尿道下裂相关miRNA差异表达分析的初步探讨
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摘要
目的通过筛选尿道下裂、术前经HCG治疗的尿道下裂以及正常包皮组织中miRNA差异表达,探究其与尿道下裂的发生以及术前HCG治疗影响预后的可能途径,为探究尿道下裂病因提供新思路。方法选取9例尿道下裂(其中3例术前进行HCG治疗)以及3例包茎患儿(对照组)为研究对象,分别取包皮组织抽提总RNA,利用芯片技术筛选各组差异性表达的miRNAs,并用RT-PCR技术对筛选结果进行验证。结果芯片筛选结果:与对照组比较,尿道下裂患儿的包皮组织里差异性表达的miRNAs中上调的19个,下调的15个。与术前行HCG治疗组的患儿相比,术前未行HCG治疗的尿道下裂患儿包皮组织里差异性表达的miRNA中上调的19个,下调的25个; RT-PCR验证结果:在差异性表达的miRNA中选择10个miRNA进行验证,其中5个miRNA与芯片结果相符,分别是let-7b-3p、miR-145-5p、miR-376a-3p、miR-377-3p和miR-1-3p。其中let-7b-3p、miR-145-5p、miR-376a-3p、miR-377-3p在尿道下裂组中低表达,在对照组和术前HCG治疗的尿道下裂组中高表达; miR-1-3p在术前HCG治疗的尿道下裂患儿中较未用药组中高表达。结论首次建立尿道下裂患儿miRNAs的基因表达谱,并筛选出在术前行HCG治疗尿道下裂、术前未行HCG治疗尿道下裂与包茎之间存在差异表达的miRNAs。根据筛选出的差异性表达的miRNAs,推测其与尿道下裂的关系,为今后疾病的预防以及提高治疗效果提供新的线索。
Objective To detect the differential expression of miRNAs in foreskin tissue related to hypospadiac deformation in etiology,explore the possible role for preoperative human chorionic gonadotropin( HCG)treatment for facilitating surgical operation and reduce the complications. Methods Foreskin tissue was collected for total RNA extraction from 9 cases of hypospadiac children including 3 cases with preoperative HCG treatment and 3 children with phimosis as control. Chip was used for screening the differential expression of miRNA and the results were verified by real-time polymerase chain reaction( RT-PCR). Results Results of miRNA chips: Compared to children with phimosis,the number of up-regulated miRNA was 19 while down-regulated miRNA was 15 in foreskin tissue of hypospadiacs. As compared to hypospadiacs with preoperative HCG treatment,the number of up-regulated miRNA was 19 while down-regulated miRNA was 25 in foreskin tissue of those without preoperative treatment. Results of RT-PCR: Among 10 differentially expressed miRNAs,half of them were verified,i. e. let-7 b-3 p,miR-145-5 p,miR-376 a-3 p,miR-377-3 p and miR-1-3 p. And let-7 b-3 p,miR-145-5 p,miR-376 a-3 p,miR-377-3 p were down-regulated in hypospadias group and up-regulated in phimosis and hypospadias with preoperative HCG treatment. Yet miR-1-3 p was up-regulated in hypospadias with preoperative HCG treatment as compared to hypospadias group. Conclusion This study has established the miRNA expression profiles of hypospadias for the first time. Differentially expressed miRNAs are screened among children with hypospadias,those with phimosis and those with hypospadias with preoperative HCG treatment. Future studies of differentially expressed miRNAs may provide new etiological clues for hypospadias.
Objective To detect the differential expression of miRNAs in foreskin tissue related to hypospadiac deformation in etiology,explore the possible role for preoperative human chorionic gonadotropin( HCG)treatment for facilitating surgical operation and reduce the complications. Methods Foreskin tissue was collected for total RNA extraction from 9 cases of hypospadiac children including 3 cases with preoperative HCG treatment and 3 children with phimosis as control. Chip was used for screening the differential expression of miRNA and the results were verified by real-time polymerase chain reaction( RT-PCR). Results Results of miRNA chips: Compared to children with phimosis,the number of up-regulated miRNA was 19 while down-regulated miRNA was 15 in foreskin tissue of hypospadiacs. As compared to hypospadiacs with preoperative HCG treatment,the number of up-regulated miRNA was 19 while down-regulated miRNA was 25 in foreskin tissue of those without preoperative treatment. Results of RT-PCR: Among 10 differentially expressed miRNAs,half of them were verified,i. e. let-7 b-3 p,miR-145-5 p,miR-376 a-3 p,miR-377-3 p and miR-1-3 p. And let-7 b-3 p,miR-145-5 p,miR-376 a-3 p,miR-377-3 p were down-regulated in hypospadias group and up-regulated in phimosis and hypospadias with preoperative HCG treatment. Yet miR-1-3 p was up-regulated in hypospadias with preoperative HCG treatment as compared to hypospadias group. Conclusion This study has established the miRNA expression profiles of hypospadias for the first time. Differentially expressed miRNAs are screened among children with hypospadias,those with phimosis and those with hypospadias with preoperative HCG treatment. Future studies of differentially expressed miRNAs may provide new etiological clues for hypospadias.
