miR-302d Competitively Binding with the lncRNA-341 Targets TLE4 in the Process of SSC Generation

MicroRNAs (miRNAs) are essential factors in the reproductive process of poultry. Here, we found miR-302d is a potential differentiation and negative factor of chicken embryonic stem cells (ESCs) into spermatogonia stem cells (SSCs). The competition mechanism was carried out for the preliminary exploration to determine the relationship among miR-302d, lncRNA-341(interacting with miR-302d), and target gene TLE4. The results showed that lncRNA-341 can competitively bind to miR-302d to decrease the targeted binding of miR-302d and TLE4 which promotes the differentiation of chicken SSCs. Moreover, it is suggested that miR-302d may participate in the Wnt signaling pathway through TLE4.


Introduction
The reproductive performance of chicken production is affected by many factors, and genetic breeding is one of the most important factors. How to make use of genetic improvement to increase the production of chicken breeding has been the direction that many scholars have devoted to. At the same time, in the modern commercial production, artificial insemination (AI) has replaced natural and artificial auxiliary mating, greatly improving the production efficiency, to adapt to the development of modernization; thus, how to efficiently obtain the male germ cells of the cock has become one of the key techniques. miRNA constitutes of a single endogenous small RNA with a length of about 22 nt that is widely found in humans, animals, plants, and viruses. Mature miRNA does not participate in protein coding, but it is an important gene regulatory factor involved in numerous fields. miRNA generally achieves posttranscriptional regulation by degrading target mRNA or blocking the translation of target mRNA affecting development, such as in cardiovascular formation [1], neural development [2], stem cell differentiation [3], apoptosis [4], and tumor formation [5].
Many reports about miRNA in mammalian germ cells are playing a crucial role in gonadal sex determination, male reproductive stem cell meiosis, spermatogonial cell differentiation, and other aspects. Fernández analyzed the miRNA expression patterns of embryonic male and female mouse primordial germ cells (PGCs), as well as gonadal cells [6], and found that the differences in the expression regulation of miR-199-214, miR-182-183-96, miR-34c-5p, and other key miRNA clusters all have a clear role in gonadal sex determination. Liu found that miR-34c may regulate the meiosis of male reproductive stem cells of milk goats and inhibit their proliferation [7]. miRNA can also coordinate with related signaling pathways to regulate the production of germ cells. Hiromitsu analyzed RNA-seq data to identify specific miRNA (miR-741-3p, miR-871-3p, and miR-880-3p) to mouse germ cells that are contiguous and adjacent to each other on the X chromosome [8] termed XmiRs (X carelinked miRNA). MiR-871 and miR-880 work together with WNT/beta-catenin signaling pathways to regulate the occurrence of testicular germ cells. Fu et al. found that miR-31-5p regulates the proliferation [9], DNA synthesis, and apoptosis of human SSCs through pak1-jazf1-cyclin A2 pathway.
However, the specific mechanism of miRNA function in birds is not fully understood in how it regulates germ cell development; thus, there are still many key miRNAs to be discovered and explored in poultry germ cells. Therefore, there are still a lot of gaps in the research on miRNA regulating the differentiation of avian male germ cells.
In the preliminary work, our lab has established the system for inducing the differentiation of ESCs into SSCs in vitro by RA (retinoic acid) and has been working on ways to improve the induction efficiency of this process. Therefore, we need to explore the important regulatory factors in this process, RNA-seq was performed in previous work, and the differential miR-302d were screened out. In this study, the function of miR-302d and its target gene were verified in vivo and in vitro, and the effect on Wnt signaling pathway has also been preliminarily explored. The ceRNA competition mechanism was also preliminarily explored to determine the relationship among miRNA, lncRNA, and target gene interactions. This study fills in the gap of miRNA in regulating the differentiation of avian male germ cells and improves reproductive modelling efficiency.

