多囊卵巢综合征患者血清外泌体中差异表达microRNAs及其功能分析
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摘要
目的分析多囊卵巢综合征(PCOS)患者血清外泌体中差异表达microRNAs(miRNAs),并进一步分析血清外泌体中差异表达miRNAs的功能。方法 PCOS患者30例为实验组,因单纯输卵管因素不孕患者30例为对照组。分别于月经周期第2～4天抽取两组外周血,提取、纯化血清外泌体,高通量测序定性、定量检测外泌体miRNAs,分析两组差异表达(差异表达倍数≥2且P <0. 01)血清外泌体miRNAs。生物信息学分析差异表达外泌体miRNAs的功能。取两组外周血,qRT-PCR法定量检测两组差异表达外泌体miRNAs。结果 PCOS患者血清外泌体中差异表达miRNAs共有243条,其中hsa-miR-378d、hsa-miR-378b、hsa-miR-4772-3p等表达上调115条、hsa-miR-5090、hsa-miR-3680-3p、hsa-miR-6836-5p等表达下调128条。PCOS患者差异表达血清外泌体miRNAs与类固醇激素的合成有关,可能参与调节胰岛素信号受体通路。与对照组比较,实验血清外泌体miR-146a-5p、miR-25-5p、miR-27a-3p、miR-93-5p、miR-23a-3p、miR-223-5p、miR-25-3p、miR-483-5p、miR-383-5p相对表达量升高(P均<0. 05)。结论PCOS患者血清外泌体hsa-miR-378d、hsa-miR-378b、hsa-miR-4772-3p等表达上调,hsa-miR-5090、hsa-miR-3680-3p、hsa-miR-6836-5p等表达下调。PCOS患者差异表达血清外泌体miRNAs与类固醇激素的合成有关,可参与调节胰岛素信号受体通路。
Objective To analyze the differentially expressed microRNAs( miRNAs) in serum exosomes of patients with polycystic ovarian syndrome( PCOS) and to identify their functions. Methods We selected 30 women with PCOS( group A) and 30 infertile women with tube factors( group B). The peripheral blood was collected on day 2-4 of the menstrual cycle. Then the serum exosomes were extracted and purified. The differentially expressed exosome miRNAs in the two groups( differential expression fold ≥ 2 and P < 0. 01) were analyzed by the high-throughput sequencing. Bioinformatics analysis predicted the functions of those differential miRNAs which were further validated by quantitative real-time PCR analysis( qRT-PCR). Results There were 243 differentially expressed miRNAs,115( hsa-miR-378 d,hsa-miR-378 b,hsa-miR-4772-3 p,et al) of which were up-regulated and 128( hsa-miR-5090 and hsa-miR-3680-3 p,hsa-miR-6836-5 p. et al) were down-regulated in the group A. These miRNAs were associated with the synthesis of steroid hormones,which were involved in the regulation of the insulin receptor signaling pathway. Compared with the group B,the relative expression levels of miR-146 a-5 p,miR-25-5 p,miR-27 a-3 p,miR-93-5 p,miR-23 a-3 p,miR-223-5 p,miR-25-3 p,miR-483-5 p,and miR-383-5 p increased in the group A( all P < 0. 05). Conclusions The expression levels of serum exosome miRNAs such as miR-378 d,miR-378 b,and miR-4772-3 p are up-regulated,and hsa-miR-5090,hsa-miR-3680-3 p,and hsa-miR-6836-5 p are down-regulated in PCOS patients. Differentially expressed exosome miRNAs are associated with the synthesis of steroid hormones and may be involved in the regulation of insulin signaling pathway.
Objective To analyze the differentially expressed microRNAs( miRNAs) in serum exosomes of patients with polycystic ovarian syndrome( PCOS) and to identify their functions. Methods We selected 30 women with PCOS( group A) and 30 infertile women with tube factors( group B). The peripheral blood was collected on day 2-4 of the menstrual cycle. Then the serum exosomes were extracted and purified. The differentially expressed exosome miRNAs in the two groups( differential expression fold ≥ 2 and P < 0. 01) were analyzed by the high-throughput sequencing. Bioinformatics analysis predicted the functions of those differential miRNAs which were further validated by quantitative real-time PCR analysis( qRT-PCR). Results There were 243 differentially expressed miRNAs,115( hsa-miR-378 d,hsa-miR-378 b,hsa-miR-4772-3 p,et al) of which were up-regulated and 128( hsa-miR-5090 and hsa-miR-3680-3 p,hsa-miR-6836-5 p. et al) were down-regulated in the group A. These miRNAs were associated with the synthesis of steroid hormones,which were involved in the regulation of the insulin receptor signaling pathway. Compared with the group B,the relative expression levels of miR-146 a-5 p,miR-25-5 p,miR-27 a-3 p,miR-93-5 p,miR-23 a-3 p,miR-223-5 p,miR-25-3 p,miR-483-5 p,and miR-383-5 p increased in the group A( all P < 0. 05). Conclusions The expression levels of serum exosome miRNAs such as miR-378 d,miR-378 b,and miR-4772-3 p are up-regulated,and hsa-miR-5090,hsa-miR-3680-3 p,and hsa-miR-6836-5 p are down-regulated in PCOS patients. Differentially expressed exosome miRNAs are associated with the synthesis of steroid hormones and may be involved in the regulation of insulin signaling pathway.
