A Regulatory Circuit Orchestrated by Novel-miR-3880 Modulates Mammary Gland Development

Milk casein and triglyceride content are important production traits in goats. Studies on mechanisms in milk casein secretion and mammary gland development is essential for milk goat breeding. miRNAs play an important role in goat lactation. While novel-miR-3880 is highly expressed at goat peak lactation stage, its molecular mechanism has not been studied. The purpose of the present study was to explore the relationship between novel-miR-3880 and lactation, as well as to construct a network among novel-miR-3880, ciRNA13761, and E74 like ETS transcription factor 2 (ELF2), thus further exploring their potential roles in milk components and mammary gland development. ELF2 was previously proven to be important in cell survival and proliferation, and 3′-UTR of ELF2 was predicted to have binding sites of novel-miR-3880. Our study found that the overexpression of novel-miR-3880 exerted anti-apoptotic and proliferative roles in GMEC, induced a boost in triglyceride synthesis, and caused a decrease in α s1-, α s2-, and β-casein, but an increase in κ-casein secretion. Furthermore, treatment in mice indicated that novel-miR-3880 could promote mammary gland development and extend the lactation period, while novel-miR-3880 expression was found to be suppressed by ciRNA13761 as a miRNA sponge. The present study explores a mechanism of triglyceride synthesis and casein secretion, and reveals a crosstalk between ciRNA13761/novel-miR-3880/ELF2 axis and PI3K/AKT/mTOR/S6K1 pathway, to gain a better understanding of lactation traits in dairy goats.


INTRODUCTION
Goat's milk is regarded as a hypoallergenic milk type with therapeutic functions and is accepted as a major functional food (Roncada et al., 2002) when casein, including κ-, β-, αs1,and αs2-casein, is the main protein in goat's milk (Singh and Singh, 1980;Chatchatee et al., 2001). MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs of approximately 23 nt in length. They are reported to regulate gene expression by repressing translation or facilitating mRNA degradation (Ambros, 2004) and play an important role in goat lactation (Ji et al., 2017). Novel-miR-3880 was highly expressed during goat peak lactation period based on our previous sequencing data . The novel-miR-3880 expression data was selected from Supplementary File S1 . Our study explored effects of novel-miR-3880-related molecules on mammary gland development in vivo and in vitro, thus providing a theoretical molecular basis for breeding and breast care.
Our study found that the overexpression of novel-miR-3880 induced anti-apoptotic and proliferative effects, promoted triglyceride synthesis, and caused a decrease in α s1-, α s2-, and β-casein but an increase in κ-casein secretion. Further, the regulation pattern was studied. Firstly, negative control (NC) and novel-miR-3880 were transfected into goat mammary epithelial cell (MEC) and RT-qPCR was applied to detect the expression of miRNAs, thus ensuring the efficiency of RNA sequencing. Differentially expressed genes (DEGs) of novel-miR-3880 were screened, and 67 downstream genes were found. E74 like ETS transcription factor 2 (ELF2), which is proven to be important in cell survival and proliferation (Qiu et al., 2008), might be essential for mammary gland development and lactation, and was found in the downregulated DEG list of sequencing results.
CircRNAs are another class of endogenous non-coding RNAs from non-classical alternative splicing that can expropriate miRNAs as a sponge to block miRNAs from binding to target genes (Hansen et al., 2013;Vicens and Westhof, 2014;Meng et al., 2017). To explore how novel-miR-3880 expression was regulated, circRNAs that have binding sites of novel-miR-3880 were screened according to circRNA sequencing data our laboratory acquired before. CircRNA13761 (ciRNA13761), whose structure is shown in Supplementary File S2, was selected and verified as a novel-miR-3880 sponge, while its source gene DOCK1 is reported to be involved in mammary gland involution (Bagci et al., 2014). Then, the network among ciRNA13761, novel-miR-3880, and ELF2 was constructed, and the role of DOCK1 in the network was detected.
As is known, mTOR is a central modulator in protein/lipid synthesis and cell growth processes and plays important roles in milk production (Osorio et al., 2016;Saxton and Sabatini, 2017). It serves as a crucial downstream signal of PI3K/AKT pathway to form a functional compound (Yang et al., 2014), which participates in lactation initiation (Chen et al., 2012). S6K1, a downstream effector of mTOR, is also critical for promoting protein and lipid synthesis (Yang et al., 2014); its activation relies on phosphorylation mediated by mTOR (Magnuson et al., 2012). Our study explored whether and how novel-miR-3880 regulates PI3K/AKT/mTOR/S6K1 pathway and participates in MEC biological processes and mammary gland development. In addition, MEC anti-apoptosis signaling was evaluated by the protein expression ratio of Bcl-2 and Bax, which is regarded as a cell survival signal (Basu and Haldar, 1998).
In this study, roles of ciRNA13761, novel-miR-3880, and ELF2 on mammary gland development and lactation traits were studied to provide a basis for molecular breeding of dairy goats. More precisely, the interaction among ciRNA13761, novel-miR-3880, and ELF2, and their effects on MEC triglyceride synthesis, lipid formation, casein secretion, viability, proliferation, and apoptosis were explored, as well as their participation in PI3K/AKT/mTOR/S6K1 pathway and Bcl-2/Bax pathway. Novel-miR-3880 and siELF2 were injected into C57BL/6 mice through the tail vein to examine the participation of PI3K/AKT/mTOR/S6K1 pathway in vivo and to observe the development of mammary glands affected by novel-miR-3880 and siELF2 with ultramicroscopic technique, to judge the availability of the molecular experiments and provide a theoretical basis for practice in dairy goat breeding and breast care.