Materials and Methods
2.1. Bioinformatics Analysis. The ESCs at specific days (day 0, day 4, and day 10) induced by RA were collected, and RNA sequencing was performed. According to the RNA-seq data, differential miRNAs were screened (|log 2:Fold change | >8 was the screening criteria). miR-302d, which plays an important role in germ cell differentiation, was identified as the research object. The target genes of gga-miR-302d (referred to as miR-302d) were analyzed and screened with online bioprediction software: http://www.targetscan.org/vert_71/ (Targetscan); http://www.mirdb.org/(miRDB); we performed a Venn analysis of the screening results from these two databases and identified four common target genes. lncRNA transcripts were screened by exon number screening, length screening, known annotation screening, expression volume screening, and encoding potential screening of CNCI (Coding-Non-Coding Index), CPC (Coding Potential Calculator), PFAM (pfamscan), etc., lncRNA and miR-302d interaction analysis online bioprediction software: http://www.mirbase .org/(miRBase).

Vector
Construction. miR-302d lentivirus expression vector and interference vector are provided by the Genomeditech Company (Shanghai, China). The expression vector was named miR-302d mimics, and the interference vector was named miRNA-inhibitor. Sequences were listed in supporting Table 1 and Table 2. The construction methods of TLE4 3′UTR wild-type and mutant pMIR-REPORT vector refer to the vector system protocol. pMIR-REPORT vector was purchased from Applied Biosystems (http://www .appliedbiosystems.com). Sequences were listed in supporting Table s8. 2.3. ESC Isolation and Induction Culture. ESCs were isolated from freshly fertilized eggs as previously described [10,11]. ESCs were induced by medium containing 10 -5 mol/L RA, and viruses were added with a titer of 5 × 10 8 TU/mL on D0 induction. Cell morphology was observed every 2 days, and cells were collected from day 0, day 4, and day 10 for RNA, and cells were collected from day 4 and day 10 for immunofluorescence (IF) and flow cytometry (FCM). 30 eggs per treatment in 3 separate repeats of the experiment.

Embryo Injection Experiment.
When the chicken embryos developed normally for 2.5 days, the blunt end was sterilized with 70% ethanol, and embryos were opened for microinjection into vessels. Each embryo was injected with 5 × 10 8 TU/mL virus diluent of 2 μL in total. After injection, 20 μL of 1% penicillin and streptomycin were dropped into the injection site, sealed with medical tape, and incubated under normal conditions. The embryonic genital ridges were collected at 4.5 days, and testes separated at 18.5 days after incubation for RNA, Periodic Acid-Schiff stain (PAS), FCM, Western blot and other tests.  2.10. Statistical Analyses. Differences between groups were examined for statistical significance using Student's t-test or one-way ANOVA. P value < 0.05 was regarded as significant, and P value < 0.01 was regarded as extremely significant.

Screening of miRNA-302d during RA-Induced ESC Differentiation into SSCs.
To explore the miRNA in RAinduced ESC differentiation into SSCs, ESCs, and SSCs, ESCs were treated with RA for 10 days. Cells were then processed for RNA sequencing. It was found that the miRNAs involving in the differentiation process were classified into four categories (Figures 1(a) and 1(b)). To identify specific groups involved in SSC differentiation, GO analysis was performed, and we found that the miRNA in the Cluster1 can be significantly enriched in the relevant GO items of germ cell development ( Figure 1(c)). Comparing with other groups, miRNA in the Cluster1 mainly involved in the formation of SSCs. To further clarify the functions of miRNA in various groups, we conducted KEGG pathway enrichment analysis. We found that the miRNA in the Cluster1 is significantly enriched in the SSC formation of related signaling pathways, such as Wnt signaling pathway [12], JAK-STAT signaling pathway [13], and MAPK signaling pathway [14] ( Figure 1(d)), showing that among the four miRNA clusters, miRNAs in the Cluster1 were more likely to participate in the SSC formation process. Meanwhile, we found these signaling pathways are regulated by miRNA302d target genes. And qRT-PCR showed that the expression of miRNA302d in ESCs was higher than that of SSCs ( Figure 1(e)), suggesting that miR-NA302d may play an important role in SSC formation.

