Application of miR-148a-3p in regulating reproductive traits

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to the application of miR-148a-3p in regulating reproductive traits. The present invention provides the application of miR-148a-3p in regulating reproductive traits, wherein the miR-148a-3p includes pig miR-148a-3p, and the sequence of the pig miR-148a-3p is 5'-UCAGUGCACUACAGAACUUUGU-3'. The present invention provides a new gene that can be used to regulate reproductive traits, provides a new means for effectively regulating reproductive traits, and has good industrial application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to the application of miR-148a-3p in regulating reproductive traits. Background Art

[0002] Reproductive traits are one of the important economic traits of pig breeding. Breeding sows with high reproductive capacity is of great significance for promoting the healthy development of the pig industry and innovation in the seed industry. Reproductive traits are low-heritability traits, and the traditional phenotypic selection genetic progress is slow. At present, researchers have identified several major genes that affect pig fertility, such as prolactin receptor (PRLR), estrogen receptor (ESR), follicle-stimulating hormone-β (FSHβ) and retinol-binding protein 4 (RBP4). The molecular mechanism of these major genes regulating reproductive traits has not yet been elucidated. With the in-depth study of non-coding RNA (ncRNA), studying the molecular regulatory mechanism of sow reproductive traits from the perspective of the interaction between ncRNA and mRNA molecules, thereby obtaining more reliable molecular markers, has become one of the effective means to improve breeding efficiency. However, the current research on genes that can be used to regulate reproductive traits is still insufficient. How to develop more relevant genes to regulate relevant reproductive traits as needed is an urgent need for research. Summary of the invention

[0003] In view of the defects of the prior art, the object of the present invention is to provide the application of miR-148a-3p in regulating reproductive traits.

[0004] As shown in the embodiments of the present invention, the inventors found that the mature sequence of miR-148a-3p was highly conservative in multiple species when analyzing the conservation of miR-148a-3p, which indicates that the probability of mutation of the mature sequence is low during the evolution process, and the early transcriptome sequencing analysis found that miR-148a-3p was highly expressed in ovarian tissue, and it was speculated that it may have a biological function of regulating reproductive traits. Functional enrichment analysis was performed on the target genes of miR-148a-3p predicted by different databases, and GO annotation analysis showed that the target genes were mainly distributed in items such as positive regulation of transferase activity, phosphorylation, and enzyme-linked receptor protein signaling pathways. Transferase has the function of catalyzing the transfer of chemical groups or free radicals from one molecule to another, which is crucial for embryonic development. Insufficient transferase activity can lead to abnormal pregnancy, embryonic development, and growth restriction; dormant and growing oocytes are inhibited for a long time in the early stage of meiosis I, and phosphorylation-mediated inhibition of Cdk1 activity is one of the key factors in maintaining long-term stagnation of oocytes, which is crucial for maintaining female reproductive function; enzyme-linked receptor protein signaling pathways are involved in regulating oocyte maturation and development. KEGG enrichment showed that the target genes were significantly enriched in pathways related to reproduction, such as PI3K-Akt, FoxO, TGF-β, HIF-1 and MAPK. The PI3K-Akt signaling pathway is related to ovarian function and can regulate follicle formation, oocyte development and granulosa cell function; the PI3K-Akt-FoxO signaling pathway is a central pathway that controls the growth and metabolism of all cells and is also involved in regulating the development of primordial follicles; researchers in this field have found that the HIF-1α / VEGF signaling pathway is one of the important mechanisms for regulating ovarian corpus luteum development in mammals; TGF-β and MAPK signaling pathways are both involved in regulating follicle development and are the main signaling pathways affecting ovarian function. These GO annotations and pathways further suggest that miR-148a-3p plays an important role in regulating reproductive function. At present, the research on miR-148a-3p in the ovary is focused on ovarian cancer, and there is little research on the regulatory mechanism of ovarian function in livestock and poultry. Researchers in this field have found that when miRNA sequencing was performed on pig oocytes of large and small follicles, the top ten miRNAs expressed in large and small follicles included miR-148a-3p; and miR-205, miR-16 and miR-148a-3p together accounted for 51.8% and 56.5% of the miRNA readings in the large follicle group and the small follicle group, respectively. This is consistent with the inventor's previous research, and miR-148a-3p is abundantly expressed in follicles. The present invention found that miR-148a-3p is expressed in all tissues, and the highest expression level is in the ovary.

