Tian Donghao,Xu Wenyao,Yu Hui,et al.Experimental study of miR-939 regulating the phenotypic features of ENCCs by targeting SOX4 in Hirschsprung’s disease[J].Journal of Clinical Pediatric Surgery,2021,20(03):222-229.[doi:10.12260/lcxewkzz.2021.03.005]
先天性巨结肠中miR-939靶向调控SOX4表达抑制肠神经嵴前体细胞功能表型的实验研究
- Title:
- Experimental study of miR-939 regulating the phenotypic features of ENCCs by targeting SOX4 in Hirschsprung’s disease
- 关键词:
- 先天性巨结肠/病因学; 肠神经系统; 细胞增值
- 分类号:
- R574;R393;R329.2+8
- 摘要:
- 目的 检测MicroRNA-939（miR-939）和性别决定区相关HMG族盒蛋白4（Sry-related HMG box 4，SOX4）在先天性巨结肠（Hirschsprung’s disease，HSCR）中的表达水平，评估miR-939在SOX4调控肠神经嵴前体细胞（enteric neural crest cells，ENCCs）增殖、凋亡和迁移过程中的作用，并探讨miR-939/SOX4在HSCR干预治疗方面的潜在价值。方法 采用RT-PCR、Western blot检测HSCR和肠套叠患者肠组织中miR-939、SOX4表达水平；从常规孕鼠肠管取材，体外培养ENCCs，通过Nestin、GFAP双免疫荧光染色鉴定。使用miR-939模拟物、SOX4-siRNA干预，并分别通过CCK8法、流式细胞仪和Transwell小室检测ENCCs增殖、凋亡和迁移。结果 与肠套叠组患者相比，HSCR患者肠组织中miR-939的表达水平显著提高，而SOX4表达水平显著降低。miR-939模拟物可显著抑制ENCCs中SOX4的表达水平；上调miR-939和下调SOX4均能够显著降低ENCCs增殖和迁移，并促进细胞凋亡。结论 HSCR患者肠组织miR-939、SOX4均表达异常，负向调控ENCCs增殖、凋亡和迁移可能参与HSCR发病过程，靶向miR-939/SOX4的干预策略可为后续HSCR干预治疗提供潜在的治疗参考。
- Abstract:
- Objective To detect the expression levels of miR-939 and SOX4 in Hirschsprung’s disease (HSCR),evaluate the role of miR-939 in SOX4 regulating enteric neural crest cells (ENCCs) and explore the potential value of miR-939/SOX4 in the intervention of HSCR.Methods Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were employed for measuring the expression levels of miR-939 and SOX4 in intestinal tissues of HSCR children with intussusception.ENCCs from pregnant Sprague Dawley (SD) rats were routinely cultured and identified by Nestin/GFAP immunofluorescent staining.And miR-939 mimics and SOX4 siRNA were applied to ENCCs and cellular proliferation,apoptosis and migration detected by CCK8,flow cytometry and Transwell chamber assay.Results As compared with intussusception group,the expression level of miR-939 significantly increased in HSCR group while the expression level of SOX4 markedly declined.And miR-939 mimics significantly inhibited the expression of SOX4 in ENCCs.The up-regulation of miR-939 and the down-regulation of SOX4 significantly reduced the proliferation and migration of ENCCs,and promoted apoptosis of ENCCs. Conclusion Conclusion The expression of miR-939/SOX4 is abnormal in HSCR children.A negative regulation of the proliferation,apoptosis and migration of ENCCs may be involved in the pathogenesis of HSCR.Targeted intervention of miR-939/SOX4 signaling pathway provides potential therapeutic targets for subsequent intervention of HSCR.
参考文献/References:
1 朱天琦.先天性巨结肠的诊断及治疗专家共识[J].中华小儿外科杂志,2017,38(11):805-815.DOI:10.3760/cma.j.issn.0253-3006.2017.11.002. Zhu TQ.Expert consensus of diagnosis and treatment of Hirschspung’s disease[J].Chin J Pediatr Surg,2017,38(11):805-815.DOI:10.3760/cma.j.issn.0253-3006.2017.11.002.
