miRNA-148a-3p的生物信息学分析及其在小鼠不同组织中的表达水平检测

doi:10.11843/j.issn.0366-6964.2022.02.009

摘要/Abstract

摘要： 旨在研究小鼠不同组织中miR-148a-3p的表达情况，并对其进行生物信息学分析，构建miR-148a-3p基因相关网络，进一步阐明其在机体中的作用。本研究以6周龄雄性C57BL/6 J小鼠胃、肺、脾、皮肤、肝、心和肾7个组织为研究材料，每组3个重复，进行3次平行试验。利用qRT-PCR技术比较小鼠不同组织中miR-148a-3p的表达水平。在miBase上对miR-148a-3p在不同物种间的保守性进行分析，并通过Promoter Scan和TRANSFAC预测其转录因子结合位点。利用TargetScan、PicTar和miRanda预测miR-148a-3p的靶基因并取交集，并进行GO和KEGG分析。利用Cytoscape构建TF-miRNA-mRNA互作网络。qRT-PCR结果显示，在所有组织中，miR-148a-3p在脾中的表达水平最高（P<0.001），肝次之（P<0.001），在皮肤、胃、心、肾、肺中的表达水平逐渐降低。经预测bata-pol、CREB、E4F1、NF-S等多个转录因子可以与miR-148a-3p结合并调控其表达。GO富集和KEGG通路分析表明，miR-148a-3p的靶基因主要参与离子转运、乙酰胆碱激活的阳离子选择性通道活性、构成细胞质膜等功能；并参与TGF-β、肥厚性心肌病和黏附连接等通路。由70个点和70条边构成了TF-miRNA-mRNA互作网络，包含5个转录因子和63个mRNAs。结果表明，miR-148a-3p在小鼠脾组织中高表达且受到多种转录因子调控，与癌症发展密切相关。

关键词: 生物信息学, miR-148a-3p, 表达, 功能, 癌症

Abstract: The study aimed to detect the miR-148a-3p expression in different tissues of mice, conduct bioinformatics analysis and construct gene network to further clarify the role of miR-148a-3p in the body. The stomach, lung, spleen, skin, liver, heart and kidney of 6-week-old male C57BL/6 J mice were used as research materials, and 3 parallel experiments were conducted with 3 replicates in each group. The qRT-PCR was used to analyze the miR-148a-3p expression in different tissues of mice. miBase was used to analyze the conservation of miR-148a-3p among different species, and to predict its transcription factor binding site through Promoter Scan and TRANSFAC. TargetScan, PicTar and miRanda were used to predict the target genes of miR-148a-3p, then the target genes intersection was took, and GO and KEGG analysis was performed. Cytoscape was used to construct a TF-miRNA-mRNA interaction network. The results of qRT-PCR showed that among all tissues, the expression level of miRNA-148a-3p was the highest in spleen (P<0.001), followed by liver (P<0.001), and gradually decreased in skin, stomach, heart, kidney and lung. Transcription factors such as bata-pol, CREB, E4F1 and NF-S could bind to miR-148a-3p and regulate its expression. GO enrichment and KEGG pathway analysis showed that the target genes of miR-148a-3p mainly involved in the functions of ion transport, acetylcholine-activated cation-selective channel activity, and external side of plasma membrane. Target genes were mainly involved in TGF-β, hypertrophic cardiomyopathy and adhesion pathways. The TF-miRNA-mRNA interaction network was composed of 70 sites and 70 edges, including 5 transcription factors and 63 mRNAs. The results indicate that miR-148a-3p is highly expressed in mouse spleen tissues and is regulated by a variety of transcription factors, which is closely related to cancer development.

Key words: bioinformatics, miR-148a-3p, expression, function, cancer

中图分类号:

S813.1

刘芳君, 蓝吴涛, 马悦悦, 李鹏飞, 张磊, 朱芷葳. miRNA-148a-3p的生物信息学分析及其在小鼠不同组织中的表达水平检测[J]. 畜牧兽医学报, 2022, 53(2): 414-422.

LIU Fangjun, LAN Wutao, MA Yueyue, LI Pengfei, ZHANG Lei, ZHU Zhiwei. Bioinformatics Analysis of miRNA-148a-3p and Detection of Its Expression Level in Different Tissues of Mice[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(2): 414-422.