[0005] PRLR is a member of the cytokinin receptor superfamily and is expressed in various tissues of mammals, such as the corpus luteum, ovary, testis, and liver. PRLR is one of the major genes that affect the reproductive performance of sows. A large number of studies have confirmed that the polymorphism of PRLR is closely related to the number of litters and the number of live piglets, and it is used as a candidate gene to improve litter performance. PRLR plays an important role in regulating reproductive function. PRLR knockout can lead to loss of corpus luteum function, further causing a decrease in ovulation, oocyte deformity, and failure of fertilized egg implantation. Researchers in this field have found that the expression of PRLR in various tissues of small-tailed Han sheep during the follicular phase is significantly lower than that during the luteal phase, suggesting that the gene may be related to the conversion of the reproductive cycle; it is also found that PRLR has the highest expression level in the matrix and wall cells of follicles with a diameter of less than 2 mm, and the expression level gradually decreases with the maturation of the follicles, indicating that the high expression of PRLR may have an inhibitory effect on reproductive activity. PRLR has expression differences in the in vitro culture of granulosa cells of goose follicles of different sizes, indicating that PRLR is involved in the regulation of granulosa cell function during follicular development. The research on PRLR and miRNA mainly focuses on mammary gland development and milk secretion. miR-142-3p regulates milk synthesis and mouse mammary gland structure through multiple signaling pathways mediated by PRLR; miR-135a was overexpressed and silenced in cultured primary mammary epithelial cells, and PRLR was verified as a direct target gene of miR-135a by dual luciferase reporter assay, indicating that miR-135a / PRLR is involved in regulating animal mammary gland development and lactation function. circ-CYP24A1 regulates cell proliferation and apoptosis by binding to and inhibiting miR-224, resulting in increased PRLR expression, thereby regulating recurrent miscarriage. However, the molecular mechanism of PRLR and miRNA regulating ovarian function has not been reported.

[0006] Most miRNAs regulate the biological processes of the body by binding to target genes. For example, miR-148a-3p specifically binds to PPARγ in goose ovarian granulosa cells, thereby affecting progesterone synthesis and reducing steroid hormone secretion in granulosa cells. In pig granulosa cells, miR-574 targets tissue inhibitor of metalloproteinase 3 (TIMP3) and inhibits gene expression, resulting in reduced ERK1 / 2 phosphorylation, thereby promoting estradiol production. miR-146b targets cytochrome P450 aromatase (CYP19A1), a key enzyme in estradiol synthesis signaling, to inhibit estradiol secretion and induce apoptosis in pig granulosa cells. The present invention found that there is a binding site between miR148a-3p and PRLR, and the stability of the binding site between miR-148a-3p and PRLR CDS region is higher than that of the binding site between miR-148a-3p and PRLR 3'UTR region. Although the negative correlation between the expression levels of miR-148a-3p and PRLR in the luteal phase was not significant, there was a negative correlation trend. It was speculated that miR-148a-3p may regulate the reproductive traits of sows by targeting PRLR. Based on this, PRLR was studied in depth as the most likely target gene of miR-148a-3p. This study found that the fluorescence activity of co-transfected miR-148a-3p mimics and PRLR wild type was significantly reduced, which preliminarily revealed that miR-148a-3p and PRLR have a targeting relationship, laying the foundation for further in-depth exploration of the biological function of miR-148a-3p and the regulatory mechanism of the target gene PRLR. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 is the vector map;

[0008] Figure 2 is the phylogenetic tree of miR-148a-3p;

[0009] Figure 3 Analysis of miR-148a-3p target genes by three prediction softwares;

[0010] Figure 4 GO annotation for potential target genes of miR-148a-3p;

[0011] Figure 5 KEGG pathway enrichment analysis for miR-148a-3p target gene set;

[0012] Figure 6 Prediction for the binding of miR-148a-3p to PRLR;

[0013] Figure 7Analysis of the expression of miR-148a-3p in major pig tissues;

[0014] Figure 8 Verification of miR-148a-3p target genes. DETAILED DESCRIPTION

[0015] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by technical personnel in this field based on the above invention content still fall within the scope of protection of the present invention.

