miR-375通过靶基因Sp1调控大鼠肾上腺髓质儿茶酚胺的合成和分泌
发布时间：2018-08-12 14:34
【摘要】：肾上腺是动物机体非常重要的内分泌器官,其髓质合成和分泌的儿茶酚胺类激素(Catecholamines, CATs)在机体维持内稳态平衡过程中起着关键的作用。已有的研究证明,包括miR-375在内的miRNAs在调节众多内分泌激素(如胰岛素、雌激素)的合成和分泌过程中起着非常关键的作用,但到目前为止,miRNAs对于CATs合成和分泌的研究较少。作者实验室前期研究发现miR-375高表达于肾上腺,但其在肾上腺中的相关生物学功能尚不明确。因此,本研究的主要目标是研究miR-375在肾上腺中的功能及相关机理。首先,利用实时定量PCR (real-time quantitative PCR,qPCR)检测到miR-375高表达于大鼠的肾上腺组织,并且在大鼠胚胎肾上腺的发育过程中,miR-375表达呈现逐渐升高趋势,在成年时达到最高;原位杂交(in situ hybridization, ISH)方法证明了 miR-375特异性表达于肾上腺髓质嗜铬细胞,皮质细胞中未检测到阳性信号。为了进一步研究miR-375在大鼠肾上腺髓质细胞中的功能,我们采用大鼠束缚应急应激模型(immobilization stress, IMO),检测miR-375在应急应激反应中的表达水平及其对CATs的影响。结果发现在不同的应急应激过程中,肾上腺中miR-375的表达下调,而大鼠血浆中CATs的水平上升。随后我们利用人T.合成的miR-375抑制剂(miR-375-in)和模拟物(miR-375-mi)分别抑制和超表达大鼠原代髓质细胞和肾上腺髓质瘤细胞系PC12细胞中的miR-375,结果证明miR-375-in能促进细胞CATs的分泌;而miR-375-mi可以显著抑制细胞分泌CATs。另外,miR-375-in可显著上调髓质细胞中酪氨酸羟化酶(tyrosine hydroxylase, TH)和多巴胺-β-羟化酶(dopamine-β-hydroxylase, DBH)的表达;而miR-375-mi明显下调细胞中TH和DBH的表达。同时,本研究的结果证明miR-375不参与CATs的降解及去甲肾上腺素(norepinephrine,NE)对CATs的负反馈过程。此外,采用BrdU细胞免疫化学、TUNEL检测及qPCR的方法均发现miR-375不影响PC12细胞的增殖和凋亡。以上实验结果说明了 miR-375在肾上腺CATs合成和分泌过程中起着非常重要的调节作用。另外,通过生物信息学方法预测miR-375的靶基因为特异性蛋白 1 (specificity protein 1,Sp1),并通过双荧光素酶报告系统和蛋白免疫印迹等方法验证了 Sp1是miR-375在大鼠肾上腺中的直接靶基因。在PC12细胞中,通过RNAi干扰实验和Sp1过表达实验证明了 Sp1参与了 miR-375对TH和DBH调节的信号通路。在IMO应急应激反应中,Sp1蛋内表达显著上调与miR-375的表达下调呈负相关,再次验证了 Sp1为miR-375的靶基因并参与了大鼠的应急应激反应过程。综上所述,本论文证明了 miR-375在大鼠的肾上腺发育过程中持续表达,并且特异地高表达于髓质嗜铬细胞。miR-375参与大鼠的IMO应急应激反应。miR-375通过直接作用于靶基因Sp1调控TH和DBH的表达,进而影响大鼠CATs的合成和分泌。上述研究为了解miRNAs在CATs合成和分泌调控机制提供了新的依据。
[Abstract]:Adrenal gland is an important endocrine organ in animal. The synthesis and secretion of catecholamine hormone (Catecholamines, CATs) in medulla play a key role in maintaining homeostasis. It has been proved that miRNAs including miR-375 plays a key role in regulating the synthesis and secretion of many endocrine hormones (such as insulin estrogen). However so far little has been done on the synthesis and secretion of CATs by miRNAs. We found that miR-375 is highly expressed in adrenal gland, but its biological function in adrenal gland is unclear. Therefore, the main objective of this study is to study the function and mechanism of miR-375 in adrenal gland. Firstly, the high expression of miR-375 was detected in the adrenal tissue of rats by real-time quantitative PCR (real-time quantitative PCR), and the expression of mmiR-375 increased gradually during the development of embryonic adrenal gland, and reached the highest level in adulthood. In situ hybridization (in situ hybridization, ISH) showed that miR-375 was specifically expressed in chromaffin cells of adrenal medulla, but no positive signal was detected in cortical cells. In order to further study the function of miR-375 in adrenal medullary cells of rats, the expression of miR-375 in emergency stress response and its effect on CATs were detected by rat restraint emergency stress model (immobilization stress, IMO),). The results showed that the expression of miR-375 was down-regulated in adrenal gland and the level of CATs in plasma was increased during different emergency stress. Then we use the human T. The synthesis of miR-375 inhibitor (miR-375-in) and analogue (miR-375-mi) inhibited and overexpressed miR-375in primary rat medullary cells and adrenal medulloma cell line PC12, respectively. The results showed that miR-375-in could promote the secretion of CATs, while miR-375-mi could significantly inhibit the secretion of CATs. In addition, miR-375-in significantly up-regulated the expression of tyrosine hydroxylase (tyrosine hydroxylase, TH) and dopamine 尾 -hydroxylase (DBH) in medullary cells, while miR-375-mi significantly down-regulated the expression of th and DBH in medullary cells. At the same time, the results showed that miR-375 did not participate in the degradation of CATs and the negative feedback of norepinephrine (NE) on CATs. In addition, miR-375 did not affect the proliferation and apoptosis of PC12 cells by BrdU cell immunocytochemistry and qPCR assay. These results suggest that miR-375 plays an important role in the regulation of adrenal CATs synthesis and secretion. In addition, the target gene of miR-375 was predicted by bioinformatics because of its specific protein 1 (specificity protein 1 Sp1, and the direct target gene of miR-375 in rat adrenal gland was confirmed by double luciferase report system and Western blot. In PC12 cells, RNAi interference experiments and Sp1 overexpression experiments demonstrated that Sp1 participated in the signal pathway regulated by miR-375 on th and DBH. There was a negative correlation between the up-regulation of SP1 expression and the down-regulation of miR-375 expression in the IMO emergency stress response, which confirmed that Sp1 was the target gene of miR-375 and participated in the process of emergency stress response in rats. To sum up, this paper proves that miR-375 is expressed continuously during the development of adrenal gland in rats. MiR-375 was specifically overexpressed in medullary chromaffin cells. MiR-375 participated in the IMO emergency stress response of rats. MiR-375 regulated the expression of th and DBH by directly acting on the target gene Sp1, and then affected the synthesis and secretion of CATs in rats. These studies provide a new basis for understanding the regulatory mechanism of miRNAs in the synthesis and secretion of CATs.