营养水平对后备母猪卵巢microRNA-378表达影响及microRNA-378作用机制研究
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摘要
营养可通过调控雌二醇、孕酮等激素的水平影响后备母猪的繁殖性能。在转录水平或翻译水平上调控雌二醇合成的限速酶芳香化酶及孕酮受体对雌二醇的合成分泌和孕酮功能的发挥具有重要作用。Micro-RNAs (miRNAs)是一种21～25nt长度的单链小分子RNA,在基因表达的转录后水平调控发挥着重要功能。研究发现miR-378在牛卵巢的黄体退化过程存在表达,生物信息学预测发现miR-378在猪芳香化酶、孕酮受体3’端非翻译区(3'UTR)上存在靶定位点,可能影响芳香化酶和孕酮受体的表达。因此,本研究考察了营养水平对后备母猪卵巢miR-378表达的影响,并通过体外培养后备母猪卵巢颗粒细胞研究了miR-378的功能和作用机制。本研究包括以下四部分：
试验一营养水平对后备母猪卵巢miR-378表达的影响
本试验通过不同营养水平(采食水平)饲喂正常发情一次后的后备母猪,研究营养水平对后备母猪卵巢miR-378表达的影响。8头LY(Landrace×York)后备母猪第一次发情后,随机分为两个处理,每个处理4个重复,每个重复1头猪,按正常采食水平(2.5kg/d)和过度限饲(0.5kg/d)分别饲喂。后备母猪第三个情期时屠宰取样。
试验结果表明不同营养水平显著(P<0.05)影响后备母猪发情率,过度限饲组母猪出现全部不发情现象。营养水平影响后备母猪卵巢miR-378的表达水平,正常营养水平组显著(P<0.05)低于过度限饲组。正常水平组大、小卵泡的颗粒细胞均表达miR-378,且大卵泡颗粒细胞的表达量显著(P<0.05)低于小卵泡颗粒细胞,说明miR-378在卵泡发育过程中可能有重要作用。
试验二慢病毒介导的miR-378对猪颗粒细胞的影响
试验一发现营养水平显著影响卵巢miR-378的表达,同时也影响了后备母猪的繁殖性能,结合生物信息学预测结果miR-378在多种与繁殖相关基因的3’UTR上存在靶定位点,推测营养对繁殖性能影响的途径之一可能是通过miR-378而实现。为此本试验拟通过慢病毒转导的方式将miR-378导入颗粒细胞,研究miR-378对颗粒细胞功能的影响。
试验通过TA克隆并酶切获得H1-miR-378片段,片段与含相同酶切位点的骨架质粒连接获得目的质粒pL-SIN-Lenti-H1-miR-378-EF1α-GFP。随后分别应用脂质体转染和磷酸钙共沉淀法转染HEK293FT细胞制备miR-378慢病毒,并选取合理的方法批量生产病毒。体外原代培养大、小卵泡的颗粒细胞,当细胞生长汇合达40-50%时转导miR-378慢病毒,使miR-378在颗粒细胞中过量表达,研究miR-378对颗粒细胞增殖、抗凋亡、基因表达、蛋白表达和雌二醇分泌的影响。试验结果表明：
1.试验成功构建了用于包装慢病毒的miR-378质粒pL-SIN-Lenti-H1-miR-378-EF1α-GFP。
2.脂质体转染和磷酸钙共沉淀法转染均成功制备了miR-378慢病毒。分离培养后备母猪卵巢颗粒细胞,转导两种方法制备的慢病毒,发现转导效率相当,且磷酸钙共沉淀法实验成本低于脂质体转染法,因而确定批量生产miR-378慢病毒采用磷酸钙共沉淀法。
3.转导miR-378慢病毒后,miR-378的表达量显著(P<0.05)高于对照组,达到过量表达miR-378的目的。过表达miR-378促进颗粒细胞的增殖和抗凋亡能力。miR-378在转录水平上对C-kit,Ret,GDNF,aromatase,FSHR,GFRa-1, MAPK, Star, LHR, PGR, Bmi-1和Cx43基因的表达差异均未达显著水平(P>0.05),却显著抑制(P<0.05)了PGR和a(?)romatase蛋白的合成,说明miR-378在转录后水平或翻译水平对PGR和(?)aromatase起着调控作用；试验同时研究了miR-378对FSHR、GDNF、和MAPK蛋白表达的影响,但差异均未达显著水平(P>0.05)；过表达miR-378抑制(P<0.05)颗粒细胞雌二醇的分泌。
试验三miR-378调控芳香化酶、孕酮受体表达的分子机制
生物信息学预测发现miR-378在aromatase, FSHR, c-Kit, MAPK和(?)PGR3'UTR上存在靶定位点,试验二在细胞水平上发现miR-378影响axomatase和PGR蛋白的表达,miR-378对这两种蛋白的抑制作用是否因为靶定了其3’UTR,为此开展了试验三的研究。
本试验首先克隆PGR和(?)aromatase3'UTR及miR-378靶定位点突变的3’UTR进入荧光素酶报告基团质粒。随后体外培养大卵泡颗粒细胞,细胞达40～50%汇合时,转导miR-378病毒,24h后分别瞬时转染3’UTR及突变的3’UTR荧光素酶报告基团质粒和内参质粒,从正反两方面研究miR-378是否通过靶定PGR和(?)aromatase3’UTR而影响荧光素酶的表达。试验结果表明：
1.试验成功构建了PGR和(?)aromatase3'UTR的荧光素酶报告基团质粒：p-miR-Report+WT miR-378BS in PGR3'UTR, p-miR-Report+WT miR-378BS in PGRb3'UTR, p-miR-Report+WT miR-378BS in aromatase3'UTR, p-miR-Report+conUTR以及PGR和aromatase3'UTR特定位点突变的荧光素酶报告基团质粒:p-miR-Report+MU miR-378BS in PGRb3'UTR, p-miR-Report+MUA miR-378BS in aromatase3'UTR, p-miR-Report+MUB miR-378BS in aromatase3'UTR和p-miR-Report+MUC miR-378BS in aromatase3'UTR。
2. PGR3'UTR显著(P<0.05)降低了荧光素酶的活力,靶定位点突变后荧光素酶的活力较未突变组显著(P<0.05)升高,并与对照组无差异(P>0.05),由此说明PGR3'UTR上的预测位点+4062-+4082即为miR-378的靶定位点；aromatase3'UTR也显著降(P<0.05)低了荧光素酶的活力,miR-378在aromatase3’UTR存在三个靶定位点,分别突变这三个位点,发现突变位点A和B突变后荧光素酶的活力较未突变组显著(P<0.05)升高,并与对照组无差异(P>0.05),而突变位点C仍然降低(P<0.05)荧光素酶的活力,说明预测位点A(+1589+1609)和B(+1607-+1627)是(?)niR-378发挥作用的靶定位点,而位点C(+1627-+1647)则不发挥作用。