[0016] Example 1

[0017] 1.1 Experimental Materials Three two-month-old Köhler pigs raised under the same conditions were selected, and brain, heart, liver, spleen, lung, kidney, ileum, longissimus dorsi and ovarian tissues were collected; luteal phase ovaries were collected from sows on the 14th day after estrus, and follicular phase ovaries were collected from sows on the 20th day after estrus, washed with physiological saline, and quickly frozen in liquid nitrogen, and then stored in a -80℃ refrigerator for RNA sample extraction.

[0018] 1.2 Main reagents and instruments Trizol, PrimeScript RT reagent Kit with gDNA Eraser reagent, TB Green Premix Ex TaqⅡ reagent and Mir-X miRNA First-Strand Synthesis Kit were purchased from TaKaRa (Dalian); Hela cells were purchased from the Stem Cell Bank of the Chinese Academy of Sciences (Shanghai); fetal bovine serum and DMEM / F12 culture medium were purchased from Gibco (USA); Lipofectamine TM 3000 transfection reagent was purchased from Thermo Fisher (USA); ssc-miR-148a-3pmimics and mimics NC were purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd.; dual-luciferase reporter gene detection system kit was purchased from Promega (USA); NanoDrop ONE / C ultra-micro spectrophotometer (Thermo, USA), fluorescence quantification instrument (Eppendorf, Germany), microplate reader (Thermo, USA), Olympus IX53 fluorescence microscope (Olympus, Japan) were used.

[0019] 1.3 Analysis of miR-148a-3p sequences in different species The mature sequence of porcine miR-148a-3p was obtained from the miRBase database (http: / / www.mirb ase.org / ) and compared with the miR-148a-3p sequences of 13 species, including human (Homo sapiens), mouse (Mus musculus), macaque (Macaca mulatta), rat (Rattus norvegicus), and goat (Capra hircus). A phylogenetic tree was constructed using Mega7 software to analyze the relationship between species.

[0020] 1.4 Prediction of miR-148a-3p target genes and functional enrichment analysis The target genes of miR-148a-3p were predicted using miRWalk (http: / / mirwalk.umm.uniheidelberg.de / ), miRDB (http: / / mirdb.org / ) and TargetScan (https: / / www.targetscan.org / ). The intersection of the prediction results of the three databases was drawn using Draw VeenDiagram (http: / / bioinformatics.psb.ugent.be / webtools / V enn / ), and then the intersection was taken with the experimentally verified target genes in miRTarbase (http: / / mirtarbase.mbc.nctu.edu.tw / php / index.php). The merged target genes were used for the next step of analysis. Metascape (https: / / metascape. scape.org / ) performed GO annotation classification and KEGG signaling pathway enrichment analysis on the target gene set predicted by miR-148a-3p, and used P < 0.05 as the significance threshold to obtain the GO entries and KEGG signaling pathways with statistical significance compared with the background genes in the gene set.

[0021] 1.5 Structural analysis of miR-148a-3p and target gene PRLR The binding sites of miR-148a-3p and porcine PRLR mRNA sequence were analyzed by RNAhybrid (http: / / bibiserv.techfak.unibielefeld.de / rnahybri d / submission.html), and the secondary structure was predicted. At the same time, the conservation of the target gene binding sites among different species was analyzed.

[0022] 1.6 Total RNA extraction and reverse transcription TRIzol method was used to extract total RNA from each tissue, and the RNA concentration and purity were detected by spectrophotometer. Gel electrophoresis was used to detect RNA integrity. mRNA reverse transcription was performed according to the instructions of PrimeScript RT reagent Kit with gDNA Eraser Kit, and the total reaction system was 20μL: the first step reaction was 8X gDNA Eraser Premix 2μL, RNA sample 1μL, RNase Free H2O 13μL, and the reaction procedure was 42℃2min, 4℃; the second step reaction: the previous step reaction mixture 16μL, 5X RT Premix 4μL, the reaction procedure was 37℃10min, 85℃5s, 4℃; after the reaction, 80μLddH2O was added to the final volume of 100μL, and stored in a -20℃ refrigerator for later use. According to the instruction manual of Mir-X miRNA First-Strand Synthesis Kit, the miRNA was reverse transcribed. The total reaction system was 10 μL: mRQ Buffer (2x) 5 μL, RNA sample 3.75 μL, mRQ Enzyme 1.25 μL, and the reaction procedure was incubation at 37°C for 1 h and 85°C for 5 s. After the reaction, 90 μL ddH2O was added to the final volume of 100 μL and stored in a -20°C refrigerator for later use.

