, et al. miR-302a-5p/367-3p-HMGA2 axis regulates m-alignant processes during endometrial cancer development[J]. Cancer Res, 2018, 37(1): 19-36. DOI:10.1186/s13046-018-0686-6
[8]
XU J, LIN H, LI G, et al. MiR-367-3p increases sorafenib chemotherapy eff-icacy to suppress hepatocellular carcinoma metastasis through altering thean-drogen receptor signals[J]. EBioMedicine, 2016, 12(1): 55-67. DOI:10.1016/j.ebiom.2016.07.013