由此表明miR-378通过靶定结合aromatase和(?)PGR mRNA的3’UTR+1589-+1609,+1607-+1627和+4062-+4082位点在转录后水平发挥调控作用。阐明了miR-378抑制(?)aromatase和PGR蛋白表达的机制。试验四过表达aromatase3'UTR及miR-378抑制剂对颗粒细胞的影响
雌二醇是后备母猪初情启动卵巢卵母细胞发育的关键激素,芳香化酶是颗粒细胞内合成雌二醇的关键酶,为了进一步明确miR-378对芳香化酶表达和雌二醇分泌的影响,开展了试验四的研究。
本试验首先构建了过表达aromatase3'UTR的质粒。颗粒细胞转导miR-378慢病毒后转染aromatase3'UTR过表达质粒,转染该质粒后颗粒细胞过量表达aromatase3'UTR,这类aromatase3'UTR可竞争性耗竭颗粒细胞内过表达的miR-378,逆转miR-378与内源aromatase3'UTR靶定结合引起的aromatase表达降低现象。本试验同时还研究了miR-378抑制剂对大、小卵泡颗粒细胞内源miR-378表达的影响；转导miR-378(?)病毒同时转染miR-378抑制剂研究miR-378抑制剂对颗粒细胞芳香化酶蛋白表达和雌激素分泌的影响；转导miR-378病毒同时转染miR-378抑制剂和aromatase3'UTR荧光素酶报告基团质粒,研究miR-378抑制剂对miR-378引起的aromatase3'UTR荧光素酶活力降低的影响。试验结果表明：
1.试验成功构建了过表达aromatase3'UTR的质粒CMV-Aro-3'UTR以及对照组质粒CMV-con-3'UTR。
2.转染过表达aromatase3'UTR质粒后,aro-3'UTR的表达显著(P<0.05)高于miR-378组和UTR对照组,达到过量表达aro-3'UTR的目的。
3.颗粒细胞转导miR-378病毒后转染aromatase3'UTR,发现aromatase3’UTR显著(P<0.05)逆转了miR-378导致的颗粒细胞aromatase蛋白表达和雌二醇分泌的抑制作用。
4.转染miR-378抑制剂于未转导miR-378病毒的颗粒细胞中,发现miR-378抑制剂显著(P<0.05)抑制颗粒细胞内源miR-378的表达。
5.颗粒细胞转导miR-378病毒后转染miR-378抑制剂,发现miR-378抑制剂显著(P<0.05)逆转了miR-378抑制颗粒细胞aromatase蛋白表达和雌二醇的分泌。
6.颗粒细胞转导miR-378病毒后转染miR-378抑制剂和aromatase3'UTR荧光素酶报告基团质粒,发现miR-378抑制剂逆转了(P<0.05)miR-378导致的aromatase3'UTR荧光素酶活力降低的现象。
综上所述,营养水平显著影响后备母猪卵巢miR-378的表达,限制营养水平下miR-378的表达显著高于正常营养水平。miR-378通过靶定结合PGR3’UTR+4062-+4082和(?)aromatase3'UTR+1589-+1609和+1607-+1627位点,在转录后水平上调控这两种蛋白的表达,阐明了miR-378在猪卵巢颗粒细胞中的作用机制。Aromatase是雌二醇合成关键酶,aromatase蛋白水平的变化直接影响着雌二醇的合成,雌二醇是影响繁殖内分泌的重要激素,由此阐明了营养影响卵巢miR-378的表达是实现营养调控繁殖内分泌的途径之一。
Nutrition would affect reproductive performance of gilts through hormone which like estradiol and progesterone. Reglulation of the estradiol synthesis enzyme aromatase and progesterone receptor at the transcriptional or translational level are important for estradiol secretion and progesterone function implementation. Micro-RNAs (miRNAs) are small (19-25nucleotides) single-stranded RNAs that were found diversely regulate gene expression through post-transcriptional level. MiR-378regulated the corpus luteum regression in bovine ovarian. The predication of bioinformatics showed that miR-378has binding sites at3'untranslated region (3'UTR) of aromatase and progesterone receptor that might affect the expression of them. Thus, in this study we investigate the effects of nutrition on the expression of miR-378in gilt ovaries, and study the physiological function and mechanism of miR-378in gilt ovary granulosa cells (GCs) in vitro. This study consists of four parts as followings:
Exp.1Effects of nutrient level on the miR-378expression in gilt ovary
Through different nutrient levels (feed intake levels) feeding replacement gilts which already have once estrus, to study the effects of nutritional levels on the expression of miR-378in ovary. After first estrus a total of8Landrace×Yorksire (LY) gilts were randomly assigned to1of2treatments of4gilts in each treatment. The two feed intake level were1) normal (2.5kg/d) and2) restriction (0.5kg/d). At the third estrus slaughter the gilts and collect the sample.
