奶山羊卵巢颗粒细胞中TIMP3基因的表达调控及功能研究
发布时间：2018-08-22 10:01
【摘要】：基质金属蛋白酶(MMPs)在卵泡发生、发育、排卵和黄体形成等生物过程中发挥着重要的功能。TIMP3作为MMPs的抑制剂可能参与卵泡周期中各生命活动间的相互协调,而关于TIMP3在奶山羊卵泡中是否表达,表达规律如何,表达调控信号通路及其表达产物调节卵泡激素合成和颗粒细胞凋亡的分子机制等方面的研究,尚未见报道。本研究以关中奶山羊卵泡及其颗粒细胞为研究对象,在克隆和分析TIMP3基因cDNA序列的基础上,分析TIMP3在卵泡周期中的表达规律,探究LH/hCG和mi RNA对TIMP3基因表达、TIMP3基因表达产物对卵泡激素合成、胞外基质重构和颗粒细胞凋亡等生物过程相关基因表达的分子调控机制,以及TIMP3基因表达产物对颗粒细胞凋亡的影响,阐明TIMP3基因在卵泡周期中的生物功能,主要获得以下研究结果:1.TIMP3基因cDNA的克隆及其序列的生物信息学分析采用RACE技术从奶山羊卵巢中获得TIMP3基因的cDNA,通过测序和序列分析发现:奶山羊TIMP3基因cDNA序列全长2208 bp,其中5'非翻译区(5'UTR)长348 bp,CDS区长636 bp(编码211氨基酸的蛋白),3'UTR长1224 bp;预测的氨基酸序列与绵羊、牛、猪、人、小鼠和大鼠的序列的相似度分别为100%、99%、100%、99%、97%和96%;蛋白质结构预测发现其蛋白质由一个N端结构域和一个C端结构域构成,其中每个结构域中包含6个保守的半胱氨酸残基。2.TIMP3基因在奶山羊卵泡周期中的表达规律分析通过Real-time PCR分析发现,TIMP3基因在奶山羊各组织中的表达存在差异,其中在输卵管中的相对表达量最高;在卵泡中,TIMP3基因mRNA的丰度随着卵泡的发育成熟逐渐升高,且在黄体中达到最高;比较分析发现,TIMP3在多羔奶山羊(连续3胎每胎产羔3-4只)卵巢组织中的表达量显著高于单羔奶山羊(连续3胎每胎产羔1-2只),显示TIMP3基因与奶山羊产羔数可能密切相关。3.LH/hCG对TIMP3基因表达的分子调控机制分析采用hCG对培养的颗粒细胞进行处理,Real-time PCR分析发现,TIMP3基因mRNA的丰度分别在处理后4 h和24 h出现峰值;Western Blot分析发现,TIMP3蛋白丰度随着处理时间延长而增加,24 h时达到最大;采用抑制剂进行阻断分析,发现hCG诱导的TIMP3的增加受PKA,PKC,MAPK和PI3K信号通路的影响。进一步对TIMP3基因转录调控区进行分析,发现TIMP3基因上游-1至-122bp的区域内,包含3个Sp1结合位点,定点突变分析,发现-67~-58和-74~-65的突变明显降低TIMP3的转录;采用cAMP激活剂FSK处理颗粒细胞,促进Sp1表达,发现TIMP3转录增强,该结果与EMSA,ChIP以及Sp1过表达和沉默分析相一致,由此确定cAMP通过调节转录因子Sp1来调节TIMP3表达。4.miRNA对TIMP3基因表达的分子调控机制分析依据生物信息学分析的结果,构建荧光素酶报告载体并与miRNAs共转进颗粒细胞中,发现miR-21,miR-221,miR-222和miR-181b均显著的抑制了荧光素酶的活性;采用Real-time PCR分析发现,与NC组相比miR-21和miR-181b显著减少了颗粒细胞中TIMP3基因mRNA的丰度,而miR-221和miR-222组差异不显著;Western Blot分析发现,与NC组相比miR-21,miR-181b,mi R-221和miR-222都显著减少了TIMP3蛋白的丰度,表明miR-21,miR-221,miR-222和mi R-181b与TIMP3基因3'UTR区特异位点结合,调控TIMP3基因的表达,其中miR-21和miR-181b通过促进mRNA降解调节TIMP3基因表达,而miR-221和mi R-222通过抑制翻译调节TIMP3的表达,且miR-21,miR-221,miR-222和miR-181b可促进颗粒细胞的活力。5.TIMP3基因表达产物对颗粒细胞凋亡和激素合成的分子调控机制分析采用腺病毒介导TIMP3基因过表达和siRNA沉默TIMP3基因表达,发现TIMP3基因过表达可抑制颗粒细胞的活力,促进细胞凋亡;TIMP3下调可抑制hCG诱导类固醇激素合成酶StAR,p450scc,HSD3B基因的表达,降低孕酮水平;同时TIMP3抑制ADAM17基因表达,促进ECM蛋白相关基因FN和DCN基因的表达。以上研究表明,LH/hCG与颗粒细胞上的受体LHR结合,激活cAMP信号通路,促进转录因子Sp1表达,Sp1与TIMP3基因Sp1应答元件结合,促进TIMP3基因表达,TIMP3蛋白通过促进StAR,p450scc,HSD3B基因的表达调节孕酮的合成,促进FN和DCN基因表达调节ECM的重构,抑制ADAM17基因表达,抑制颗粒细胞的活力,促进颗粒细胞凋亡参与卵泡周期的调控。这些研究结果,为进一步阐明TIMP3参与调控卵泡周期的分子机理,揭示卵泡发育成熟和排卵的分子调控机制提供理论和试验依据。
[Abstract]:Matrix metalloproteinases (MMPs) play an important role in folliculogenesis, development, ovulation and luteal formation. As an inhibitor of MMPs, TIMP3 may participate in the coordination of various life activities during the follicular cycle. However, whether TIMP3 is expressed in the follicles of dairy goats, how it is expressed, the signaling pathway and its regulation are also involved. The expression products regulate the synthesis of follicular hormones and the molecular mechanism of apoptosis of granulosa cells. In this study, the follicles and granulosa cells of Guanzhong Dairy Goat were selected as the research object. On the basis of cloning and analyzing the cDNA sequence of TIMP3 gene, the expression of TIMP3 in follicular cycle was analyzed, and the effects of LH/hCG and MIRNA on TIMP were explored. 3 gene expression, TIMP3 gene expression products on follicular hormone synthesis, extracellular matrix remodeling and granulosa cell apoptosis and other biological processes related gene expression of molecular regulatory mechanisms, as well as TIMP3 gene expression products on granulosa cell apoptosis, to clarify the biological function of TIMP3 gene in the follicular cycle, the main results are as follows: 1. The cloning and bioinformatics analysis of the MP3 gene cDNA were carried out by RACE. The TIMP3 gene cDNA was obtained from the ovary of dairy goats. By sequencing and sequence analysis, it was found that the total length of