引文
1 Antonio M,Atila R,Valdemar O.Hypospadias[J].Current Opinion in Urology,2012,22(6):447-452.DOI:10.1097/MOU.0b013e328357bc62.
2 van der Zanden LF,van Rooij IA,Feitz WF,et al.Aetiology of hypospadias:a systematic review of genes and environment[J].Hum Reprod Update,2012,18(3):260-283.DOI:10.1093/humupd/dms002.
3 Springer A,Van vom den Heijkant M,Baumann S.Worldwide prevalence of hypospadias[J].J Pediatr Urol,2016,12(3):152.e1-152.e7.DOI:10.1016/j.jpurol.2015.12.002.
4张潍平.尿道下裂手术治疗的热点与难点问题[J].临床小儿外科杂志,2016,15(5):417-419.DOI:10.3969/j.issn.1671-6353.2016.05.001.Zhang WP.The hot and difficult points of surgical treatment in hypospadias[J].J Clin Ped Sur,2016,15(5):417-419.DOI:10.3969/j.issn.1671-6353.2016.05.001.
5 Wang N,Zhou Z,Liao X,et al.Role of microRNAs in cardiac hypertrophy and heart failure[J].IUBMB Life,2009,61(6):566-571.DOI:10.1002/iub.204.
6 O'Reilly S.MicroRNAs in fibrosis:opportunities and challenges[J].Arthritis Res Ther,2016,18(1):11.DOI:10.1186/s13075-016-0929-x.
7 Hu YB,Li CB,Song N,et al.Diagnostic value of microRNAfor Alzheimer's disease:a systematic review and meta-analysis[J].Front Aging Neuroscie,2016,8:13.DOI:10.3389/fnagi.2016.00013.
8 Hawkes JE,Nguyen GH,Fujita M,et al.microRNAs in Psoriasis[J].J Invest Dermatol,2016,136(2):365-371.DOI:10.1038/JID.2015.409.
9 Wu L,Zhou H,Zhang Q,et al.DNA methylation mediated by a microRNA pathway[J].Mol Cell,2010,38(3):465-475.DOI:10.1016/j.molcel.2010.03.008.
10 Hata A,Kashima R.Dysregulation of microRNA biogenesis machinery in cancer[J].Crit Rev Biochem Mol Biol,2016,51(3):121-134.DOI:10.3109/10409238.2015.1117054.
11 Sun T,Yang M,Kantoff PW,et al.Role of microRNA-221/-222 in cancer development and progression[J].Cell Cycle,2009,8(15):2315-2316.DOI:10.4161/cc.8.15.9221.
12 Netto JM,Ferrarez CE,Schindler,et al.Hormone therapy in hypospadias surgery:A systematic review[J].J Pediatr Urol,2013,9(6):971-979.DOI:10.1016/j.jpurol.2013.03.009.
13 Zhao W,Yin J,Yang Z,et al.Meta-analysis of androgen insensitivity in preoperative hormone therapy in hypospadias[J].Uroly,2015,85(5):1166.DOI:10.1016/j.urology.2015.01.035.
14 Han XB,Yang XF,Mi ZG.Human chorionic gonadotrophin regulates epidermal growth factor in the phallus of hypospadias mice[J].Zhonghua Nan Ke Xue,2008,14(10):884-887.
15李德彪.HCG调控兔先天性尿道下裂阴茎组织VEGF浓度的研究[D].山西:山西医科大学,2007.Li DB.Stimulation of VEGF in rabbit phallus with hypospadias:role of HCG[D].Shanxi:Shanxi Medical Unive-rsity,2007.
16 Kim D,Lee J,Johnson AL.Vascular endothelial growth factor and angiopoietins during Hen ovarian follicle development[J].Gen Comp Endocrinol,2016,232:25-31.DOI:10.1016/j.ygcen.2015.11.017.
17 Lei P,Xie J,Wang L,et al.microRNA-145 inhibits osteosarcoma cell proliferation and invasion by targeting ROCK1[J].Mol Med Rep,2014,10(1):155-160.DOI:10.3892/mmr.2014.2195.
18 Sakr M,Takino T,Sabit H,et al.miR-150-5p and miR-133a suppress glioma cell proliferation and migration through targeting membrane-type-1 matrix metalloproteinase[J].Gene,2016,587(2):155-162.DOI10.1016/j.gene.2016.04.058.