Results showed that nutrition level significantly (P<0.05) affects gilts' estrus rate, there's no estrus phenomenon in the restriction group. Expression of miR-378in normal nutrition group was significantly (P<0.05) lower than the restriction group in the ovaries. In normal group, miR-378also expressed physiologically in estrus gilts' GCs which derived from small and large follicles, and miR-378expression level in the GCs derived from large follicles was lower (P<0.05) than in the GCs derived from small follicles.
Exp.2Effects of microRNA-378on the porcine granulosa cells
Experiment one found nutritional levels significantly affect ovarian miR-378expression, and also affected the reproductive performance of gilts, combination of bioinformatics prediction results of miR-378which has target binding site in reproduction related genes'3'UTR, we presume that one way of nutrition affect the reproduction performance achieving through miR-378. MiR-378might be influence GCs functions to achieve reproductive performance regulation in ovary. For this study we proposed transduct miR-378by lentivirus to study the effects of miR-378on GCs' physiological function.
This experiment, firstly, through TA cloning and restriction enzyme digest obtained H1-miR-378fragment, and ligation with the backbone plasmid which containing the same restriction sites, then got the plasmid pL-SIN-Lenti-Hl-miR-378-EFla-GFP. Application the lipofectamine transfection and calcium phosphate co-precipitation transfection to transfect the HEK293FT cells for miR-378virus'preparation, then select a reasonable method to prepare the large volume of the lentivirus for the next experiments. In vitro cultured GCs which derived from small and large follicles, at40-50%confluence, GCs were transducted miR-378lentivirus thus over-express miR-378intra-cellular, and then study the effects of miR-378on GCs'proliferation, apoptosis, gene expression, protein expression and estradiol secretion. The results showed that:
1. Successfully constructed the miR-378plasmid pL-SIN-Lenti-H1-miR-378-EF1a-GFP which for the lentivirus' packaging.
2. Appling both lipofectamine transfection and calcium phosphate co-precipitation transfection methods successfully prepared the miR-378lentivurus. Isolated and cultured porcine GCs, by transduction of miR-378which prepared from the two methods, found there's no difference with the transduction efficiency, but the cost of calcium precipitation method was less than the lipofectamine transfection method, thus confirming to use the calcium phosphate coprecipitation to prepare the large volume of lentivirus for the next experiments.
3. After miR-378lentivirus transduction, the miR-378expression was significantly higher (P<0.05) than control group. Over expression of miR-378promoted GCs' proliferation and improved the ability of anti-apoptosis. Although miR-378didn't significant (P>0.05) affect the transcriptional level of C-kit, Ret, GDNF, aromatase, FSHR, GFRa-1, MAPK, Star, LHR, PGR, Bmi-1and Cx43gene, at the translation level PGR and aromatase protein were inhibited (P<0.05), indicating that miR-378regulate the PGR and the aromatase at the post-transcriptional level or translation level. For the effects of miR-378on the protein expression of FSHR, GDNF and MAPK, the results showed that there's no difference (P>0.05). In addition, we found over-expression of miR-378inhibited (P<0.05) secretion of estradiol in GCs.
Exp.3miR-378regulate aromatase and PGR expression through its'3'UTR
Bioinformatics prediction show that miR-378have binding sites at3'UTR of gene aromatase, FSHR, c-Kit, MAPK and PGR, and experiment two found that miR-378influence the aromatase and PGR proteins's expression at the cellular level. MiR-378suppresses the two proteins'expression might by target its3'UTR, therefore carried out experiment three.
For this experiment first cloned PGR and aromatase3'UTR and mutations of miR-378target binding site in the3'UTR into luciferase reporter plasmid. Subsequently cultured GCs derived from large follicles, when GCs reached40-50%confluence in vitro, transducted miR-378lentivirus,24h later transient transfect plasmids of above separately and the intro-control, from positive and negative aspects to investigate whether the miR-378influence the luciferase activity by targeting PGR and aromatase3'UTR. Results showed that:
1. Successfully constructed the PGR and the aromatase3'UTR luciferase reporter plasmids:p-miR-Report+WT miR-378BS in PGR3'UTR, p-miR-Report+WT miR-378BS in PGRb3'UTR, p-miR-Report+WT miR-378BS in aromatase3'UTR, p-miR-Report+conUTR and specific mutation of PGR and aromatase3'UTR luciferase reporter plasmids:p-miR-Report+MU miR-378BS in PGRb3'UTR, p-miR-Report+MUA miR-378BS in aromatase3'UTR, p-miR-Report+MUB miR-378BS in aromatase3'UTR and p-miR-Report+MUC miR-378BS in aromatase3'UTR.
2. PGR3'UTR significant decrease (P<0.05) the luciferace activity, and increased (P<0.05) the luciferace activity after the binding site mutated. So it identified that miR-378affect the PGR protein expression at the site+4062-+4082in its3'UTR. The aromatase3'UTR also decreased (P<0.05) the luciferace activity. MiR-378have three binding sites on the aromatase3'UTR, mutated them separately found the site A and B reversed (P<0.05) the luciferace activity repress effect, but site C still decreased (P<0.05) the luciferace activity. That meaned miR-378affects the aromatase protein expression at the site+1589-+1609and+1607-+1627in aromatase3'UTR.
These showed that by targeting aromatase and PGR mRNA3'UTR+1589-+1609,+1607-+1627and+4062-+4082sites, miR-378regulate aromatase and PGR protein expression at the posttranscriptional level.
Exp.4Effects of aromatase3'UTR over-expression and miR-378inhibitor on the granulosa cells
Estradiol is the key hormone for the gilt puberty and the development of ovarian oocyte. Aromatase is the key enzymes for the estradiol synthesis in GCs. In order to deeply clarify the effects of miR-378on the expression of aromatase and secretion of estradiol, we carried out the experiment four.
