International Journal of Molecular Sciences Article Prolactin-Releasing Peptide Differentially Regulates Gene Transcriptomic Profiles in Mouse Bone Marrow-Derived Macrophages Yulong Sun 1,2,* , Zhuo Zuo 1,2 and Yuanyuan Kuang 1,2 1 School of Life Sciences, Northwestern Polytechnical University, Xi’an 710072, China; [email protected] (Z.Z.); [email protected] (Y.K.) 2 Key Laboratory for Space Biosciences & Biotechnology, Institute of Special Environmental Biophysics, School of Life Sciences, Northwestern Polytechnical University, Xi’an 710072, China * Correspondence: [email protected]; Tel.: +86-29-8846-0332 Abstract: Prolactin-releasing Peptide (PrRP) is a neuropeptide whose receptor is GPR10. Recently, the regulatory role of PrRP in the neuroendocrine field has attracted increasing attention. However, the influence of PrRP on macrophages, the critical housekeeper in the neuroendocrine field, has not yet been fully elucidated. Here, we investigated the effect of PrRP on the transcriptome of mouse bone marrow-derived macrophages (BMDMs) with RNA sequencing, bioinformatics, and molecular simulation. BMDMs were exposed to PrRP (18 h) and were subjected to RNA sequencing. Differentially expressed genes (DEGs) were acquired, followed by GO, KEGG, and PPI analysis. Eight qPCR-validated DEGs were chosen as hub genes. Next, the three-dimensional structures of the proteins encoded by these hub genes were modeled by Rosetta and Modeller, followed by molecular dynamics simulation by the Gromacs program. Finally, the binding modes between PrRP Citation: Sun, Y.; Zuo, Z.; Kuang, Y. and hub proteins were investigated with the Rosetta program. PrRP showed no noticeable effect on Prolactin-Releasing Peptide the morphology of macrophages. A total of 410 DEGs were acquired, and PrRP regulated multiple Differentially Regulates Gene BMDM-mediated functional pathways. Besides, the possible docking modes between PrRP and hub Transcriptomic Profiles in Mouse proteins were investigated. Moreover, GPR10 was expressed on the cell membrane of BMDMs, which Bone Marrow-Derived Macrophages. Int. J. Mol. Sci. 2021, 22, 4456. increased after PrRP exposure. Collectively, PrRP significantly changed the transcriptome profile https://doi.org/10.3390/ijms22094456 of BMDMs, implying that PrRP may be involved in various physiological activities mastered by macrophages. Academic Editor: Sarath Chandra Janga Keywords: prolactin-releasing peptide; GPR10; bone marrow-derived macrophage; RNA sequencing; bioinformatics; transcriptomic profiles Received: 2 March 2021 Accepted: 20 April 2021 Published: 24 April 2021 1. Introduction Publisher’s Note: MDPI stays neutral Prolactin-releasing peptide (PrRP) is a neuropeptide originally extracted from the with regard to jurisdictional claims in bovine hypothalamus [1,2]. PrRP has two biologically active isoforms, PrRP20 and PrRP31, published maps and institutional affil- which share identical C-terminal Arg-Phe-amide sequences [3,4]. GPR10 (G-protein cou- iations. pled receptor 10, GPCR 10) is considered as the endogenous receptor for PrRP [2,5]. Both isoforms PrRP20 and PrRP31 bind to GPR10 with high affinity [6]. PrRP belongs to a family named RF-amide, which includes neuropeptide FF (NPFF), gonadotropin-inhibitory hormone (GnIH), kisspeptin, and pyroglutamylated RFamide (QRFP) [7]. Copyright: © 2021 by the authors. The original biological function of PrRP is to promote the release of prolactin from cul- Licensee MDPI, Basel, Switzerland. tured pituitary cells [2]. Then, follow-up research results show that PrRP has an important This article is an open access article role in the neuroendocrine system, including reducing food intake in fasting and free- distributed under the terms and eating rat models [8], regulating energy homeostasis and increasing energy expenditure [9], conditions of the Creative Commons mediating the anorexia effect of nerves [10], protecting neuron on several rodent neurode- Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ generative disorder models [11], and participating in stress response [12]. However, the 4.0/). effect of PrRP on macrophages has not been reported yet. Int. J. Mol. Sci. 2021, 22, 4456. https://doi.org/10.3390/ijms22094456 https://www.mdpi.com/journal/ijms Int.Int. J.J. Mol.Mol. Sci. 2021,, 22,, 4456x FOR PEER REVIEW 22 of of 4651 The regulatory effect of PrRPPrRP on leukocytes has been investigated. Romeroa and col- leagues findfind that PrRPPrRP mRNA isis expressed inin leukocytesleukocytes fromfrom thethe headhead kidneykidney andand bloodblood of Salmon salar.. Moreover, syntheticsynthetic PrRPPrRP promotespromotes thethe expressionexpression ofof pro-inflammatorypro-inflammatory cytokines (IL-12,(IL-12, IL-6,IL-6, IL-8,IL-8, and and IL-1 IL-1ββ),), suggesting suggesting that that PrRP PrRP may may be be a local a local neurotransmit- neurotrans- termitter of innate of innate immune immune processes processes in leukocytesin leukocytes from fromSalmon Salmon salar salar[13 [13].]