Effects Of Nutrition Level On The Expression Of MicroRNA-378in The Gilts’ Ovary And The Mechanism Investigation Of MicroRNA-378’s Physiological Functions

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 ovaryThrough 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’UTRBioinformatics 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 cellsEstradiol 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.