This experiment constructed the over-expression of aromatase3'UTR plasmid. After GCs transducted miR-378, transfected the over-expression of aromatase3'UTR plasmid to over-express aromatase3'UTR which would competitive exhaustion of the over-expression miR-378in GCs, thus reverse the depression effects of miR-378on aromatase expression through binding endogenous aromatase3'UTR. This experiment also examined the effects of miR-378inhibitor on the endogenetic miR-378expression in GCs which derived from small and large follicles. Next investigated the effects of miR-378inhibitor on the aromatase protein expression and estradiol secretion after transducted miR-378. After miR-378transducting, transfected miR-378inhibitor and aromatase3'UTR luciferase reporter plasmid, we investigated the effects of miR-378on aromatase3'UTR-linked luciferase activity. The results showed that:
1. Successfully constructed the over-expression of aromatase3'UTR plasmid named CMV-Aro-3'UTR, and control plasmid named CMV-con-3'UTR.
2. After plasmid transfecting, the Aro-3'UTR expression was significantly higher (P<0.05) than miR-378treated group and conUTR group, thus achieved the purposes of over-expression Aro-3'UTR.
3. Transfected3'UTR plasmids after miR-378lentivirus transduction, results showed that over-expression of aromatase3'UTR reversed the effects of miR-378suppresses (P<0.05) aromatase protein expression and estradiol secretion.
4. Transfected the miR-378inhibitor to the GCs without the miR-378lentivirus transduction, results showed that miR-378inhibitor repressed (P<0.05) the expression of endogenetic miR-378in GCs.
5. Transfected miR-378inhibitor after miR-378lentivirus transduction, experimental results showed that miR-378inhibitors reversed the effects of miR-378suppresses (P<0.05) aromatase protein expression and estradiol secretion.
6. MiR-378inhibitor also reversed (P<0.05) the phenomenon of miR-378induced the aromatase3'UTR reduced luciferase activity.
In summary, nutrition affects the expression of miR-378in gilt ovarian. The expression of miR-378was significant higher in the lower nutrition group than the normal nutrition group. By targeting3'UTR+4062-+4082of PGR and3'UTR+1589-+1609and+1607-+1627of aromatase, miR-378regulate the PGR and aromatase protein expression at the post-transcriptional level, thus clarified the molecular mechanism of miR-378on the GCs' effects. Aromatase is a key enzyme for estradiol synthesis, aromatase protein levels directly affect the synthesis of estradiol, and the estradiol is an important indicator in reproduction, it indicates that nutrition influence the expression of miR-378in ovary is one way of nutritional regulation on reproduction.
试验一营养水平对后备母猪卵巢miR-378表达的影响
本试验通过不同营养水平(采食水平)饲喂正常发情一次后的后备母猪,研究营养水平对后备母猪卵巢miR-378表达的影响。8头LY(Landrace×York)后备母猪第一次发情后,随机分为两个处理,每个处理4个重复,每个重复1头猪,按正常采食水平(2.5kg/d)和过度限饲(0.5kg/d)分别饲喂。后备母猪第三个情期时屠宰取样。
试验结果表明不同营养水平显著(P<0.05)影响后备母猪发情率,过度限饲组母猪出现全部不发情现象。营养水平影响后备母猪卵巢miR-378的表达水平,正常营养水平组显著(P<0.05)低于过度限饲组。正常水平组大、小卵泡的颗粒细胞均表达miR-378,且大卵泡颗粒细胞的表达量显著(P<0.05)低于小卵泡颗粒细胞,说明miR-378在卵泡发育过程中可能有重要作用。
试验二慢病毒介导的miR-378对猪颗粒细胞的影响
试验一发现营养水平显著影响卵巢miR-378的表达,同时也影响了后备母猪的繁殖性能,结合生物信息学预测结果miR-378在多种与繁殖相关基因的3’UTR上存在靶定位点,推测营养对繁殖性能影响的途径之一可能是通过miR-378而实现。为此本试验拟通过慢病毒转导的方式将miR-378导入颗粒细胞,研究miR-378对颗粒细胞功能的影响。
试验通过TA克隆并酶切获得H1-miR-378片段,片段与含相同酶切位点的骨架质粒连接获得目的质粒pL-SIN-Lenti-H1-miR-378-EF1α-GFP。随后分别应用脂质体转染和磷酸钙共沉淀法转染HEK293FT细胞制备miR-378慢病毒,并选取合理的方法批量生产病毒。体外原代培养大、小卵泡的颗粒细胞,当细胞生长汇合达40-50%时转导miR-378慢病毒,使miR-378在颗粒细胞中过量表达,研究miR-378对颗粒细胞增殖、抗凋亡、基因表达、蛋白表达和雌二醇分泌的影响。试验结果表明：
1.试验成功构建了用于包装慢病毒的miR-378质粒pL-SIN-Lenti-H1-miR-378-EF1α-GFP。
2.脂质体转染和磷酸钙共沉淀法转染均成功制备了miR-378慢病毒。分离培养后备母猪卵巢颗粒细胞,转导两种方法制备的慢病毒,发现转导效率相当,且磷酸钙共沉淀法实验成本低于脂质体转染法,因而确定批量生产miR-378慢病毒采用磷酸钙共沉淀法。
3.转导miR-378慢病毒后,miR-378的表达量显著(P<0.05)高于对照组,达到过量表达miR-378的目的。过表达miR-378促进颗粒细胞的增殖和抗凋亡能力。miR-378在转录水平上对C-kit,Ret,GDNF,aromatase,FSHR,GFRa-1, MAPK, Star, LHR, PGR, Bmi-1和Cx43基因的表达差异均未达显著水平(P>0.05),却显著抑制(P<0.05)了PGR和a(?)romatase蛋白的合成,说明miR-378在转录后水平或翻译水平对PGR和(?)aromatase起着调控作用；试验同时研究了miR-378对FSHR、GDNF、和MAPK蛋白表达的影响,但差异均未达显著水平(P>0.05)；过表达miR-378抑制(P<0.05)颗粒细胞雌二醇的分泌。
试验三miR-378调控芳香化酶、孕酮受体表达的分子机制