2 沈涤华,施诚仁,吴晔明,等.先天性巨结肠患儿肠道内Smoothelin的表达[J].中华小儿外科杂志,2017,38(4):288-291.DOI:10.3760/cma.j.issn.0253-3006.2017.04.010. Shen DH,Shi CR,Wu YM,et al.Expression levels of smoothelin in colonic tissues from patients with Hirschs-prung’s disease[J].Chin J Pediatr Surg,2017,38(4):288-291.DOI:10.3760/cma.j.issn.0253-3006.2017.04.010.
3 张建军,刘丰丽,唐维兵,等.深化的加速康复外科理念在婴儿先天性巨结肠症围手术期的应用[J].中华小儿外科杂志,2019,40(3):245-248.DOI:10.3760/cma.j.issn.0253-3006.2019.03.011. Zhang JJ,Liu FL,Tang WB,et al.Promoting perioperative application of enhanced recovery after surgery for infantile Hirschsprung’s disease[J].Chin J Pediatr Surg2019,40(3):245-248.DOI:10.3760/cma.j.issn.0253-3006.2019.03.011.
4 Borrego S,Ruiz-Ferrer M,Fernandez RM,et al.Hirschs-prung’s disease as a model of complex genetic etiology[J].Histology & Histopathology,2013,28(9):1117-1136.DOI:10.14670/HH-28.1117.
5 牛鑫,景雅,李海荣,等.神经嵴细胞和胰岛因子-1阳性细胞与小鼠胚胎心动脉端的发育[J].解剖学杂志,2012,35(5):553-557.DOI:10.3969/j.issn.1001-1633.2012.05.001. Niu X,Jing Y,Li HR,et al.Distribution patterns of neural crest cells and ISL-1 positive cells during the development of arterial pole of murine embryonic heart[J].Chinese Journal of Anatomy,2012,35(5):553-557.DOI:10.3969/j.issn.1001-1633.2012.05.001.
6 Luzón-Toro B,Gui H,Ruiz-Ferrer M,et al.Exome sequencing reveals a high genetic heterogeneity on familial Hirschsprung disease[J].Scientific Reports,2015,5:16473.DOI:10.1038/srep16473.
7 Nishikawa R,Hotta R,Shimojima N,et al.Migration and differentiation of transplanted enteric neural crest-derived cells in murine model of Hirschsprung’s disease[J].Cytotechnology,2015,67(4):661-670.DOI:10.1007/s10616-014-9754-8.
8 Rogers JM.Search for the missing lncs:gene regulatory networks in neural crest development and long non-coding RNA biomarkers of Hirschsprung’s disease[J].Neurogastroenterol Motil,2016,28(2):161-166.DOI:10.1111/nmo.12776.
9 Feng A,Yuan X,Li X.MicroRNA-345 inhibits metastasis and epithelial-mesenchymal transition of gastric cancer by targeting FOXQ1[J].Oncology Reports,2017,38(5):2752-2760.DOI:10.3892/or.2017.6001.
10 Liu Y,Wang Y,Xu Q,et al.Prognostic evaluation of microRNA-210 in various carcinomas[J].Medicine,2017,96(43):e8113.DOI:10.1097/MD.0000000000008113.
11 Zhang Z,Wang J,Li J,et al.MicroRNA-150 promotes cell proliferation,migration,and invasion of cervical cancer through targeting PDCD4[J].Biomed Pharmacother,2017,97:511-517.DOI:10.1016/j.biopha.2017.09.143.
12 Sharan A,Zhu H,Xie H,et al.Down-regulation of miR-206 is associated with Hirschsprung disease and suppresses cell migration and proliferation in cell models[J].Scientific Reports,2015,5(1):9302.DOI:10.1038/srep09302.
13 Gunadi,Budi Nyp,Kalim As,et al.Aberrant expressions of miRNA-206 target,FN1,in multifactorial Hirschsprung disease[J].Orphanet J Rare Dis,2019,14(1):5.DOI:10.1186/s13023-018-0973-5.
14 Zhu D,Xie H,Li H,et al.Nidogen-1 is a common target of microRNAs MiR-192/215 in the pathogenesis of Hirschs-prung’s disease[J].Journal of Neurochemistry,2015,134(1):39-46.DOI:10.1111/jnc.13118.
15 Du G,Wang X,Wu Y,et al.Downregulation of miR-140-5p affects the pathogenesis of HSCR by targeting EGR2[J].Pediatr Surg Int,2020,36(8):883-890.DOI:10.1007/s00383-020-04686-0.