Animals and Ethics
Three-year old female Guanzhong dairy goats 90-day postpartum (peak lactation period) in a research-animal-keeping farm near Northwest A&F University of Shaanxi province in China were selected and anesthetized. Then, one cubic centimeter of mammary gland tissue was removed to PBS with penicillin/streptomycin (100 U/mL, Harbin Pharmaceutical Group, China) from the middle part of the mammary gland with a scalpel. The mammary gland tissue was used to isolate MECs. The wound was sewn and sterilized immediately and animals recovered after the surgical line was removed a week later. C57BL/6 mice used in this study were of a similar age, weight, parity, and litter size, and delivered newborns on the same day. The mice were raised in an SPF environment with natural drink and food in separate nests. Each group had six nests of mice. Injective novel-miR-3880 agomir and siELF2 with 2 OMe and 5 Chol in vivo modification were bought from Ribobio (Guangzhou, China). All surgical procedures conformed to institutional and national guidelines and were approved by the Animal Care and Use Committee of the Northwest A&F University (China).

RNA-Sequencing
Goat MEC RNA quality was analyzed with an Agilent bioanalyzer 2100, and mRNA was captured using the Poly (A) mRNA Magnetic Isolation Module (NEBNext, United States). The library was constructed using an Ultra RNA Library Prep Kit for Illumina (NEBNext, United States). After the library was purified by Agencourt AMPure XP beads (Beckman, United States), the quality of the library was detected again using an Agilent Bioanalyzer 2100 and Qubit. In addition, before Illumina HiSeq, cBOT automatic clustering was conducted using the TruSeq PE Cluster Kit v4.

Dual-Luciferase Reporter Assays
Luciferase activities were measured in accordance with the protocol of Dual-Luciferase Reporter Assay System (Promega, Madison, WI, United States). The ratio of hRluc and hluc + activities was used to calculate the relative luciferase activity.

Immunohistochemistry
Slides cut from paraffin-embedded tissues underwent drying, rehydration, antigen retrieval, and permeation before the samples were blocked in goat serum for 20 min and incubated in the primary antibody then in the second antibody. Color development was performed with a DAB Substrate kit (Solarbio, DA1010, Beijing, China) and counterstained with Hematoxylin (Solarbio, H8070, Beijing, China).

Flow Cytometry
MEC apoptotic rate was evaluated using the Annexin V-FITC/PI apoptosis kit (7Sea Biotech, Shanghai, China) and MEC cell cycle was measured by Cell cycle staining Kit (MultiSciences, Hangzhou, China) according to the manufacturer's instructions.