生物信息学预测发现miR-378在aromatase, FSHR, c-Kit, MAPK和(?)PGR3'UTR上存在靶定位点,试验二在细胞水平上发现miR-378影响axomatase和PGR蛋白的表达,miR-378对这两种蛋白的抑制作用是否因为靶定了其3’UTR,为此开展了试验三的研究。
本试验首先克隆PGR和(?)aromatase3'UTR及miR-378靶定位点突变的3’UTR进入荧光素酶报告基团质粒。随后体外培养大卵泡颗粒细胞,细胞达40～50%汇合时,转导miR-378病毒,24h后分别瞬时转染3’UTR及突变的3’UTR荧光素酶报告基团质粒和内参质粒,从正反两方面研究miR-378是否通过靶定PGR和(?)aromatase3’UTR而影响荧光素酶的表达。试验结果表明：
1.试验成功构建了PGR和(?)aromatase3'UTR的荧光素酶报告基团质粒：p-miR-Report+WT miR-378BS in PGR3'UTR, p-miR-Report+WT miR-378BS in PGRb3'UTR, p-miR-Report+WT miR-378BS in aromatase3'UTR, p-miR-Report+conUTR以及PGR和aromatase3'UTR特定位点突变的荧光素酶报告基团质粒:p-miR-Report+MU miR-378BS in PGRb3'UTR, p-miR-Report+MUA miR-378BS in aromatase3'UTR, p-miR-Report+MUB miR-378BS in aromatase3'UTR和p-miR-Report+MUC miR-378BS in aromatase3'UTR。
2. PGR3'UTR显著(P<0.05)降低了荧光素酶的活力,靶定位点突变后荧光素酶的活力较未突变组显著(P<0.05)升高,并与对照组无差异(P>0.05),由此说明PGR3'UTR上的预测位点+4062-+4082即为miR-378的靶定位点；aromatase3'UTR也显著降(P<0.05)低了荧光素酶的活力,miR-378在aromatase3’UTR存在三个靶定位点,分别突变这三个位点,发现突变位点A和B突变后荧光素酶的活力较未突变组显著(P<0.05)升高,并与对照组无差异(P>0.05),而突变位点C仍然降低(P<0.05)荧光素酶的活力,说明预测位点A(+1589+1609)和B(+1607-+1627)是(?)niR-378发挥作用的靶定位点,而位点C(+1627-+1647)则不发挥作用。
由此表明miR-378通过靶定结合aromatase和(?)PGR mRNA的3’UTR+1589-+1609,+1607-+1627和+4062-+4082位点在转录后水平发挥调控作用。阐明了miR-378抑制(?)aromatase和PGR蛋白表达的机制。试验四过表达aromatase3'UTR及miR-378抑制剂对颗粒细胞的影响
雌二醇是后备母猪初情启动卵巢卵母细胞发育的关键激素,芳香化酶是颗粒细胞内合成雌二醇的关键酶,为了进一步明确miR-378对芳香化酶表达和雌二醇分泌的影响,开展了试验四的研究。
本试验首先构建了过表达aromatase3'UTR的质粒。颗粒细胞转导miR-378慢病毒后转染aromatase3'UTR过表达质粒,转染该质粒后颗粒细胞过量表达aromatase3'UTR,这类aromatase3'UTR可竞争性耗竭颗粒细胞内过表达的miR-378,逆转miR-378与内源aromatase3'UTR靶定结合引起的aromatase表达降低现象。本试验同时还研究了miR-378抑制剂对大、小卵泡颗粒细胞内源miR-378表达的影响；转导miR-378(?)病毒同时转染miR-378抑制剂研究miR-378抑制剂对颗粒细胞芳香化酶蛋白表达和雌激素分泌的影响；转导miR-378病毒同时转染miR-378抑制剂和aromatase3'UTR荧光素酶报告基团质粒,研究miR-378抑制剂对miR-378引起的aromatase3'UTR荧光素酶活力降低的影响。试验结果表明：
1.试验成功构建了过表达aromatase3'UTR的质粒CMV-Aro-3'UTR以及对照组质粒CMV-con-3'UTR。
2.转染过表达aromatase3'UTR质粒后,aro-3'UTR的表达显著(P<0.05)高于miR-378组和UTR对照组,达到过量表达aro-3'UTR的目的。
3.颗粒细胞转导miR-378病毒后转染aromatase3'UTR,发现aromatase3’UTR显著(P<0.05)逆转了miR-378导致的颗粒细胞aromatase蛋白表达和雌二醇分泌的抑制作用。
4.转染miR-378抑制剂于未转导miR-378病毒的颗粒细胞中,发现miR-378抑制剂显著(P<0.05)抑制颗粒细胞内源miR-378的表达。
5.颗粒细胞转导miR-378病毒后转染miR-378抑制剂,发现miR-378抑制剂显著(P<0.05)逆转了miR-378抑制颗粒细胞aromatase蛋白表达和雌二醇的分泌。
6.颗粒细胞转导miR-378病毒后转染miR-378抑制剂和aromatase3'UTR荧光素酶报告基团质粒,发现miR-378抑制剂逆转了(P<0.05)miR-378导致的aromatase3'UTR荧光素酶活力降低的现象。
综上所述,营养水平显著影响后备母猪卵巢miR-378的表达,限制营养水平下miR-378的表达显著高于正常营养水平。miR-378通过靶定结合PGR3’UTR+4062-+4082和(?)aromatase3'UTR+1589-+1609和+1607-+1627位点,在转录后水平上调控这两种蛋白的表达,阐明了miR-378在猪卵巢颗粒细胞中的作用机制。Aromatase是雌二醇合成关键酶,aromatase蛋白水平的变化直接影响着雌二醇的合成,雌二醇是影响繁殖内分泌的重要激素,由此阐明了营养影响卵巢miR-378的表达是实现营养调控繁殖内分泌的途径之一。
Nutrition would affect reproductive performance of gilts through hormone which like estradiol and progesterone. Reglulation of the estradiol synthesis enzyme aromatase and progesterone receptor at the transcriptional or translational level are important for estradiol secretion and progesterone function implementation. Micro-RNAs (miRNAs) are small (19-25nucleotides) single-stranded RNAs that were found diversely regulate gene expression through post-transcriptional level. MiR-378regulated the corpus luteum regression in bovine ovarian. The predication of bioinformatics showed that miR-378has binding sites at3'untranslated region (3'UTR) of aromatase and progesterone receptor that might affect the expression of them. Thus, in this study we investigate the effects of nutrition on the expression of miR-378in gilt ovaries, and study the physiological function and mechanism of miR-378in gilt ovary granulosa cells (GCs) in vitro. This study consists of four parts as followings:
Exp.1Effects of nutrient level on the miR-378expression in gilt ovary
Through different nutrient levels (feed intake levels) feeding replacement gilts which already have once estrus, to study the effects of nutritional levels on the expression of miR-378in ovary. After first estrus a total of8Landrace×Yorksire (LY) gilts were randomly assigned to1of2treatments of4gilts in each treatment. The two feed intake level were1) normal (2.5kg/d) and2) restriction (0.5kg/d). At the third estrus slaughter the gilts and collect the sample.