miR-302d Inhibits the Differentiation of Chicken ESCs into SSCs.
To further determine the role of miRNA-302d in regulating ESC differentiation into SSCs, we also used RA induction system which could induce ESC differentiation into SSCs. We observed cell morphology every 2 days, and marker genes were detected during embryonic bodies (EBs) and SSC-like cell formation (Figure 2(a)). Since the EBs appear on day 4 and the SSC-like cell appears on day 10 in vitro, we choose these two timepoints. Briefly describe: we treated ESCs with miRNA-302d inhibitor or miRNA-302d mimics and measure maker gene expression. EBs in the inhibitor group were significantly more than other groups, but the mimics group showed the opposite results ( Figure 2(b)). The expression of reproductive marker genes was significantly increased in the cells treated by miRNA-302d inhibitor and decreased in the cells treated with miRNA-302d mimics ( Figure 2(c), Table s1). In IF, the expression of marker proteins in the mimics group decreased, while in the inhibitor group, it was increased ( Figure 3(a)). Flow cytometry analysis (FCM) showed that the positive cell rate in the mimics group was significantly lower than that in the control group, while the inhibitor group had no significant difference compared with the control group but had an upward trend ( Figure 3(b)). The above results indicated that miR-302d inhibits the differentiation of chicken ESCs into SSCs in vitro.
We then injected miRNA-302d inhibitor or miRNA-302d mimics viruses into 2.5-day-old chicken embryo vessels (Figure 4(a)). 4.5-day-old chicken embryos were harvested for PAS. Genital ridges were observed and developed normally. The number of PGCs increased in the inhibitor group    Cell morphology every two days, control: inducing by RA; miR-302d inhibitor: based on RA induction, miR-302d inhibitor lentivirus was infected on day 0; miR-302d mimics: based on RA induction, miR-302d mimics lentivirus was infected on day 0. (c) mRNA expression levels of marker genes were detected on day 4 and day 10; cvh and c-kit were used as PGC maker genes in chicken, integrin α6 and integrin β1 were SSC maker genes, and β-actin was the reference gene.

Stem Cells International
and decreased in the mimics group compared with the control group (Figure 4(b)). We harvested 4.5-day-old genital ridges and 18.5-day-old testes and examined the mRNA expression of reproduction-related genes cvh, c-kit, integrin α6, and integ-rinβ1. The miRNA-302d mimics significantly inhibited the expression of these marker genes (Figure 4(c), Table s2). Likewise, FCM showed that the number of marker-proteinpositive cells in the mimics group was significantly lower than that in the control group (Figure 4(d)). The above results indicated that miR-302d had an inhibitory effect on SSC differentiation in chicken.

miR-302d
Can Bind to the 3′UTR of Target Gene TLE4 and Regulate Its Expression. According to gga-miR-302d (MIMAT0003360) sequence, UAAGUGCUUCCAUGUU UUAGUU, we used multiple online biological software to analyze target genes and ranked them by scores ( Figure 5(a)). Among these, four common target genes were identified: MYT1L, ELAVL2, TLE4, and HLF (Table s3).
TLE4, which has the highest target score of 96, is a target gene that maintains stem cell pluripotency. We speculated that it may play an important role in the process of chicken SSC differentiation. Therefore, we carried out targeted verification on TLE4.
According to the expression profile results ( Figure 5(b)), miR-302d and TLE4 showed opposite expression trends in ESCs, PGCs, and SSCs. miR-302d had the highest expression in ESCs, but TLE4 expressed more in SSCs than ESCs and PGCs. In the dual-luciferase reporting assay ( Figure 5(c)). The results indicated that miR-302d inhibited the expression of TLE4 mRNA, but when the binding site of miR-302d and TLE4 was deleted, reducing the binding of miR-302d and TLE4 and making up for the inhibition of TLE4 mRNA expression. In conclusion, TLE4 is the target gene of miR-302d, and miR-302d suppresses its expression.      Stem Cells International in vivo. We treated ESCs with virus for overexpression and inhibition. We then observed cell morphology every two days, and marker genes were detected. EBs in overexpression (OE) group were significantly more than that of other groups (Figure 6(a)). The qRT-PCR results also showed that the expression of marker genes in the OE group was significantly higher than that in other groups, while the SH group was significantly downregulated (Figure 6(b), Table s4). In IF, the expression of marker proteins in SH group was decreased, while that in the OE group was increased (Figure 7(a)). FCM showed that the amount of CVh + and integrin + cells in the SH group was significantly lower than that in the control group and OE group, while the OE group was significantly higher than that in the control group on day 10 (Figure 7(b)). The above results indicated that TLE4 promotes the differentiation of chicken ESCs into SSCs in vitro.