Triglyceride Content Detection and Oil Red O Staining
Triglyceride content was measured by Tissue/Cell triglyceride assay kit (Applygen, Beijing, China) and normalized to protein concentration, which was tested by BCA protein assay kit. Oil red O used in staining was purchased from Solarbio (Beijing, China). Relative density of lipid drops was analyzed by Image-Pro Plus 6.0 Software.

Cell Counting Kit-8 (CCK-8) Assay
Ten microliter of CCK-8 solution (7Sea Biotech, Shanghai, China) was added into MECs seeded in 96-well plates and incubated at 37 • C for 4 h to evaluate MEC viability. Then, the optical density value was measured at 450 nm with a Microplate Reader (Bio Tek, United States).

Statistical Analysis
Sequencing data quality was evaluated using FastQC v0.10.1 and data filtering was performed with Cutadapt 1.9.1. Short read alignment was conducted using Hisat v2.0.14. SNV and InDel information was acquired from Samtools v0.1.18 analysis. The weight of female offspring in every nest was averaged and the data in six nests were set as one group. The acquired data of three groups were applied to analyze. Experiments in this study were conducted in three independent repeats. SPSS 22.0 was used to conduct one-way ANOVA and Student t-test. * p < 0.05, which was considered significant, and * * p < 0.01, which was considered highly significant.

Novel-miR-3880 Directly Suppresses the Expression of ELF2
After novel-miR-3880 was transfected into MECs with NC as control, RNA sequencing was conducted and DEGs are shown in Supplementary File S6. There were 57 downregulated DEGs and 10 upregulated DEGs in novel-miR-3880 group (Figure 1A). ELF2 selected from DEGs of RNA-sequencing was verified via RT-qPCR and western blotting to evaluate the sequencing accuracy ( Figure 1B).
A schematic diagram of dual luciferase reporter vectors was described in Figure 1C. It is shown in Figure 1D that the relative luciferase activity was lowered when novel-miR-3880 was co-transfected with psiCHECK2-ELF2-1 3 -UTR Wt vector. However, the relative luciferase activity stayed the same level when either NC or novel-miR-3880 were co-transfected with ELF2-2 3 -UTR Wt vector or psiCHECK2-ELF2-1 3 -UTR Mu vector ( Figure 1D). In addition, the expression of ELF2 in different goat tissues was detected ( Figure 1E) and pcDNA3.1-ELF2 overexpression vector efficiency is provided in Figures 1F,G. CiRNA13761 Sponges Novel-miR-3880 and Induces a Regulation in ciRNA13761/Novel-miR-3880/ELF2 Network Relative luciferase activity shown in Figure 2A illustrates that the relative luciferase activity of Wt psiCHECK2-ciRNA13761 vector was reduced, but that of the Mu vector was not changed, which indicates novel-miR-3880 was absorbed by ciRNA13761. To explore the regulatory pattern of ciRNA13761, the network among ciRNA13761, novel-miR-3880, and ELF2 was established, and the role of ciRNA13761 source gene-DOCK1 in the network was determined. The efficiency of si-and pcDNA3.1 # -ciRNA13761 is clarified in Figures 2B,C; it could be seen in Figures 2C,D that novel-miR-3880 decreased the expression of ciRNA13761. Combined with the effect of ciRNA13761 on novel-miR-3880 shown in Figure 2E, it showed a mutual inhibition between novel-miR-3880 and ciRNA13761. Furthermore, ciRNA13761 induced an increase in ELF2 expression, while si-ciRNA13761 decreased ELF2 expression (Figures 2F,G). To determine the function of ELF2 in the network, the efficiency of siELF2 was verified (Figures 2H,I). It is evident that ELF2 inhibited novel-miR-3880 ( Figure 2J), but promoted ciRNA13761 and DOCK1 expression (Figures 2K,L). Otherwise, novel-miR-3880 improved but ciRNA13761 restrained the expression of DOCK1 (Figures 2M,N). Interestingly, as DOCK1 was knocked down (Figure 2O), novel-miR-3880 and ciRNA13761 expression were enhanced while ELF2 expression was not affected (Figures 2P,R).