the TIMP3 gene was 2208 bp, of which the 5'untranslated region (5'UTR) was 348 bp, the CDS region was 636 BP (encoded 211 amino acid protein), and the 3'UTR was 1224 bp. The similarity of amino acid sequences with sheep, cattle, pigs, humans, mice and rats was 100%, 99%, 100%, 99%, 97% and 96% respectively. Protein structure prediction revealed that the protein was composed of a N terminal domain and a C terminal domain, each containing 6 conserved cysteine residues.2.TIMP3 gene in dairy goat follicles. The expression pattern of TIMP3 gene in different tissues of dairy goats was analyzed by Real-time PCR, and the relative expression of TIMP3 gene was the highest in oviduct, and the abundance of TIMP3 gene mRNA in follicle increased gradually with the development and maturation of follicle, and reached the highest level in corpus luteum. The expression of TIMP3 gene in ovaries of multiple lambs dairy goats (3-4 lambs per litter) was significantly higher than that of single lamb dairy goats (1-2 lambs per litter of three consecutive fetuses). It was suggested that TIMP3 gene might be closely related to litter size of dairy goats. 3. The molecular regulation mechanism of TIMP3 gene expression by LH / hCG was analyzed by hCG in cultured granulosa cells, Real-time P CR analysis showed that the mRNA abundance of TIMP3 gene peaked at 4 h and 24 h after treatment respectively; Western Blot analysis showed that the protein abundance of TIMP3 increased with the prolongation of treatment time and reached the maximum at 24 h; blocking analysis showed that the increase of TIMP3 induced by hCG was affected by PKA, PKC, MAPK and PI3K signaling pathways. The transcriptional regulatory region of IMP3 gene was analyzed. Three Sp1 binding sites were found in the region from - 1 BP to - 122 bp upstream of TIMP3 gene. Site-directed mutagenesis analysis showed that mutations of - 67 ~ - 58 and - 74 ~ - 65 significantly reduced the transcription of TIMP3. The granulosa cells were treated with cAMP activator FSK to promote the expression of Sp1, and the transcription of TIMP3 was enhanced with EMSA, ChIP and Sp. The results of bioinformatics analysis showed that the luciferase reporter vector was constructed and co-transfected with microRNAs into granulosa cells. It was found that the expression of TIMP3 was regulated by cAMP through regulating the transcription factor Sp1. Luciferase activity was significantly inhibited; Real-time PCR analysis showed that compared with NC group, microarray-21 and microarray-181b significantly reduced the abundance of TIMP3 gene mRNA in granulosa cells, while microarray-221 and microarray-222 groups showed no significant difference; Western Blot analysis showed that compared with NC group, microarray-21, microarray-181b, microarray-221 and microarray-222 significantly reduced TIMP3 protein. Mi-21, Mi-221, Mi-222 and Mi-R-181b bind to specific sites in the 3'UTR region of the TIMP3 gene to regulate the expression of TIMP3 gene. Mi-21 and Mi-181b regulate the expression of TIMP3 gene by promoting the degradation of mRNA, while Mi-221 and Mi-R-222 regulate the expression of TIMP3 by inhibiting translation, and Mi-21, Mi-221, Mi-222 and Mi-181b promote granules. Cell viability. 