湖羊羔皮毛囊候选miRNA在不同花纹间的表达与毛囊发育特性关联的研究

doi:10.3864/j.issn.0578-1752.2018.14.017

中国农业科学 ›› 2018, Vol. 51 ›› Issue (14): 2814-2824.doi: 10.3864/j.issn.0578-1752.2018.14.017

• 畜牧·兽医·资源昆虫 • 上一篇下一篇

湖羊羔皮毛囊候选miRNA在不同花纹间的表达与毛囊发育特性关联的研究

金澄艳¹，吕晓阳¹，高雯¹，王悦¹，陈炜昊¹，盛水兴²，陈玲²，林杰³，孙伟¹

¹扬州大学动物科学与技术学院，江苏扬州225009；²苏州市畜牧兽医站，江苏苏州215200；³丹阳市皇塘镇动物防疫站，江苏丹阳213027

收稿日期:2017-12-13 出版日期:2018-07-16 发布日期:2018-07-16
通讯作者: 孙伟，E-mail： dkxmsunwei@163.com
作者简介:金澄艳，E-mail：1342315339@qq.com。吕晓阳，E-mail：420333142@qq.com。金澄艳与吕晓阳为同等贡献作者。
基金资助:
科技部家养动物平台项目、江苏省高校自然科学研究重大项目（17KJA230001）、江苏高校优势学科建设工程资助项目、江苏省苏北科技计划（BN2014003，BN2015013，BN2015014）、江苏省农业科技自主创新项目（CX（14）2073）和江苏省六大高峰人才项目、扬州大学研究生创新工程项目（XKYCX17_060）

Study on the Relationship Between the Expression of Candidate miRNAs and the Developmental Characteristics in Different Patterns in Hu Sheep Lambskin

JIN ChengYan¹, LÜ XiaoYang¹, GAO Wen¹, WANG Yue¹, CHEN WeiHao¹, SHENG ShuiXing², CHEN Ling², LIN Jie³, SUN Wei¹

¹College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, Jiangsu；² Suzhou Animal Husbandry and Veterinary Station，Suzhou 215200, Jiangsu；³ Danyang Huangtang Town Animal Epidemic Prevention Station，Danyang 213027, Jiangsu

Received:2017-12-13 Online:2018-07-16 Published:2018-07-16

摘要/Abstract

摘要： 【目的】通过前期高通量测序技术结合生物信息学分析研究湖羊大花、中花和小花羔皮毛囊间miRNA的差异表达，筛选出了14个候选miRNA，现进一步研究14个候选miRNA在不同花纹组间的表达量与毛囊各指标的相关性，以了解候选miRNA与毛囊发育特性间的关联，筛选用于后期功能验证的miRNA。【方法】利用高通量测序技术筛选湖羊大花、中花、小花不同花纹羔皮毛囊中的差异表达miRNA。利用与高通量测序同一批样品所提取的RNA进行RT-RCR验证，随机选取高通量测序结果中的5个差异表达miRNA，验证高通量测序结果的可靠性。通过荧光定量PCR技术检测14个候选miRNA在不同组别间的表达量，制作不同花纹羔皮组织的石蜡切片以评估不同毛囊的结构，并结合组织学观察与显微观测技术，采用SPSS 17.0分析14个候选miRNA的表达水平与毛囊组织学性质的相关性。【结果】湖羊毛囊成群分布，以3毛囊群的居多，直径较大的为中心初级毛囊，直径较小的为侧部次级毛囊。在显微镜相同视野中，湖羊大花、中花和小花3种不同类型花纹羔皮的初级毛囊数差异不显著（P＞0.05），小花组的次级毛囊数多于大花组和中花组的次级毛囊数且差异极显著（P＜0.01），中花组与小花组二者次级毛囊数相仿，差异不显著（P＞0.05），在相同的单位面积中，小花的毛囊总数要高于大花组和中花组；大花组的初级毛囊直径比中花组和小花组的初级毛囊直径要大的多，与中花组和小花组的初级毛囊直径差异极显著（P＜0.01），大花组的次级毛囊直径也比中花组、小花组的次级毛囊直径大，同时也与二者均呈极显著差异（P＜0.01），中花组和小花组二者的初级毛囊直径以及次级毛囊直径均差异不显著（P＞0.05）；在已知的miRNA中，miRNA-143在大花组中的表达量与小花组差异极显著（P＜0.01），miRNA-10a在小花组的表达量与大花和中花组差异显著（P＜0.05），let-7i在大花组的表达量与中花组差异显著（P＜0.05）；在测序新预测的差异miRNA中，NW_004080184.1_6326在中花组的表达量与大花组差异显著（P＜0.05）、与小花组差异极显著（P＜0.01），NW_004080165.1_8572在中花组的表达量与大花组和小花组差异极显著（P＜0.01），NW_004080181.1_3961在中花组的表达量与小花组差异极显著（P＜0.01），NW_004080190.1_13733在中花组的表达量与大花组差异极显著（P＜0.01），其余miRNA在大、中、小花中的表达差异不显著。14个miRNA的相对表达量均与毛囊部分指标存在相关性，表明I4个miRNA均有可能参与毛囊的生长发育。【结论】 miRNA-143、miRNA-10a、let-7i、NW_004080184.1_6326、NW_004080165.1_8572、NW_004080181.1_3961以及NW_004080190.1_13733 等7个miRNA可作为对湖羊羔皮毛囊发育具有重要影响的候选miRNA，在后续试验中将进一步验证。