(Supplementary
while an interval of three days and four days was applied alternately. Mammary gland tissue and blood samples were collected at day 22. The phosphorylation levels of PI3K, AKT, mTOR, and S6K1 in mammary gland tissue were shown in Figures 3B,C, which showed a higher phosphorylation of PI3K/AKT/mTOR/S6K1 in novel-miR-3880 and siELF2 groups. In MEC, novel-miR-3880 and siELF2 enhanced the phosphorylation of PI3K, AKT, mTOR, and S6K1, and novel-miR-3880 balanced the reduction of phosphorylation caused by ELF2 overexpression (Figures 3D,E). Then PI3K, AKT, mTOR, and S6K1 inhibitors dissolved in DMSO were applied to demonstrate the role of novel-miR-3880 and siELF2 in PI3K/AKT/mTOR/S6K1 pathway. In Figure 3F, PI3K, AKT, mTOR, and S6K1 inhibitors show a negative effect to the phosphorylation of each other, and the level of Bcl2/Bax was decreased when PI3K, AKT, mTOR, and S6K1 were inhibited. The results indicated that novel-miR-3880 and siELF2 could alleviate the reduction of phosphorylated-PI3K/AKT/mTOR/S6K1 and Bcl2/Bax levels (Figures 3G-J). However, Figures 3K,L show that ciRNA13761 played a negative role in PI3K/AKT/mTOR/S6K1 pathway activation, but PI3K/AKT/mTOR/S6K1 pathway was reactivated when novel-miR-3880 was co-transfected or ciRNA13761/DOCK1 was knocked down.
Novel-miR-3880 Enhances MEC Proliferation Through PI3K/AKT/mTOR/S6K1 and Bcl-2/Bax Pathway via Targeting ELF2 The results show that MEC proliferation would be suppressed when PI3K, AKT, mTOR, or S6K1 were inhibited (Supplementary Files S8a,b), but the participation of novel-miR-3880 and siELF2 played a positive part in freeing MEC to proliferate (Supplementary Files S8a,c-g).
In addition, results shown in Supplementary Files S8hk provide evidence for the restraint of ciRNA13761 and ELF2 overexpression on MEC proliferation and the restraint was alleviated by novel-miR-3880, and further affirmed the modulation of ciRNA13761, novel-miR-3880, and ELF2 on MEC proliferation.

ELF2 Contributes to the Modulation of Novel-miR-3880 to MEC Lipid Formation, Triglyceride Synthesis and Casein Secretion
The Oil Red O staining and triglyceride content test revealed that novel-miR-3880, si-ciRNA13761, and siELF2 enhanced while ciRNA13761 and ELF2 overexpression reduced MEC lipid formation (Supplementary Files S9a-c) and triglyceride synthesis (Supplementary Files S9d-f), with the reduction weakened by novel-miR-3880 (Supplementary Files S9b,c,e,f). The participation of PI3K/AKT/mTOR/S6K1 pathway is illustrated in Supplementary File S9g; it is shown that MEC triglyceride content declined significantly when PI3K, AKT, mTOR, or S6K1 was inhibited. It is worthwhile to mention that novel-miR-3880 and siELF2 improved the triglyceride content in mouse mammary glands but did not change blood triglyceride content (Supplementary Files S9h,i).

Novel-miR-3880 Promotes Mammary Gland Development, Extends Lactation Period and Benefits Offspring Growth
The ultrastructure of collected mammary gland tissues was observed. It is significant that lipid droplets became larger but fewer, as the acini cavities turned smaller in the control group ( Figure 4A). However, animals injected with novel-miR-3880 or siELF2 were still lactating with plump acini filled with secreta and had more abundant mammary ducts (Figures 4B,C). More images can be found in Supplementary File S11. The weight of all female offspring in six nests of each group was measured, and it was found that newborns fed by mothers in novel-miR-3880 or siELF2 groups gained more weight (Figure 4D).