19徐丽平,李佳,沈雷,等.微小RNA-518b在乳腺浸润性导管癌中的表达及其功能[J].中华实验外科杂志,2012,29(5):833-836.DOI:10.3760/cma.j.issn.1001-9030.2012.05.019.Xu LP,Li J,Shen L,et al.Expression pattern and function of microRNA-518b in invasive ductal breast cancer[J].Chin J Exp Surg,2012,29(5):833-836.DOI:10.3760/cma.j.issn.1001-9030.2012.05.019.
20胡超,沈世强,崔忠惠.下调微小RNA-145表达对人肝癌血管内皮细胞生物学行为的影响[J].中华实验外科杂志,2014,31(10):2108-2111.DOI:10.3760/cma.j.issn.1001-9030.2014.10.008.Hu C,Shen SQ,Cui ZH.Effects of suppressing the expression of microRNA-1 on biological behaviors in the vascular endothelial cells of human hepatocellular carcinoma[J].Chin J Exp Surg,2014,31(10):2108-2111.DOI:10.3760/cma.j.issn.1001-9030.2014.10.008.
21范良,殷铁军,李岽健,等.微小RNA145对人脐静脉血管内皮细胞增殖迁移的影响[J].中华实验外科杂志,2013,30(2):262-264.DOI:10.3760/cma.j.issn.1001-9030.2013.02.021.Fan L,Yin TJ,Li DJ,et al.Effects of microRNA145 on proliferation and migration of human umbilical vein endothelial cells[J].Chin J Exp Surg,2013,30(2):262-264.DOI:10.3760/cma.j.issn.1001-9030.2013.02.021.
22 Albinsson S,Suarez Y,Skoum A,et al.MiRNAs are necessary for vascular smooth muscle growth,differentiation and function[J].Arterioscler Thromb Vasc Biol,2010,30(6):1118-1126.DOI:10.1161/ATVBAHA.109.200873.
23 Cordes KR,Sheehy NT,White MP,et al.miR-145 and miR-143 regulate smooth muscle cell fate and plasticity[J].Nature,2009,460(7256):705-710.DOI:10.1038/nature08195.
24 Mo ZH,Wu XD,Li S,et al.Expression and clinical significance of microRNA-376a in colorectal cancer[J].Asian Pac J Cancer Prev,2014,15(21):9523-9527.
25 Wen Z,Huang W,Feng Y,et al.MicroRNA-377 regulates mesenchymal stem cell-induced angiogenesis in ischemic hearts by targeting VEGF[J].PLo S One,2014,9(9):e104666.DOI:10.1371/journal.pone.0104666.
2 van der Zanden LF,van Rooij IA,Feitz WF,et al.Aetiology of hypospadias:a systematic review of genes and environment[J].Hum Reprod Update,2012,18(3):260-283.DOI:10.1093/humupd/dms002.
3 Springer A,Van vom den Heijkant M,Baumann S.Worldwide prevalence of hypospadias[J].J Pediatr Urol,2016,12(3):152.e1-152.e7.DOI:10.1016/j.jpurol.2015.12.002.
4张潍平.尿道下裂手术治疗的热点与难点问题[J].临床小儿外科杂志,2016,15(5):417-419.DOI:10.3969/j.issn.1671-6353.2016.05.001.Zhang WP.The hot and difficult points of surgical treatment in hypospadias[J].J Clin Ped Sur,2016,15(5):417-419.DOI:10.3969/j.issn.1671-6353.2016.05.001.
5 Wang N,Zhou Z,Liao X,et al.Role of microRNAs in cardiac hypertrophy and heart failure[J].IUBMB Life,2009,61(6):566-571.DOI:10.1002/iub.204.
6 O'Reilly S.MicroRNAs in fibrosis:opportunities and challenges[J].Arthritis Res Ther,2016,18(1):11.DOI:10.1186/s13075-016-0929-x.
7 Hu YB,Li CB,Song N,et al.Diagnostic value of microRNAfor Alzheimer's disease:a systematic review and meta-analysis[J].Front Aging Neuroscie,2016,8:13.DOI:10.3389/fnagi.2016.00013.
8 Hawkes JE,Nguyen GH,Fujita M,et al.microRNAs in Psoriasis[J].J Invest Dermatol,2016,136(2):365-371.DOI:10.1038/JID.2015.409.
9 Wu L,Zhou H,Zhang Q,et al.DNA methylation mediated by a microRNA pathway[J].Mol Cell,2010,38(3):465-475.DOI:10.1016/j.molcel.2010.03.008.