16 Li H,Zhou L,Zhi Z,et al.Lipopolysaccharide upregulates miR-132/212 in Hirschsprung-associated enterocolitis,facilitating pyroptosis by activating NLRP3 inflammasome via targeting Sirtuin 1 (SIRT1)[J].Aging (Albany NY),2020,12(18):18588-18602.DOI:10.18632/aging.103852.
17 Situ J,Zhang H,Jin Z,et al.MicroRNA-939 directly targets HDGF to inhibit the aggressiveness of prostate cancer via deactivation of the WNT/β-Catenin pathway[J].Onco Targets Ther,2020,13:4257-4270.DOI:10.2147/OTT.S250101.
18 Zhao X,Li J,Yu D.MicroRNA-939-5p directly targets IGF-1R to inhibit the aggressive phenotypes of osteosarcoma through deactivating the PI3K/Akt pathway[J].Int J Mol Med,2019,44(5):1833-1843.DOI:10.3892/ijmm.2019.4333.
19 Chen G,Du C,Shen Z,et al.MicroRNA-939 inhibits cell proliferation via targeting LRSAM1 in Hirschsprung’s disease[J].Aging (Albany NY),2017,9(12):2471-2479.DOI:10.18632/aging.101331.
20 Kavyanifar A,Turan S,Lie DC.SoxC transcription factors:multifunctional regulators of neurodevelopment[J].Cell Tissue Res,2018,371(1):91-103.DOI:10.1007/s00441-017-2708-7.
21 Pan W,Yu H,Zheng B,et al.Upregulation of MiR-369-3p suppresses cell migration and proliferation by targeting SOX4 in Hirschsprung’s disease[J].J Pediatr Surg,2017,52(8):1363-1370.DOI:10.1016/j.jpedsurg.2017.04.002.
22 肖莉,刘勇,高亚.小鼠胚胎肠神经嵴干细胞的分离培养及鉴定[J].西安交通大学学报(医学版),2007,28(5):595-597.DOI:10.3969/j.issn.1671-8259.2007.05.034. Xiao L,Liu Y,Gao Y.Isolation,culturing and identification of murine embryonic enteric neural crest stem cells[J].Journal of Xi’an Jiaotong University,2007,28(5):595-597.DOI:10.3969/j.issn.1671-8259.2007.05.034.
23 Fattahi F,Steinbeck JA,Kriks S,et al.Deriving human ENS lineages for cell therapy and drug discovery in Hirschsprung disease[J].Nature,2016,531(7592):105-109.DOI:10.1038/nature16951.
24 肖俊,李智.肠神经系统发育的研究进展[J].中华小儿外科杂志,2018,39(12):952.DOI:10.3760/cma.j.issn.0253-3006.2018.12.019. Xiao J,Li Z.Recent advances in the development of enteric nervous system[J].Chin J Pediatr Surg,2018,39(12):952.DOI:10.3760/cma.j.issn.0253-3006.2018.12.019.
25 Li H,Tang J,Lei H,et al.Decreased MiR-200a/141 suppress cell migration and proliferation by targeting PTEN in Hirschsprung’s disease[J].Cellular Physiology and Biochemistry,2014,34(2):543-553.DOI:10.1159/000363021.
26 Lv X,Li Y,Li H,et al.Molecular function predictions and diagnostic value analysis of plasma exosomal miRNAs in Hirschsprung’s disease[J].Epigenomics,2020,12(5):409-422.DOI:10.2217/epi-2019-0190.
27 Yoon TM,Kim SA,Cho WS,et al.SOX4 expression is associated with treatment failure and chemoradioresistance in oral squamous cell carcinoma[J].BMC Cancer,2015,15(1):888.DOI:10.1186/s12885-015-1875-8.
28 Wang B,Li Y,Tan F,et al.Increased expression of SOX4 is associated with colorectal cancer progression[J].Tumor Biology,2016,37(7):9131-9137.DOI:10.1007/s13277-015-4756-5.
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备注/Memo
收稿日期:2020-12-15。
基金项目:国家自然科学基金（编号：NSFC81701501，NSFC82071692，NSFC81770513）；陕西省科技新星（编号：2019KJXX-044）；陕西省自然科学基金（编号：2020JM-407）
通讯作者:高亚,Email:ygao@xjtu.edu.cn;潘伟康,Email:weikang915@mail.xjtu.edu.cn