DISCUSSION
This research aimed to study the effects of novel-miR-3880 and relative molecules on mammary gland development and lactation traits, thus gaining a better understanding of lactation in dairy goats. A general view of our work is presented in Supplementary File S12. Novel-miR-3880 was selected from our previous study  for its higher expression in peak lactation stage. The present study reveals that novel-miR-3880 targeted ELF2 and was sponged by ciRNA13761. CiRNA13761, novel-miR-3880, and ELF2 as well as DOCK1 had an interaction with each other, although siDOCK1 did not directly affect the expression of ELF2. It indicates that the molecules we studied have a mutual regulation to keep a steady state in MEC. Novel-miR-3880 combined with ELF2 3 -UTR to suppress the expression of ELF2, while ciRNA13761 sponged novel-miR-3880 and reduced the chance of combination between novel-miR-3880 and ELF2, and ELF2 inhibited the expression of novel-miR-3880 to protect itself from too low expression. Interestingly, novel-miR-3880 also presented resistance to ciRNA13761 to ensure its own function in the network. DOCK1, as the source gene of ciRNA13761, improved both expression of novel-miR-3880 and ciRNA13761 when it was knocked down, while ELF2 exerted a facilitated role in DOCK1 expression. Our findings suggest that the network participates in MEC triglyceride synthesis, casein secretion, cell viability, and proliferation, as well as anti-apoptosis regulation, and plays an important part in PI3K/AKT/mTOR/S6K1 and Bcl-2/Bax pathways.
Novel-miR-3880 improved MEC triglyceride synthesis, casein secretion, cell viability, proliferation, and anti-apoptotic ability via targeting ELF2. Novel-miR-3880 would offset the reduction caused by ciRNA13761 and ELF2 overexpression. This confirms the mutual regulation among ciRNA13761, novel-miR-3880, and ELF2 on MEC. Besides, the results shown in Figure 3 not only demonstrate that ciRNA13761, novel-miR-3880, ELF2, and DOCK1 engaged in PI3K/AKT/mTOR/S6K1 pathway and Bcl2/Bax signaling, but also certify a close relationship among PI3K, AKT, mTOR, and S6K.Once any of them was inhibited, all of them would face a decrease in phosphorylation level as well as a decline in the expression of Bcl2/Bax. Fortunately, novel-miR-3880 and siELF2 could activate PI3K/AKT/mTOR/S6K1 and Bcl2/Bax pathways, and possesses the ability to alleviate the decline of PI3K/AKT/mTOR/S6K1 phosphorylation levels and Bcl2/Bax expression ratio, which might be a reason for the mitigative effects of novel-miR-3880 and siELF2 on reduced MEC proliferation induced by PI3K, AKT, mTOR, or S6K1 inhibitor. It thus appears that the results are consistent with previous research that PI3K/AKT/mTOR/S6K1 pathway activation benefits cell growth (Park et al., 2011;Zhang et al., 2019), and provide sufficient evidence for the participation of novel-miR-3880 and ELF2 in the modulation of PI3K/AKT/mTOR/S6K1 pathway both in vitro and in vivo. In addition, the role of ciRNA13761 and DOCK1 in PI3K/AKT/mTOR/S6K1 pathway and Bcl2/Bax pathway was elaborated, which increases evidence for the regulation of ciRNA13761/novel-miR-3880/ELF2 to PI3K/AKT/mTOR/S6K1 and Bcl2/Bax pathway.
The results provide evidence that novel-miR-3880 is a key molecule in regulating the effects of ciRNA13761 and ELF2 on MEC function, illustrating the role of their mutual regulation in MEC cellular processes. It is worth mentioning that novel-miR-3880 decreased content of most casein (α s1-, α s2-, and β-casein), which makes it closer to human milk protein composition (Kunz and Lonnerdal, 1992;Hao-Feng, 2012), and increased κ-casein content to make milk products easier to process (Brigid et al., 2003).
Importantly, it was confirmed that novel-miR-3880 and siELF2 work well in facilitating mammary gland development, improving triglyceride content in mammary glands but not in blood, extending lactation days, and promoting newborn growth through PI3K/AKT/mTOR/S6K1 pathway. Therefore, novel-miR-3880 application is promising in the goat breeding and milk industry.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/Supplementary Material.

ETHICS STATEMENT
The animal study was reviewed and approved by the Animal Care and Use Committee of Northwest A&F University.