TLE4
We harvested 4.5-day-old chicken embryos for PAS staining. At high magnification, the number of PGCs increased in the OE group and decreased in the SH group compared with the control group (Figure 8(a)). We detected the mRNA expression of reproduction-related genes, and the results showed that the SH group significantly inhibited the expression of marker genes, while the OE group promoted the expression (Figure 8(b)), and the qRT-PCR data is showed in Table s5. Likewise, FCM showed that the rate of marker-protein-positive cells in the SH group was significantly lower than that in the OE group (Figure 8(c)). The above results indicated that TLE4 had a positive effect on SSC differentiation in chicken.

miR-302d Can Affect the Expression of Node Genes
Directly or Indirectly Interacting with TLE4 on the Wnt Signaling Pathway. It has been shown that TLE4 plays an important role in the signaling pathway related to germ cell differentiation [15]. Proteins expressed in certain nodes of the Wnt pathway have been shown in mice to bind directly or indirectly to the TLE4 protein [15][16][17]. To explore whether miR-302d can target TLE4 and affect the expression  TTTTTTTTTC  TTTTTACCTT  ACTGTCAGAT  TTTAATTATG  GAACTGACTA  GTGTTTTTGC  GCTACACTAA   CCCCTTTTCT  CTTAAATTGC  AGCTATTTGG  TATTATAGCT  AATAAAGCTG  TAGTGGATAA  ATAGGCTTGT   AAACTAAGAA  TATTGATTTG  GAGAAGATTG  GTAATGTATT  TCTGGCCCGG  ATTGGGGATT  AGTTTCTATT   CTTAAATGCT  TGAATGCTCA  AAAAAGCATA  TATTTTGTGT  CTTTAGTTTA  TTGGAGAAGG  TAAAAAAAA   GATCGCAGTT  TTAAGAACTT  CTGAACAGTG  AAAGAAGGCT  AAAGGTGCAT  ATGTTCAAGA   GTGGAATTTG  GTGATACCAA  AACTTGACTC  TTCTAACAAT  TGAATACTTT   (c) Dual-luciferase reporting assay; G1: pmiR-tle4-wt group (use TLE4 3′UTR wild-type pmiR-REPORT vector to transfect DF-1 cells), G2: pmiR-tle4-wt+miR-302d mimics group (use TLE4 3′UTR wild-type pmiR-REPORT vector and miR-302d mimics to transfect DF-1 cells), G3: pmiR-tle4-mt+miR-302d mimics group (use TLE4 3′UTR mutant-type pmiR-REPORT vector and miR-302d mimics to transfect DF-1 cells, TLE4 3′UTR mutant-type vector lacks binding site: AGCACTT). 8 Stem Cells International of node genes in the signaling pathway, related node genes in Wnt signaling were selected for analysis, including Tcf, Lef, β-catenin, Axin1, and Apc. 4-day and 10-day cells were harvested and analyzed for mRNA and protein expression involving miR-302d functional verification in vitro. On day 4, the expression of Lef and β-catenin was significantly upregulated in miR-302d mimics group compared with the control group, Apc was significantly downregulated in mimics group compared with the control group, and Tcf and Axin1 had no significant difference (Figure 9(a); Table s6). Western blot showed that the expression of β-catenin in the mimics group was more than the control group (Figure 9(e)). On day 10, Tcf, Lef, and βcatenin were significantly upregulated in the inhibitor group compared with the control group, but Axin1 and Apc had no significant difference (Figure 9(b); Table s6). Western blot test showed that the expression of β-catenin in the mimics group to be far less than the control (Figure 9(f)).