[0023] 1.7 Real-time fluorescence quantitative PCR analysis Primer Premier 3.0 software was used to design PCR quantitative primers for related genes according to the basic principles of primer design, and primer specificity was verified by Primer-BLAST in NCBI. The primers (Table 1) were synthesized by Chongqing Qingke Biotechnology Co., Ltd. 20μL reaction system: cDNA 1μL, upstream and downstream primers 1μL each, TBGreen Premix Ex TaqⅡ10μL, ddH2O 7μL. Reaction procedure: 95℃ pre-denaturation for 30s, 1 cycle; 95℃5s, 60℃30s, 40 cycles. The experimental data of genes were obtained using 2 –ΔΔCt Perform relative quantitative analysis.

[0024] Table 1

[0025]

[0026] 1.8 Mimics synthesis and vector construction ssc-miR-148a-3p mimics and mimics NC (negative control) were synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd. The PRLR gene sequence containing the target site was sent to Chongqing Qingke Biotechnology Co., Ltd. for synthesis and introduced into the plasmid to construct the psiCHECK2-PRLR-CDS wild-type vector (WT) and psiCHECK2-PRLR-CDS mutant vector (MUT). The vector construction diagram is shown in the figure. Figure 1 As shown, it was constructed by Chongqing Qingke Biotechnology Co., Ltd.

[0027] 1.9 Cell culture and transfection Hela cell lines were inoculated into 96-well cell culture plates containing complete culture medium (DMEM + 10% FBS + 1% double antibody) and cultured in a cell culture incubator containing 25% CO2 and 37°C. When the cell density reached 70% to 80%, transfection was performed and divided into 4 groups: Group 1: PRLR–CDS wild type (WT) + ssc-miR-148a-3p mimics NC; Group 2: PRLR–CDS wild type (WT) + ssc-miR-148a-3p mimics; Group 3: PRLR–CDS mutant (MUT) + ssc-miR-148a-3p mimics NC; Group 4: PRLR–CDS mutant (MUT) + ssc-miR-148a-3p mimics, with 3 replicates in each group. After transfection, the cells were placed in a cell culture incubator, the culture medium was replaced after 6 hours, and the cells were cultured for 24 hours.

[0028] 1.10 Dual luciferase activity detection: 24 hours after transfection in 1.7, place the cells in a clean bench, wash with PBS three times, remove floating dead cells, add 20 μL PLB lysis buffer to each well, incubate at room temperature for 15 min, blow the cells to make a cell suspension, centrifuge and take 20 μL supernatant into a 96-well white plate, add 100 μL firefly fluorescence detection solution in a dark place, use a microplate reader to select chemiluminescence to detect firefly luciferase activity, and after the detection, add 100 μL Renilla fluorescence detection solution to detect Renilla luciferase activity.

[0029] 1.11 Data Analysis Data results are expressed as Mean ± SEM, and the t-test was used for analysis of significant differences. GraphPad Prism 9.5.1 was used for drawing.

[0030] 2 Results

[0031] 2.1 Analysis of miR-148a-3p sequence conservation The mature sequence of pig miR-148a-3p was compared with that of 13 species including humans, mice, and rats. It was found that the mature sequence of miR-148a-3p in most species was 22 nt in length, and the sequences at positions 1 to 21 were completely identical. In addition, the bases of the seed sequences at positions 2 to 8 of the 13 species were completely identical (Table 2). Figure 2 ) The results showed that porcine miR-148a-3p was closely related to humans, mice and macaques, and was most distantly related to Cichlid berghei, and its mature sequence was highly conserved among species.