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q454
[Abstract]:Adrenal gland is an important endocrine organ in animal. The synthesis and secretion of catecholamine hormone (Catecholamines, CATs) in medulla play a key role in maintaining homeostasis. It has been proved that miRNAs including miR-375 plays a key role in regulating the synthesis and secretion of many endocrine hormones (such as insulin estrogen). However so far little has been done on the synthesis and secretion of CATs by miRNAs. We found that miR-375 is highly expressed in adrenal gland, but its biological function in adrenal gland is unclear. Therefore, the main objective of this study is to study the function and mechanism of miR-375 in adrenal gland. Firstly, the high expression of miR-375 was detected in the adrenal tissue of rats by real-time quantitative PCR (real-time quantitative PCR), and the expression of mmiR-375 increased gradually during the development of embryonic adrenal gland, and reached the highest level in adulthood. In situ hybridization (in situ hybridization, ISH) showed that miR-375 was specifically expressed in chromaffin cells of adrenal medulla, but no positive signal was detected in cortical cells. In order to further study the function of miR-375 in adrenal medullary cells of rats, the expression of miR-375 in emergency stress response and its effect on CATs were detected by rat restraint emergency stress model (immobilization stress, IMO),). The results showed that the expression of miR-375 was down-regulated in adrenal gland and the level of CATs in plasma was increased during different emergency stress. Then we use the human T. The synthesis of miR-375 inhibitor (miR-375-in) and analogue (miR-375-mi) inhibited and overexpressed miR-375in primary rat medullary cells and adrenal medulloma cell line PC12, respectively. The results showed that miR-375-in could promote the secretion of CATs, while miR-375-mi could significantly inhibit the secretion of CATs. In addition, miR-375-in significantly up-regulated the expression of tyrosine hydroxylase (tyrosine hydroxylase, TH) and dopamine 尾 -hydroxylase (DBH) in medullary cells, while miR-375-mi significantly down-regulated the expression of th and DBH in medullary cells. At the same time, the results showed that miR-375 did not participate in the degradation of CATs and the negative feedback of norepinephrine (NE) on CATs. In addition, miR-375 did not affect the proliferation and apoptosis of PC12 cells by BrdU cell immunocytochemistry and qPCR assay. These results suggest that miR-375 plays an important role in the regulation of adrenal CATs synthesis and secretion. In addition, the target gene of miR-375 was predicted by bioinformatics because of its specific protein 1 (specificity protein 1 Sp1, and the direct target gene of miR-375 in rat adrenal gland was confirmed by double luciferase report system and Western blot. In PC12 cells, RNAi interference experiments and Sp1 overexpression experiments demonstrated that Sp1 participated in the signal pathway regulated by miR-375 on th and DBH. There was a negative correlation between the up-regulation of SP1 expression and the down-regulation of miR-375 expression in the IMO emergency stress response, which confirmed that Sp1 was the target gene of miR-375 and participated in the process of emergency stress response in rats. To sum up, this paper proves that miR-375 is expressed continuously during the development of adrenal gland in rats. MiR-375 was specifically overexpressed in medullary chromaffin cells. MiR-375 participated in the IMO emergency stress response of rats. MiR-375 regulated the expression of th and DBH by directly acting on the target gene Sp1, and then affected the synthesis and secretion of CATs in rats. These studies provide a new basis for understanding the regulatory mechanism of miRNAs in the synthesis and secretion of CATs.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q454
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