5. Molecular regulatory mechanisms of TIMP3 gene expression products on apoptosis and hormone synthesis in granulosa cells were analyzed. Adenovirus-mediated TIMP3 gene overexpression and siRNA silencing of TIMP3 gene expression were used. Overexpression of TIMP3 gene inhibited the viability of granulosa cells and promoted cell apoptosis; down-regulation of TIMP3 gene inhibited hCG-induced steroid hormone expression. Synthase StAR, p450scc, HSD3B gene expression and progesterone level were decreased, while TIMP3 inhibited the expression of ADAM17 gene and promoted the expression of FN and DCN genes related to ECM protein. These studies showed that LH/hCG binds to receptor LHR on granulosa cells, activates cAMP signaling pathway, promotes the expression of transcription factor Sp1, and Sp1 response element junction between Sp1 and TIMP3 gene. TIMP3 protein regulates progesterone synthesis by promoting the expression of StAR, P450scc and HSD3B genes, promotes the expression of FN and DCN genes to regulate ECM reconstruction, inhibits the expression of ADAM17 gene, inhibits the activity of granulosa cells, and promotes the apoptosis of granulosa cells to participate in the regulation of follicular cycle. The molecular mechanism involved in the regulation of follicular cycle provides theoretical and experimental basis for revealing the molecular mechanism of follicular development and maturation and ovulation.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S827
，
本文编号：2196765
[Abstract]:Matrix metalloproteinases (MMPs) play an important role in folliculogenesis, development, ovulation and luteal formation. As an inhibitor of MMPs, TIMP3 may participate in the coordination of various life activities during the follicular cycle. However, whether TIMP3 is expressed in the follicles of dairy goats, how it is expressed, the signaling pathway and its regulation are also involved. The expression products regulate the synthesis of follicular hormones and the molecular mechanism of apoptosis of granulosa cells. In this study, the follicles and granulosa cells of Guanzhong Dairy Goat were selected as the research object. On the basis of cloning and analyzing the cDNA sequence of TIMP3 gene, the expression of TIMP3 in follicular cycle was analyzed, and the effects of LH/hCG and MIRNA on TIMP were explored. 3 gene expression, TIMP3 gene expression products on follicular hormone synthesis, extracellular matrix remodeling and granulosa cell apoptosis and other biological processes related gene expression of molecular regulatory mechanisms, as well as TIMP3 gene expression products on granulosa cell apoptosis, to clarify the biological function of TIMP3 gene in the follicular cycle, the main results are as follows: 1. The cloning and bioinformatics analysis of the MP3 gene cDNA were carried out by RACE. The TIMP3 gene cDNA was obtained from the ovary of dairy goats. By sequencing and sequence analysis, it was found that the total length of the TIMP3 gene was 2208 bp, of which the 5'untranslated region (5'UTR) was 348 bp, the CDS region was 636 BP (encoded 211 amino acid protein), and the 3'UTR was 1224 bp. The similarity of amino acid sequences with sheep, cattle, pigs, humans, mice and rats was 100%, 99%, 100%, 99%, 97% and 96% respectively. Protein structure prediction revealed that the protein was composed of a N terminal domain and a C terminal domain, each containing 6 conserved cysteine residues.2.TIMP3 gene in dairy goat follicles. The expression pattern of TIMP3 gene in different tissues of dairy goats was analyzed by Real-time PCR, and the relative expression of TIMP3 gene was the highest in oviduct, and the abundance of TIMP3 gene mRNA in follicle increased gradually with the development and maturation of follicle, and reached the highest level in corpus luteum. The expression of TIMP3 gene in ovaries of multiple lambs dairy goats (3-4 lambs per litter) was significantly higher than that of single lamb dairy goats (1-2 lambs per litter of three consecutive fetuses). It was suggested that TIMP3 gene might be closely related to litter size of dairy goats. 