Results showed that nutrition level significantly (P<0.05) affects gilts' estrus rate, there's no estrus phenomenon in the restriction group. Expression of miR-378in normal nutrition group was significantly (P<0.05) lower than the restriction group in the ovaries. In normal group, miR-378also expressed physiologically in estrus gilts' GCs which derived from small and large follicles, and miR-378expression level in the GCs derived from large follicles was lower (P<0.05) than in the GCs derived from small follicles.
Exp.2Effects of microRNA-378on the porcine granulosa cells
Experiment one found nutritional levels significantly affect ovarian miR-378expression, and also affected the reproductive performance of gilts, combination of bioinformatics prediction results of miR-378which has target binding site in reproduction related genes'3'UTR, we presume that one way of nutrition affect the reproduction performance achieving through miR-378. MiR-378might be influence GCs functions to achieve reproductive performance regulation in ovary. For this study we proposed transduct miR-378by lentivirus to study the effects of miR-378on GCs' physiological function.
This experiment, firstly, through TA cloning and restriction enzyme digest obtained H1-miR-378fragment, and ligation with the backbone plasmid which containing the same restriction sites, then got the plasmid pL-SIN-Lenti-Hl-miR-378-EFla-GFP. Application the lipofectamine transfection and calcium phosphate co-precipitation transfection to transfect the HEK293FT cells for miR-378virus'preparation, then select a reasonable method to prepare the large volume of the lentivirus for the next experiments. In vitro cultured GCs which derived from small and large follicles, at40-50%confluence, GCs were transducted miR-378lentivirus thus over-express miR-378intra-cellular, and then study the effects of miR-378on GCs'proliferation, apoptosis, gene expression, protein expression and estradiol secretion. The results showed that:
1. Successfully constructed the miR-378plasmid pL-SIN-Lenti-H1-miR-378-EF1a-GFP which for the lentivirus' packaging.
2. Appling both lipofectamine transfection and calcium phosphate co-precipitation transfection methods successfully prepared the miR-378lentivurus. Isolated and cultured porcine GCs, by transduction of miR-378which prepared from the two methods, found there's no difference with the transduction efficiency, but the cost of calcium precipitation method was less than the lipofectamine transfection method, thus confirming to use the calcium phosphate coprecipitation to prepare the large volume of lentivirus for the next experiments.
3. After miR-378lentivirus transduction, the miR-378expression was significantly higher (P<0.05) than control group. Over expression of miR-378promoted GCs' proliferation and improved the ability of anti-apoptosis. Although miR-378didn't significant (P>0.05) affect the transcriptional level of C-kit, Ret, GDNF, aromatase, FSHR, GFRa-1, MAPK, Star, LHR, PGR, Bmi-1and Cx43gene, at the translation level PGR and aromatase protein were inhibited (P<0.05), indicating that miR-378regulate the PGR and the aromatase at the post-transcriptional level or translation level. For the effects of miR-378on the protein expression of FSHR, GDNF and MAPK, the results showed that there's no difference (P>0.05). In addition, we found over-expression of miR-378inhibited (P<0.05) secretion of estradiol in GCs.
Exp.3miR-378regulate aromatase and PGR expression through its'3'UTR
Bioinformatics prediction show that miR-378have binding sites at3'UTR of gene aromatase, FSHR, c-Kit, MAPK and PGR, and experiment two found that miR-378influence the aromatase and PGR proteins's expression at the cellular level. MiR-378suppresses the two proteins'expression might by target its3'UTR, therefore carried out experiment three.
For this experiment first cloned PGR and aromatase3'UTR and mutations of miR-378target binding site in the3'UTR into luciferase reporter plasmid. Subsequently cultured GCs derived from large follicles, when GCs reached40-50%confluence in vitro, transducted miR-378lentivirus,24h later transient transfect plasmids of above separately and the intro-control, from positive and negative aspects to investigate whether the miR-378influence the luciferase activity by targeting PGR and aromatase3'UTR. Results showed that:
1. Successfully constructed the PGR and the aromatase3'UTR luciferase reporter plasmids:p-miR-Report+WT miR-378BS in PGR3'UTR, p-miR-Report+WT miR-378BS in PGRb3'UTR, p-miR-Report+WT miR-378BS in aromatase3'UTR, p-miR-Report+conUTR and specific mutation of PGR and aromatase3'UTR luciferase reporter plasmids:p-miR-Report+MU miR-378BS in PGRb3'UTR, p-miR-Report+MUA miR-378BS in aromatase3'UTR, p-miR-Report+MUB miR-378BS in aromatase3'UTR and p-miR-Report+MUC miR-378BS in aromatase3'UTR.