. However, However, the the target tar- genesget genes by whichby which PrRP PrRP affects affects the leukocytesthe leukocytes have have not beennot been identified identified yet. yet. RNA-seq (RNA-sequencing)(RNA-sequencing) isis a a useful useful method method to to explore explore the the effect effect of theof the peptide peptide on theon genethe gene expression expression profile profile of macrophages of macropha fromges the from transcriptome the transcriptome level [14 –level16]. Hence, [14–16]. it wouldHence, beit would useful be to investigateuseful to investigate the PrRP-induced the PrRP-induced gene expression gene expression profile of profile macrophages of mac- byrophages using RNA-seq. by using RNA-seq. In the present In the study, present the stud effecty, of the PrRP effect on of the PrRP transcriptome on the transcriptome of BMDM wasof BMDM studied was by studied the following by the procedures:following procedures: (a)(a) PrRP31-triggeredPrRP31-triggered differentially expressed genes (DEGs) were acquired from murinemurine bonebone marrow-derived marrow-derived macrophages (BMDMs). Cells treated with 1 nM PrRP31 (18 h) werewere detected by RNA-seq. A A total of 410410 DEGsDEGs werewere obtained.obtained. In addition,addition, thethe influenceinfluence of of PrRP PrRP on on the the morphology morphology of of cells cells was was observed observed with with optical optical microscopy. microscopy. (b)(b) DEGsDEGs were were analyzed by a series of bioinf bioinformaticsormatics approaches, includingincluding GOGO analysis,analysis, functionalfunctional enrichment, enrichment, and protein–protein interaction (PPI)(PPI) studies.studies. Next,Next, eighteight hubhub genesgenes were were finally finally acquired for subsequent study. (c) The three-dimensional structures of hub proteins were studied. By using homology- (c) The three-dimensional structures of hub proteins were studied. By using homology- modeling, the structure of proteins encoded by hub genes (hub proteins) was built. modeling, the structure of proteins encoded by hub genes (hub proteins) was built. Subsequently, molecular dynamics simulations (at least 300 ns) were performed to Subsequently, molecular dynamics simulations (at least 300 ns) were performed to capture the trajectory of hub proteins. Finally, molecular dynamics-optimized protein capture the trajectory of hub proteins. Finally, molecular dynamics-optimized structures of hub proteins were obtained. protein structures of hub proteins were obtained. (d) The docking models of PrRP and hub proteins were predicted with the peptide-protein (d) The docking models of PrRP and hub proteins were predicted with the peptide- docking module of the Rosetta program. protein docking module of the Rosetta program. By studying the influence of PrRP on the gene expression of BMDMs at the tran- By studying the influence of PrRP on the gene expression of BMDMs at the transcrip- scriptome level, we provide clues for exploring the gene expression network of PrRP on tome level, we provide clues for exploring the gene expression network of PrRP on mac- macrophages, which will be helpful to investigate the immune-regulating function of PrRP inrophages, the future which (Figure will1 ).be helpful to investigate the immune-regulating function of PrRP in the future (Figure 1). Figure 1. Schematic diagram of this study. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 51 2. Results Int. J. Mol. Sci. 2021, 22, 4456 2.1. PrRP Demonstrated No Significant Effect on the Shape of BMDMs 3 of 46 As displayed in Figure 2A, the cell morphology of BMDMs was detected by electron microscopy. In addition, the purity of BMDMs was evaluated using flow cytometry with anti-CD11b and anti-F4/80 (Figure 2B). The results of flow cytometry showed that the dou- 2. Results ble-positive rate of the BMDMs control group (no antibody was added) was 0.14% (the 2.1. PrRP Demonstrated No Significant Effect on the Shape of BMDMs left side of Figure 2B), whereas the double-positive rate of BMDMs (incubated with anti- F4/80As and displayed anti-CD11b) in Figure 96.7%2A, (right the cell side morphology of Figure 2B). of BMDMs Hence, these was detected data hinted by electron that the microscopy.purity of BMDMs In addition, was acceptable. the purity of BMDMs was evaluated using flow cytometry with anti-CD11bNext, the and morphological anti-F4/80 (Figure characters2B). Theof BMDMs results before of flow and cytometry after PrRP showed exposure that were the double-positive rate of the BMDMs control