10 Hata A,Kashima R.Dysregulation of microRNA biogenesis machinery in cancer[J].Crit Rev Biochem Mol Biol,2016,51(3):121-134.DOI:10.3109/10409238.2015.1117054.
11 Sun T,Yang M,Kantoff PW,et al.Role of microRNA-221/-222 in cancer development and progression[J].Cell Cycle,2009,8(15):2315-2316.DOI:10.4161/cc.8.15.9221.
12 Netto JM,Ferrarez CE,Schindler,et al.Hormone therapy in hypospadias surgery:A systematic review[J].J Pediatr Urol,2013,9(6):971-979.DOI:10.1016/j.jpurol.2013.03.009.
13 Zhao W,Yin J,Yang Z,et al.Meta-analysis of androgen insensitivity in preoperative hormone therapy in hypospadias[J].Uroly,2015,85(5):1166.DOI:10.1016/j.urology.2015.01.035.
14 Han XB,Yang XF,Mi ZG.Human chorionic gonadotrophin regulates epidermal growth factor in the phallus of hypospadias mice[J].Zhonghua Nan Ke Xue,2008,14(10):884-887.
15李德彪.HCG调控兔先天性尿道下裂阴茎组织VEGF浓度的研究[D].山西:山西医科大学,2007.Li DB.Stimulation of VEGF in rabbit phallus with hypospadias:role of HCG[D].Shanxi:Shanxi Medical Unive-rsity,2007.
16 Kim D,Lee J,Johnson AL.Vascular endothelial growth factor and angiopoietins during Hen ovarian follicle development[J].Gen Comp Endocrinol,2016,232:25-31.DOI:10.1016/j.ygcen.2015.11.017.
17 Lei P,Xie J,Wang L,et al.microRNA-145 inhibits osteosarcoma cell proliferation and invasion by targeting ROCK1[J].Mol Med Rep,2014,10(1):155-160.DOI:10.3892/mmr.2014.2195.
18 Sakr M,Takino T,Sabit H,et al.miR-150-5p and miR-133a suppress glioma cell proliferation and migration through targeting membrane-type-1 matrix metalloproteinase[J].Gene,2016,587(2):155-162.DOI10.1016/j.gene.2016.04.058.
19徐丽平,李佳,沈雷,等.微小RNA-518b在乳腺浸润性导管癌中的表达及其功能[J].中华实验外科杂志,2012,29(5):833-836.DOI:10.3760/cma.j.issn.1001-9030.2012.05.019.Xu LP,Li J,Shen L,et al.Expression pattern and function of microRNA-518b in invasive ductal breast cancer[J].Chin J Exp Surg,2012,29(5):833-836.DOI:10.3760/cma.j.issn.1001-9030.2012.05.019.
20胡超,沈世强,崔忠惠.下调微小RNA-145表达对人肝癌血管内皮细胞生物学行为的影响[J].中华实验外科杂志,2014,31(10):2108-2111.DOI:10.3760/cma.j.issn.1001-9030.2014.10.008.Hu C,Shen SQ,Cui ZH.Effects of suppressing the expression of microRNA-1 on biological behaviors in the vascular endothelial cells of human hepatocellular carcinoma[J].Chin J Exp Surg,2014,31(10):2108-2111.DOI:10.3760/cma.j.issn.1001-9030.2014.10.008.
21范良,殷铁军,李岽健,等.微小RNA145对人脐静脉血管内皮细胞增殖迁移的影响[J].中华实验外科杂志,2013,30(2):262-264.DOI:10.3760/cma.j.issn.1001-9030.2013.02.021.Fan L,Yin TJ,Li DJ,et al.Effects of microRNA145 on proliferation and migration of human umbilical vein endothelial cells[J].Chin J Exp Surg,2013,30(2):262-264.DOI:10.3760/cma.j.issn.1001-9030.2013.02.021.
22 Albinsson S,Suarez Y,Skoum A,et al.MiRNAs are necessary for vascular smooth muscle growth,differentiation and function[J].Arterioscler Thromb Vasc Biol,2010,30(6):1118-1126.DOI:10.1161/ATVBAHA.109.200873.
23 Cordes KR,Sheehy NT,White MP,et al.miR-145 and miR-143 regulate smooth muscle cell fate and plasticity[J].Nature,2009,460(7256):705-710.DOI:10.1038/nature08195.
24 Mo ZH,Wu XD,Li S,et al.Expression and clinical significance of microRNA-376a in colorectal cancer[J].Asian Pac J Cancer Prev,2014,15(21):9523-9527.
25 Wen Z,Huang W,Feng Y,et al.MicroRNA-377 regulates mesenchymal stem cell-induced angiogenesis in ischemic hearts by targeting VEGF[J].PLo S One,2014,9(9):e104666.DOI:10.1371/journal.pone.0104666.