参考文献 41

[1]	LEUNG A K L,CALABRESE J M,SHARP P A.Quantitative analysis of Argonaute protein reveals microRNA-dependent localization to stress granules[J].Proc Natl Acad Sci U S A,2006,103(48):18125-18130.
[2]	STROVAS T J,ROSENBERG A B,KUYPERS B E,et al.MicroRNA-based single-gene circuits buffer protein synthesis rates against perturbations[J].ACS Synth Biol,2014,3(5):324-331.
[3]	BRAUN J E,HUNTZINGER E,IZAURRALDE E.The role of GW182 proteins in miRNA-mediated gene silencing[J].Adv Exp Med Biol,2013,768:147-163.
[4]	CALIN G A,SEVIGNANI C,DUMITRU C D,et al.Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers[J].Proc Natl Acad Sci U S A,2004,101(9):2999-3004.
[5]	NANA-SINKAM P,CROCE C M.MicroRNAs in diagnosis and prognosis in cancer:what does the future hold?[J]. Pharmacogenomics,2010,11(5):667-669.
[6]	YOU J,WANG X.Circ_HIPK3 knockdown inhibits cell proliferation,migration and invasion of cholangiocarcinoma partly via mediating the miR-148a-3p/ULK1 pathway[J].Cancer Manag Res,2021,13:3827-3839.
[7]	WANG Y P,HU Y N,GUO J H,et al.miR-148a-3p suppresses the proliferation and invasion of esophageal cancer by targeting DNMT1[J].Genet Test Mol Biomarkers,2019,23(2):98-104.
[8]	TAN C,LIU W,ZHENG Z H,et al.LncRNA HOTTIP inhibits cell pyroptosis by targeting miR-148a-3p/AKT2 axis in ovarian cancer[J].Cell Biol Int,2021,45(7):1487-1497.
[9]	TSAI H L,YANG I P,HUANG C W,et al.Clinical significance of microRNA-148a in patients with early relapse of stage II stage and III colorectal cancer after curative resection[J].Transl Res,2013,162(4):258-268.
[10]	GUO S L,PENG Z,YANG X,et al.miR-148a promoted cell proliferation by targeting p27 in gastric cancer cells[J].Int J Biol Sci,2011,7(5):567-574.
[11]	YAN J,GUO X Q,XIA J Z,et al.miR-148a regulates MEG3 in gastric cancer by targeting DNA methyltransferase 1[J].Med Oncol,2014,31(3):879.
[12]	HAFLIDADÓTTIR B S,BERGSTEINSDÓTTIR K,PRAETORIUS C,et al.miR-148 regulates Mitf in melanoma cells[J].PLoS One,2010,5(7):e11574.
[13]	WANG X K,ZHOU Y J,ZHAO G,et al.MiR-148a-3p suppresses cell proliferation,migration and invasion by targeting PIK3CA in human osteosarcoma cells[J].Oncotarget,2018, doi:10.18632/ONCOTARGET.23931.
[14]	WANG W,DONG J,WANG M X,et al.miR-148a-3p suppresses epithelial ovarian cancer progression primarily by targeting c-Met[J].Oncol Lett,2018,15(5):6131-6136.
[15]	QIN L M,CHEN Y S,NIU Y N,et al.A deep investigation into the adipogenesis mechanism:profile of microRNAs regulating adipogenesis by modulating the canonical Wnt/β-catenin signaling pathway[J].BMC Genomics,2010,11:320.
[16]	SONG M,LIU J,ZHENG X,et al.MiR-148a-3p targets CEMIP to suppress the genesis of gastric cancer cells[J].Biochem Biophys Res Commun,2021,575:42-49.
[17]	CHEN Q,WANG Y D,DANG H M,et al.MicroRNA-148a-3p inhibits the proliferation of cervical cancer cells by regulating the expression levels of DNMT1 and UTF1[J].Oncol Lett,2021,22(2):617.
[18]	LYU Z Z,YANG M Y,YANG T,et al.Metal-regulatory transcription factor-1 targeted by miR-148a-3p is implicated in human hepatocellular carcinoma progression[J].Front Oncol,2021,11:700649.
[19]	XU G P,ZHU Y G,LIU H J,et al.Long non-coding RNA KCNQ1OT1 promotes progression of hepatocellular carcinoma by miR-148a-3p/IGF1R axis[J].Technol Cancer Res Treat,2020,19:1-10.
[20]	ZHOU H,JIA X F,YANG F,et al.miR-148a-3p suppresses the progression of acute myeloid leukemia via targeting cyclin-dependent kinase 6(CDK6)[J].Bioengineered,2021,12(1):4508-4519.
[21]	YIN H D,HE H R,CAO X N,et al.MiR-148a-3p regulates skeletal muscle satellite cell differentiation and apoptosis via the PI3K/AKT signaling pathway by targeting Meox2[J].Front Genet,2020,11:512.
[22]	SU H B,FAN G Z,HUANG J,et al.YBX1 regulated by Runx3-miR-148a-3p axis facilitates non-small-cell lung cancer progression[J].Cell Signal,2021,85:110049.