In vivo, embryonic genital ridges were harvested for qRT-PCR test on day 4.5, Tcf, Lef, β-catenin, and Axin1 were significantly downregulated in the inhibitor group compared with the control group, but Apc had no significant difference (Figure 9(c); Table s7). Western blot analysis showed that β-catenin expression in the inhibitor group was less than the control group (Figure 9(e)). Testes were harvested for qRT-PCR test on day 18.5; Tcf, Lef, and β-catenin were significantly upregulated in the inhibitor group compared with the control group, but Axin1 and Apc had no significant difference (Figure 9(d); Table s7). Western blot analysis showed the expression of β-catenin in the mimics group was less than the control (Figure 9(f)). As a result, miR-302d can affect the expression of node genes directly or indirectly interacting with TLE4 on the Wnt signaling pathway, the overexpression of miR-302d made the expression of Lef, β-catenin, Axin1, and Tcf fluctuate during the differentiation of ESCs into SSCs.  Stem Cells International 3.6. lncRNA-341 Competes with miR-302d to Bind the Target Gene TLE4. After further RNA-seq database analysis, it was found that a total of 269 lncRNAs were correlated with the downregulation of miR-302d expression ( Figure S1A). We predicted and individually analyzed these 269 lncRNAs and found that two lncRNAs were interacting with miR-302d: lncRNA-341 and lncRNA-1784 ( Figure S1B). We verified these two lncRNAs and found no difference in the expression trend of lncRNA-1784 between ESCs, PGCs, and SSCs. The downregulation trend of lncRNA-341 expression between ESCs, PGCs, and SSCs was consistent with the sequencing results, indicating that the regulation mechanism of lncRNA-1784 expression may not be consistent in experiments in vivo and in vitro. Therefore, we chose lncRNA-341 for further investigation.
lncRNA -341 was significantly downregulated between ESCs, PGCs, and SSCs, which was consistent with the trends by time in vitro (Figure 9(g)). To further elucidate if lncRNA-341 could affect the targeted regulation of TLE4 by miR-302d, we conducted an lncRNA-341 expression vector cotransfected, respectively, with miR-302d mimics and TLE4 double-luciferase reporter vector systems. In double luciferase report analysis, two groups were set up. The 3′ UTR -wt-miR-lnc341 group was significantly upregulated compared to the 3′UTR-wt-miR group (Figure 9(h)). Combined with the previous miR-302d binding of target gene 3′ UTR experiment analysis, it showed that lncRNA-341 could bind miR-302d and block its targeted binding to TLE4, thus upregulating TLE4 expression.

Discussion
Over the past few decades, chicken has been widely used as a model animal in both commercial breeding and scientific research, contributing to the development of the global   11 Stem Cells International poultry industry and providing a wealth of information for basic research. Improving the quality and productivity of male germ cells could bring huge benefits to commercial production and could be widely applied to other species, including humans. Although many studies have shown that RA can induce different stem cell donors to differentiate into SSCs in vitro [18,19], the efficiency is not stable due to differences in the culture and induction system. The regulation mechanism of male germ cell production is complex, rendering it a challenge to harvest induced male germ cells with high purity and quality continuously in vitro, which makes it difficult to be applied in actual production. In our previous studies, we found many key genes, which may be regulated by some noncoding RNAs, such as lncRNA and miRNA. Therefore, it is necessary to explore these factors, to delineate the mechanisms and potentially improve induction efficiency.