2. PGR3'UTR significant decrease (P<0.05) the luciferace activity, and increased (P<0.05) the luciferace activity after the binding site mutated. So it identified that miR-378affect the PGR protein expression at the site+4062-+4082in its3'UTR. The aromatase3'UTR also decreased (P<0.05) the luciferace activity. MiR-378have three binding sites on the aromatase3'UTR, mutated them separately found the site A and B reversed (P<0.05) the luciferace activity repress effect, but site C still decreased (P<0.05) the luciferace activity. That meaned miR-378affects the aromatase protein expression at the site+1589-+1609and+1607-+1627in aromatase3'UTR.
These showed that by targeting aromatase and PGR mRNA3'UTR+1589-+1609,+1607-+1627and+4062-+4082sites, miR-378regulate aromatase and PGR protein expression at the posttranscriptional level.
Exp.4Effects of aromatase3'UTR over-expression and miR-378inhibitor on the granulosa cells
Estradiol is the key hormone for the gilt puberty and the development of ovarian oocyte. Aromatase is the key enzymes for the estradiol synthesis in GCs. In order to deeply clarify the effects of miR-378on the expression of aromatase and secretion of estradiol, we carried out the experiment four.
This experiment constructed the over-expression of aromatase3'UTR plasmid. After GCs transducted miR-378, transfected the over-expression of aromatase3'UTR plasmid to over-express aromatase3'UTR which would competitive exhaustion of the over-expression miR-378in GCs, thus reverse the depression effects of miR-378on aromatase expression through binding endogenous aromatase3'UTR. This experiment also examined the effects of miR-378inhibitor on the endogenetic miR-378expression in GCs which derived from small and large follicles. Next investigated the effects of miR-378inhibitor on the aromatase protein expression and estradiol secretion after transducted miR-378. After miR-378transducting, transfected miR-378inhibitor and aromatase3'UTR luciferase reporter plasmid, we investigated the effects of miR-378on aromatase3'UTR-linked luciferase activity. The results showed that:
1. Successfully constructed the over-expression of aromatase3'UTR plasmid named CMV-Aro-3'UTR, and control plasmid named CMV-con-3'UTR.
2. After plasmid transfecting, the Aro-3'UTR expression was significantly higher (P<0.05) than miR-378treated group and conUTR group, thus achieved the purposes of over-expression Aro-3'UTR.
3. Transfected3'UTR plasmids after miR-378lentivirus transduction, results showed that over-expression of aromatase3'UTR reversed the effects of miR-378suppresses (P<0.05) aromatase protein expression and estradiol secretion.
4. Transfected the miR-378inhibitor to the GCs without the miR-378lentivirus transduction, results showed that miR-378inhibitor repressed (P<0.05) the expression of endogenetic miR-378in GCs.
5. Transfected miR-378inhibitor after miR-378lentivirus transduction, experimental results showed that miR-378inhibitors reversed the effects of miR-378suppresses (P<0.05) aromatase protein expression and estradiol secretion.
6. MiR-378inhibitor also reversed (P<0.05) the phenomenon of miR-378induced the aromatase3'UTR reduced luciferase activity.
In summary, nutrition affects the expression of miR-378in gilt ovarian. The expression of miR-378was significant higher in the lower nutrition group than the normal nutrition group. By targeting3'UTR+4062-+4082of PGR and3'UTR+1589-+1609and+1607-+1627of aromatase, miR-378regulate the PGR and aromatase protein expression at the post-transcriptional level, thus clarified the molecular mechanism of miR-378on the GCs' effects. Aromatase is a key enzyme for estradiol synthesis, aromatase protein levels directly affect the synthesis of estradiol, and the estradiol is an important indicator in reproduction, it indicates that nutrition influence the expression of miR-378in ovary is one way of nutritional regulation on reproduction.
引文
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36. Kim VN. MicroRNA biogenesis:coordinated cropping and dicing. Nat Rev Mol Cell Biol,2005,6:376-385.
37. Davis CD, Ross SA. Evidence for dietary regulation of microRNA expression in cancer cells. Nutr Rev,2008,66:477-482.
38. Du T, Zamore PD. microPrimer:the biogenesis and function of microRNA. Development,2005,132:4645-4652.
39. Rana TM. Illuminating the silence:understanding the structure and function of small RNAs. Nat Rev Mol Cell Biol,2007,8:23-36.
40. Filipowicz W, Bhattacharyya SN, Sonenberg N. Mechanisms of post-transcriptional regulation by microRNAs:are the answers in sight? Nat Rev Genet,2008,9:102-114.
41. Peters L, Meister G. Argonaute proteins:mediators of RNA silencing. Mol Cell, 2007,26:611-623.
42. Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell,1993,75: 843-854.
43. Olesen CE, Yan YX, Liu B, et al. Novel methods for chemiluminescent detection of reporter enzymes. Methods Enzymol,2000,326:175-202.
44. Liu ZF, Jiang Y. The theoretical system and application of reporter gene technology. Sheng Li Ke Xue Jin Zhan,2002,33:361-363.
45. Jiang T, Xing B, Rao J. Recent developments of biological reporter technology for detecting gene expression. Biotechnol Genet Eng Rev,2008,25:41-75.
46. Koo J, Kim Y, Kim J, et al. A GUS/Luciferase Fusion Reporter for Plant Gene Trapping and for Assay of Promoter Activity with Luciferin-Dependent Control of the Reporter Protein Stability. Plant and Cell Physiology,2007,48 (8):1121-1131.