[23]	LIU N,SUN Y X.microRNA-148a-3p-targeting p300 protects against osteoblast differentiation and osteoporotic bone reconstruction[J].Regen Med,2021,16(5):435-449.
[24]	WU Y X,GU W Q,HAN X,et al.LncRNA PVT1 promotes the progression of ovarian cancer by activating TGF-β pathway via miR-148a-3p/AGO1 axis[J].J Cell Mol Med,2021,25(17):8229-8243.
[25]	ZHENG J X,YANG T,GAO S H,et al.miR-148a-3p silences the CANX/MHC-I pathway and impairs CD8+ T cell-mediated immune attack in colorectal cancer[J].FASEB J,2021,35(8):e21776.
[26]	PORSTNER M,WINKELMANN R,DAUM P,et al.miR-148a promotes plasma cell differentiation and targets the germinal center transcription factors Mitf and Bach2[J].Eur J Immunol,2015,45(4):1206-1215.
[27]	LOMBARD A P,MOOSO B A,LIBERTINI S J,et al.miR-148a dependent apoptosis of bladder cancer cells is mediated in part by the epigenetic modifier DNMT1[J].Mol Carcinog,2016,55(5):757-767.
[28]	孙小光,欧阳军.脾脏的免疫功能及脾脏对血流变影响的研究[J].现代生物医学进展,2009,9(19):3776-3778.SUN X G,OUYANG J.The research of spleen immune function and the influence of spleen to bloodcurrent[J].Progress in Modern Biomedicine,2009,9(19):3776-3778.(in Chinese)
[29]	HEY Y Y,TAN J K H,O'NEILL H C.Redefining myeloid cell subsets in murine spleen[J].Front Immunol,2016,6:652.
[30]	孙备,李承.脾脏功能的研究进展[J].临床外科杂志,2006,14(7):450-452.SUN B,LI C.Research progress of spleen function[J].Journal of Clinical Surgery,2006,14(7):450-452.(in Chinese)
[31]	XI Y M,QI Z H,MA J F,et al.PTEN loss activates a functional AKT/CXCR4 signaling axis to potentiate tumor growth and lung metastasis in human osteosarcoma cells[J].Clin Exp Metastasis,2020,37(1):173-185.
[32]	汪美容,舒宽勇.P21、P27蛋白在肿瘤中的表达研究进展[J].实用临床医学,2018,19(6):101-103,107.WANG M R,SHU K Y.Research progress on the expression of P21 and P27 proteins in tumors[J].Practical Clinical Medicine,2018,19(6):101-103,107.(in Chinese)
[33]	侯振江,张宗英.p27基因在肿瘤研究中的进展[J].国外医学临床生物化学与检验学分册,2005,26(12):960.HOU Z J,ZHANG Z Y.Progress of p27 gene in tumor research[J].Clinical Biochemistry and Laboratory Medicine,Foreign Medical Sciences,2005,26(12):960.(in Chinese)
[34]	GUO S L,PENG Z,YANG X,et al.miR-148a promoted cell proliferation by targeting p27 in gastric cancer cells[J].Int J Biol Sci,2011,7(5):567-574.
[35]	ZHANG W,LI Y.miR-148a downregulates the expression of transforming growth factor-β2 and SMAD2 in gastric cancer[J].Int J Oncol,2016,48(5):1877-1885.
[36]	祁闪闪,杨李,卢文婕,等.调控PTEN表达和活性的研究进展[J].中国病理生理杂志,2020,36(12):2289-2293.QI S S,YANG L,LU W J,et al.Advances on regulation of PTEN expression and activity[J].Chinese Journal of Pathophysiology, 2020,36(12):2289-2293.(in Chinese)
[37]	李季林,肖建,于如同.肿瘤抑制基因PTEN研究新进展[J].现代肿瘤医学,2010,18(5):1034-1037.LI J L,XIAO J,YU R T.Research progress of tumor suppressor gene PTEN[J].Journal of Modern Oncology,2010, 18(5):1034-1037.(in Chinese)
[38]	赵敬柱,郑向前,高明.肿瘤抑制基因PTEN的研究进展[J].中华耳鼻咽喉头颈外科杂志,2016,51(10):797-800.ZHAO J Z,ZHENG X Q,GAO M.Research progress of suppressor gene PTEN[J].Chinese Journal of Otorhinolaryngology Head and Neck Surgery,2016,51(10):797-800.(in Chinese)
[39]	谢振宇,程金妹,张鹏飞,等.抑癌基因PTEN研究进展[J].中国肿瘤,2008,17(1):33-37.XIE Z Y,CHENG J M,ZHANG P F,et al.Research progress on anti-oncogene PTEN[J].Bulletin of Chinese Cancer, 2008,17(1):33-37.(in Chinese)
[40]	李曼,徐宁,黄宪章.抑癌基因PTEN的研究进展[J].现代检验医学杂志,2009,24(5):12-14.LI M,XU N,HUANG X Z.Research progress of anti-oncogene PTEN[J].Journal of Modern Laboratory Medicine, 2009,24(5):12-14.(in Chinese)
[41]	黄辉云,沈二栋,唐四清,等.miR-148a-3p靶向调控PTEN基因表达对黑色素瘤细胞生物学特性的影响[J].肿瘤药学,2019,9(2):219-225,237.HUNAG H Y,SHEN E D,TANG S Q,et al.MiR-148a-3p down regulation inhibits cell proliferation,migration and invasion and promotes cell apoptosis by enhancing PTEN in melanoma[J].Anti-Tumor Pharmacy,2019,9(2):219-225,237.(in Chinese)