引文
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[11]Wang M,Sun J,Xu B,et al.Functional characterization of microrna-27a-3p expression in human polycystic ovary syndrome[J].Endocrinology,2018,159(1):297-309.
[12]Seger R,Hanoch T,Rosenberg R,et al.The ERK signaling cascade inhibits gonadotropin-stimulated steroidogenesis[J].J Biol Chem,2017,292(21):8847.
[13]Arroyo J,Price M,Straszewski-Chavez S,et al.XIAP protein is induced by placenta growth factor(PLGF)and decreased during preeclampsia in trophoblast cells[J].Syst Biol Reprod Med,2014,60(5):263-273.
[14]Li L,Hou A,Gao X,et al.Lentivirus-mediated miR-23a overexpression induces trophoblast cell apoptosis through inhibiting X-linked inhibitor of apoptosis[J].Biomed Pharm,2017,94:412-417.
[15]Nie M,Yu S,Peng S,et al.miR-23a and miR-27a promote human granulosa cell apoptosis by targeting SMAD5[J].Biol Reprod,2015,93(4):98.
[16]Zhang X,Wang X,Wu J,et al.The diagnostic values of circulating miRNAs for hypertension and bioinformatics analysis[J].Biosci Rep,2018,38(4):20180525.
[2]Teede H,Deeks A,Moran L.Polycystic ovary syndrome:a complex condition with psychological,reproductive and metabolic manifestations that impacts on health across the lifespan[J].BMCMed,2010,8:41.
[3]Zhou W,Woodson M,Ne上调ane B,et al.Exosomes serve as novel modes of tick-borne flavivirus transmission from arthropod to human cells and facilitates dissemination of viral RNA and proteins to the vertebrate neuronal cells[J].PLo S Pathog,2018,14(1):e1006764.
[4]Alvarez ML,Khosroheidari M,Kanchi Ravi R,et al.Comparison of protein,microRNA,and mRNA yields using different methods of urinary exosome isolation for the discovery of kidney disease biomarkers[J].Kidney Int,2012,82(9):1024-1032.
[5]Kanada M,Bachmann MH,Hardy JW,et al.Differential fates of biomolecules delivered to target cells via extracellular vesicles[J].Proc Natl Acad Sci U S A,2015,112(12):E1433-1442.
[6]Xu B,Zhang YW,Tong XH,et al.Characterization of microRNAprofile in human cumulus granulosa cells:Identification of microR-NAs that regulate Notch signaling and are associated with PCOS[J].Mol Cell Endocrinol,2015,404:26-36.
[7]Abak A,Abhari A,Rahimzadeh S.Exosomes in cancer:small vesicular transporters for cancer progression and metastasis,biomarkers in cancer therapeutics[J].Peer J,2018,6:e4763.
[8]Yoshikawa M,Iinuma H,Umemoto Y,et al.Exosome-encapsulated microRNA-223-3p as a minimally invasive biomarker for the early detection of invasive breast cancer[J].Oncol Lett,2018,15(6):9584-9592.
[9]Raposo G,Stoorvogel W.Extracellular vesicles:exosomes,microvesicles,and friends[J].J Cell Biol,2013,200(4):373-383.
[10]Wu D,Xi QY,Cheng X,et al.miR-146a-5p inhibits TNF-alphainduced adipogenesis via targeting insulin receptor in primary porcine adipocytes[J].J Lipid Res,2016,57(8):1360-1372.
[11]Wang M,Sun J,Xu B,et al.Functional characterization of microrna-27a-3p expression in human polycystic ovary syndrome[J].Endocrinology,2018,159(1):297-309.
[12]Seger R,Hanoch T,Rosenberg R,et al.The ERK signaling cascade inhibits gonadotropin-stimulated steroidogenesis[J].J Biol Chem,2017,292(21):8847.
[13]Arroyo J,Price M,Straszewski-Chavez S,et al.XIAP protein is induced by placenta growth factor(PLGF)and decreased during preeclampsia in trophoblast cells[J].Syst Biol Reprod Med,2014,60(5):263-273.
[14]Li L,Hou A,Gao X,et al.Lentivirus-mediated miR-23a overexpression induces trophoblast cell apoptosis through inhibiting X-linked inhibitor of apoptosis[J].Biomed Pharm,2017,94:412-417.
[15]Nie M,Yu S,Peng S,et al.miR-23a and miR-27a promote human granulosa cell apoptosis by targeting SMAD5[J].Biol Reprod,2015,93(4):98.
[16]Zhang X,Wang X,Wu J,et al.The diagnostic values of circulating miRNAs for hypertension and bioinformatics analysis[J].Biosci Rep,2018,38(4):20180525.