47. Brennecke J, Stark A, Russell R B, et al. Principles of microRNA-target recognition. PLoS Biol,2005,3:e85.
48. Miranda KC, Huynh T, Tay Y, et al. A pattern-based method for the identification of microRNA binding sites and their corresponding heteroduplexes. Cell,2006, 126:1203-1217
49. Griffiths-Jones S, Bateman A, Marshall M, et al. Rfam:an RNA family database. Nucleic Acids Res,2003,31:439-441
50. Rigoutsos I, Floratos A. Combinatorial pattern discovery in biological sequences: The TEIRESIAS algorithm. Bioinformatics,1998,14:55-67
51. Choi Y, Qin Y, Berger MF, et al. Microarray analyses of newborn mouse ovaries lacking Nobox. Biol Reprod,2007,77:312-319.
52. Landgraf P, Rusu M, Sheridan R, et al. A mammalian microRNA expression atlas based on small RNA library sequencing. Cell,2007,129:1401-1414.
53. Ro S, Song R, Park C, et al. Cloning and expression profiling of small RNAs expressed in the mouse ovary. RNA,2007,13:2366-2380.
54. Zhao H, Rajkovic A. MicroRNAs and mammalian ovarian development. Semin Reprod Med,2008,26:461-468.
55. Pasquinelli AE, Reinhart BJ, Slack F, et al. Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature,2000,408: 86-89.
56. Park SM, Shell S, Radjabi AR, et al. Let-7 prevents early cancer progression by suppressing expression of the embryonic gene HMGA2. Cell Cycle,2007,6: 2585-2590.
57. Sampson VB, Rong NH, Han J, et al. MicroRNA let-7a down-regulates MYC and reverts MYC-induced growth in Burkitt lymphoma cells. Cancer Res,2007,67: 9762-9770.
58. Yu F, Yao H, Zhu P, et al. let-7 regulates self renewal and tumorigenicity of breast cancer cells. Cell,2007,131:1109-1123.
59. Hossain MM, Ghanem N, Hoelker M, et al. Identification and characterization of miRNAs expressed in the bovine ovary. BMC Genomics,2009,10:443.
60. Tang F, Kaneda M, O'Carroll D, et al. Maternal microRNAs are essential for mouse zygotic development. Genes Dev 2007,21:644-648.
61. He L, Thomson JM, Hemann MT, et al. A microRNA polycistron as a potential human oncogene. Nature,2005,435:828-833.
62. O'Donnell KA, Wentzel EA, Zeller KI, et al. c-Myc-regulated microRNAs modulate E2F1 expression. Nature,2005,435:839-843.
63. Murchison EP, Stein P, Xuan Z, et al. Critical roles for Dicer in the female germline. Genes Dev,2007,21:682-693.
64. Schier AF. The maternal-zygotic transition:death and birth of RNAs. Science, 2007,316:406-407.
65. Amanai M, Brahmajosyula M, Perry AC. A restricted role for sperm-borne microRNAs in mammalian fertilization. Biol Reprod,2006,75:877-884.
66. Watanabe T, Takeda A, Tsukiyama T, et al. Identification and characterization of two novel classes of small RNAs in the mouse germline:retrotransposon-derived siRNAs in oocytes and germline small RNAs in testes. Genes Dev,2006,20: 1732-1743.
67. Sirotkin AV, Ovcharenko D, Grossmann R, et al. Identification of microRNAs controlling human ovarian cell steroidogenesis via a genome-scale screen. J Cell Physiol,2009,219:415-420.
68. Ambros V. The functions of animal microRNAs. Nature,2004,431:350-355.
69. Li SC, Tang P, Lin WC. Intronic microRNA:discovery and biological implications. DNA Cell Biol,2007,26:195-207.
70. Lim LP, Lau NC, Garrett-Engele P, et al. Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature,2005,433: 769-773.
71. Pan Q, Luo X, Toloubeydokhti T, et al. The expression profile of micro-RNA in endometrium and endometriosis and the influence of ovarian steroids on their expression. Mol Hum Reprod,2007,13:797-806.
72. Carleton M, Cleary MA, Linsley PS. MicroRNAs and cell cycle regulation. Cell Cycle,2007,6:2127-2132.
73. Jovanovic M, Hengartner MO. miRNAs and apoptosis:RNAs to die for. Oncogene,2006,25:6176-6187.
74. Kuehbacher A, Urbich C, Dimmeler S. Targeting microRNA expression to regulate angiogenesis. Trends Pharmacol Sci,2008,29:12-15.
75. Kasashima K, Nakamura Y, Kozu T. Altered expression profiles of microRNAs during TPA-induced differentiation of HL-60 cells. Biochem Biophys Res Commun,2004,322:403-410.
76. Landgraf P, Rusu M, Sheridan R, et al. A mammalian microRNA expression atlas based on small RNA library sequencing. Cell,2007,129:1401-1414.
77. Cho IS, Kim J, Seo HY, et al. Cloning and characterization of microRNAs from porcine skeletal muscle and adipose tissue. Mol Biol Rep,2007,37:3567-3574.
78. Mathers JC, Strathdee G, Relton CL. Induction of epigenetic alterations by dietary and other environmental factors. Adv Genet,2010,71:3-39.
79. McKay JA, Mathers JC. Diet induced epigenetic changes and their implications for health. In:Acta Physiol (Oxf),2011,202:103-118.
80. Gaedicke S, Zhang X, Schmelzer C, et al. Vitamin E dependent microRNA regulation in rat liver. FEBS Lett,2008,582:3542-3546.
81. Kutay H, Bai S, Datta J, et al. Downregulation of miR-122 in the rodent and human hepatocellular carcinomas. J Cell Biochem,2006,99:671-678.
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