[0032] Table 2

[0033]

[0034]

[0035] 2.2 Prediction and analysis of miR-148a-3p target genes The target genes of porcine miR-148a-3p were predicted using miRWalk, miRDB and TargetScan software, and 5936, 839 and 802 target genes were predicted respectively. The intersection of the three obtained a total of 270 target genes. The 199 experimentally verified target genes in the miRTargetbase database were merged, and 36 duplicate target genes were removed, resulting in a total of 433 target genes ( Figure 3 ; Table 3).

[0036] Table 3

[0037]

[0038] Note: W∩D∩T represents the intersection of the prediction results of three target gene prediction softwares: miRWalk, miRDB and TargetScan. miRTarbase∪(W∩D∩T) means that the intersection of the verified targets of miRTarbase and the three prediction softwares is taken as the target gene set for subsequent analysis.

[0039] 2.3GO annotation classification and KEGG enrichment analysis GO analysis was performed on 433 target genes, and a total of 227 GO entries were obtained, of which 172 GO entries belonged to biological processes, mainly positive regulation of transferase activity, phosphorylation, enzyme-linked receptor protein signaling pathways, etc. (P < 0.01); 31 GO entries belonged to cellular components, mainly cytoplasmic ribonucleoprotein particles, cytoplasmic perinuclear region, Golgi structure, etc. (P < 0.01); 24 GO entries belonged to molecular functions, mainly protein kinase activity, transcription factor binding, regulatory RNA binding, etc. (P < 0.01) ( Figure 4; Table 4). KEGG signal pathway enrichment analysis was performed on the target genes, and the results showed that ( Figure 5 ), the target genes of miR-148a-3p were significantly enriched in PI3K-Akt, Erb, FoxO, TGF-β, HIF-1 and MAPK signaling pathways (P<0.01), and the above pathways play an important regulatory role in ovarian function, follicle development and other processes.

[0040] Table 4

[0041]

[0042]

[0043] 2.4 Structural analysis of miR-148a-3p and PRLR RNA hybrid was used to predict the complementary pairing of the target gene PRLR and miR-148a-3p sequences. The results showed that miR-148a-3p specifically bound to the PRLR CDS region sequence with a free energy of -22.1 kcal / mol ( Figure 6 The free energy of the binding site between miR-148a-3p and PRLR 3'UTR region is -14.9 kcal / mol ( Figure 6 The free energy of the RNA double strand formed by miR-148a-3p and PRLR CDS region is lower, so this binding site was selected for subsequent experiments. In addition, the target gene binding sequences of different species were predicted, and the results showed that the binding sites were somewhat conserved in different species ( Figure 6 (Part C).

[0044] 2.5 Expression of miR-148a-3p and PRLR in tissues The expression of miR-148a-3p and PRLR in different tissues of Keller pigs was detected by RT-qPCR. Figure 7 As shown in Part A, the expression level of miR-148a-3p in the ovary was the highest and significantly higher than that in other tissues (P<0.01). Figure 7 As shown in part B, PRLR was expressed at the lowest level in the longissimus dorsi muscle and at the highest level in the kidney, followed by the lung. In the ovarian tissue during the luteal phase, the expression levels of miR-148a-3p and PRLR were negatively correlated (P < 0.05) ( Figure 7 (Part C).

[0045] 2.6 miR-148a-3p targets PRLR gene To verify the relationship between miR-148a-3p and the target gene PRLR, wild-type and mutant dual luciferase reporter vectors with PRLR and miR-148a-3p binding sites were constructed. Figure 8The results showed that overexpression of miR-148a-3p could significantly inhibit the luciferase activity of the PRLR wild-type vector (P<0.05), while the luciferase activity of the PRLR mutant vector was not affected by miR-148a-3p (P 0.05) ( Figure 8 These results indicate that PRLR is a direct target gene of miR-148a-3p.

Claims

1. Application of miR-148a-3p in regulating reproductive traits, characterized in that: The miR-148a-3p includes porcine miR-148a-3p, and the sequence of porcine miR-148a-3p is 5'-UCAGUGCACUACAGAACUUUGU-3'.

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