关键词: 湖羊羔皮, 毛囊, miRNA, 花纹, 毛囊发育

Abstract: 【Objective】 In the present study, high-throughput sequencing and bioinformatics analysis were initially employed to identify differentially expressed microRNAs (miRNAs) in hair follicles of different flower patterns with small, medium, and large waves of Hu sheep. Then 14 candidate miRNAs were screened. The purpose of this study was to study the amount of 14 nominated miRNA expression in different pattern groups and to find the correlation of different hair follicle index, and to further explore the relevance between candidate miRNAs and developmental characteristics of hair follicles and screen miRNAs for later functional validation.【Method】Firstly, utilizing high-throughput sequencing filtrated differentially expressed miRNAs in hair follicles of different flower patterns with small, medium, and large waves of Hu sheep. To confirm the reliability of sequencing results, five miRNAs were selected for RT-PCR verification. Paraffin sections of lambskin tissue were prepared to assess the structure of different hair follicles. Expression levels of 14 candidate miRNAs in different groups were analyzed by relative quantitation using fluorescent quantitative PCR. Combined with histological observation and microscopic observation technique, the correlation between expression levels of 14 candidate miRNAs and histological properties of hair follicles was analyzed by using SPSS 17.0.【Result】The distribution of hair follicles was mainly in the form of hair follicle groups, which distributed as a group of 3. The diameter of the primary follicles was larger than that of secondary follicles regardless of large, medium, or small waves. In the same field of view, no significant differences in primary follicles among the large, medium and small wave lambskins were observed (P＞0.05); the number of secondary follicles in the small waves was significantly higher than that in large and medium waves (P＜0.01), whereas the number of secondary follicles in the medium wave was similar to that in the small waves (P＞0.05). Consequently, the total number of hair follicles in the small waves was higher than that in the large and medium waves in the same unit area. The diameter of the primary and secondary follicles in the large waves were significantly higher than that in medium and small waves (P＜0.01) and the diameter of primary and secondary hair follicles were not significantly different between the medium and small waves groups (P＞0.05). The level of expression of miRNA-143 in the large waves was significantly lower than that in the large waves (P＜0.01). The expression level of miRNA-10a in the small waves was significantly higher than that in the large and medium waves (P＜0.05) and that of let-7i was significantly higher in the medium waves compared with that in the large waves (P＜0.05). In the differentially expressed novel miRNAs, the expression level of NW_004080184.1_6326 in medium waves was significantly lower than that in the large and small waves, respectively (P＜0.05; P＜0.01). The expression of NW_004080165.1_8572 in the medium waves was significantly different compared with that in the large and small waves (P＜0.01). NW_004080181.1_3961 was expressed at a higher level in small waves than in medium waves (P＜0.01). Highly significant differences in NW_004080190.1_13733 expression was observed between medium and large waves (P＜0.01). No significant differences in the expression levels of the rest of the miRNAs were observed among the large, medium, and small waves. The relative expression levels of 14 miRNAs were correlated with some indicators of hair follicles, indicating that all 14 miRNAs might be involved in the growth and development of hair follicles.【Conclusion】Overall, the present study showed that miRNA-143, miRNA-10a, let-7i, NW_004080184.1_6326, NW_004080165.1_8572, NW_004080181.1_3961, and NW_004080190.1_13733 could be considered as important candidate genes, indicating these seven miRNAs might play significant roles in hair follicle growth and development in Hu sheep lambskin, which would be confirmed in subsequent experiments.

Key words: Hu sheep lambskin, hair follicles, miRNA, floral pattern, hair follicle development

金澄艳，吕晓阳，高雯，王悦，陈炜昊，盛水兴，陈玲，林杰，孙伟. 湖羊羔皮毛囊候选miRNA在不同花纹间的表达与毛囊发育特性关联的研究[J]. 中国农业科学, 2018, 51(14): 2814-2824.

JIN ChengYan, Lü XiaoYang, GAO Wen, WANG Yue, CHEN WeiHao, SHENG ShuiXing, CHEN Ling, LIN Jie, SUN Wei. Study on the Relationship Between the Expression of Candidate miRNAs and the Developmental Characteristics in Different Patterns in Hu Sheep Lambskin[J]. Scientia Agricultura Sinica, 2018, 51(14): 2814-2824.

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湖羊羔皮毛囊候选miRNA在不同花纹间的表达与毛囊发育特性关联的研究

Study on the Relationship Between the Expression of Candidate miRNAs and the Developmental Characteristics in Different Patterns in Hu Sheep Lambskin

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