3. The molecular regulation mechanism of TIMP3 gene expression by LH / hCG was analyzed by hCG in cultured granulosa cells, Real-time P CR analysis showed that the mRNA abundance of TIMP3 gene peaked at 4 h and 24 h after treatment respectively; Western Blot analysis showed that the protein abundance of TIMP3 increased with the prolongation of treatment time and reached the maximum at 24 h; blocking analysis showed that the increase of TIMP3 induced by hCG was affected by PKA, PKC, MAPK and PI3K signaling pathways. The transcriptional regulatory region of IMP3 gene was analyzed. Three Sp1 binding sites were found in the region from - 1 BP to - 122 bp upstream of TIMP3 gene. Site-directed mutagenesis analysis showed that mutations of - 67 ~ - 58 and - 74 ~ - 65 significantly reduced the transcription of TIMP3. The granulosa cells were treated with cAMP activator FSK to promote the expression of Sp1, and the transcription of TIMP3 was enhanced with EMSA, ChIP and Sp. The results of bioinformatics analysis showed that the luciferase reporter vector was constructed and co-transfected with microRNAs into granulosa cells. It was found that the expression of TIMP3 was regulated by cAMP through regulating the transcription factor Sp1. Luciferase activity was significantly inhibited; Real-time PCR analysis showed that compared with NC group, microarray-21 and microarray-181b significantly reduced the abundance of TIMP3 gene mRNA in granulosa cells, while microarray-221 and microarray-222 groups showed no significant difference; Western Blot analysis showed that compared with NC group, microarray-21, microarray-181b, microarray-221 and microarray-222 significantly reduced TIMP3 protein. Mi-21, Mi-221, Mi-222 and Mi-R-181b bind to specific sites in the 3'UTR region of the TIMP3 gene to regulate the expression of TIMP3 gene. Mi-21 and Mi-181b regulate the expression of TIMP3 gene by promoting the degradation of mRNA, while Mi-221 and Mi-R-222 regulate the expression of TIMP3 by inhibiting translation, and Mi-21, Mi-221, Mi-222 and Mi-181b promote granules. Cell viability. 5. Molecular regulatory mechanisms of TIMP3 gene expression products on apoptosis and hormone synthesis in granulosa cells were analyzed. Adenovirus-mediated TIMP3 gene overexpression and siRNA silencing of TIMP3 gene expression were used. Overexpression of TIMP3 gene inhibited the viability of granulosa cells and promoted cell apoptosis; down-regulation of TIMP3 gene inhibited hCG-induced steroid hormone expression. Synthase StAR, p450scc, HSD3B gene expression and progesterone level were decreased, while TIMP3 inhibited the expression of ADAM17 gene and promoted the expression of FN and DCN genes related to ECM protein. These studies showed that LH/hCG binds to receptor LHR on granulosa cells, activates cAMP signaling pathway, promotes the expression of transcription factor Sp1, and Sp1 response element junction between Sp1 and TIMP3 gene. TIMP3 protein regulates progesterone synthesis by promoting the expression of StAR, P450scc and HSD3B genes, promotes the expression of FN and DCN genes to regulate ECM reconstruction, inhibits the expression of ADAM17 gene, inhibits the activity of granulosa cells, and promotes the apoptosis of granulosa cells to participate in the regulation of follicular cycle. The molecular mechanism involved in the regulation of follicular cycle provides theoretical and experimental basis for revealing the molecular mechanism of follicular development and maturation and ovulation.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S827
，
本文编号：2196765
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