LncRNA018392 promotes proliferation of Liaoning cashmere goat skin fibroblasts through up-regulation of CSF1R by binding to SPI1

doi:10.21203/rs.3.rs-2739715/v1

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LncRNA018392 promotes proliferation of Liaoning cashmere goat skin fibroblasts through up-regulation of CSF1R by binding to SPI1

https://doi.org/10.21203/rs.3.rs-2739715/v1

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The Liaoning cashmere goat has been confirmed as a valuable genetic resource breed that is prohibited from genetic outflow in China, and it achieves the highest single fleece production. Hair follicle development in the cashmere goat is regulated by melatonin and long non-coding RNAs (lnRNAs). However, the role played by lncRNAs in mediating melatonin-promoted cash-mere growth remains unclear. A novel lncRNA-lncRNA018392 with significant overexpression, which played a certain role in the melatonin-promoted proliferation of cashmere skin fibroblasts, was screened in previous research. The flow cytometry and CCK-8 results confirmed that the knockdown of lncRNA018392 reversed the effect of melatonin on cell proliferation, and the prolif-eration of cashmere skin fibroblasts was inhibited after the cells were interfered with the gene CSF1R near lncRNA018392. The dual-luciferase reporter assay further demonstrated that lncRNA018392 can positively regulate the promoter of CSF1R. Moreover, as indicated by the results of RNA-binding protein immunoprecipitation (RIP) and chromatin immunoprecipitation sequencing (ChIP-Seq), lncRNA018392 formed a complex with transcription factor SPI1, and CSF1R served as a downstream target gene regulated by SPI1. As revealed by the results of this study, melatonin-mediated novel lncRNA018392 accelerated the cell cycle, facilitated cell proliferation, and inhibited apoptosis by recruiting SPI1 to up-regulate the expression of nearby gene CSF1R. This study lays a theoretical basis for clarifying the molecular mechanism of cashmere growth and molecular breeding of cashmere goats.

Biological sciences/Cell biology

Biological sciences/Molecular biology

liaoning cashmere goat

lncRNA018392

spi1

csf1r

Cashmere refers to an unmyelinated fiber produced by secondary hair follicles of goat skin, exhibiting unique properties (e.g., softness and comfort). Skin fibroblasts in the dermis take on critical significance to hair follicle formation, development, and wound healing¹. Accordingly, the regulatory mechanism of skin fibroblast proliferation in cashmere goats should be investigated to improve the production and quality of cashmere.

Melatonin serves as a vital mediator between photoperiod and hair follicle growth, and periodic fluctuations in melatonin levels exert a direct effect on the growth of hair follicle². Melatonin can activate NF-κB signaling by up-regulating lncRNA MTC to facilitate proliferation and regulate hair follicle development in Liaoning cashmere goat fibroblasts³. There have been 64 lncRNAs with differential expression during melatonin-mediated secondary hair follicle growth in Shaanbei cashmere goats, and lncRNAs TCONS_00255106 and TCONS_00206163 may regulate COL4A2 and THBS2 expression in a cis-acting manner⁴. After melatonin implantation in live Inner Mongolian cashmere goats, six pivotal genes correlated with cashmere growth (i.e., PDGFRA, WNT5A, PPP2R1A, BMPR2, BMPR1A, and SMAD) have been identified through combinatorial differential gene expression analysis⁵. Existing research on melatonin-promoted cashmere growth has placed a major focus on the gene level⁴, whereas rare studies have reported that melatonin promotes the proliferation of cashmere skin fibroblasts by regulating non-coding RNAs.

Long non-coding RNAs are nonprotein-coding RNAs that are over 200 nucleotides in length⁶. A considerable number of lncRNAs play a certain role in hair follicle genesis in goats. For instance, lncRNA-000133 plays a role in the reconstruction of secondary hair follicles during cashmere fiber formation and growth⁷; lncRNA-HOTAIR is correctly transcribed at the anagen and resting phases of secondary hair follicles in cashmere goats, and it achieves more significant expression at the anagen phase than at the resting phase⁸; lncRNA-H19 facilitates hair follicle proliferation through the chi-miR-214-3p/β-Catenin axis, such that the proliferation of cashmere goat dermal cells is facilitated⁹. Indeed, a vital role of lncRNA is to regulate the transcription of its neighboring genes¹⁰, whereas the mechanism of the above-described phenomenon should be elucidated in depth.

A previous study has reported that 0.2 g/L melatonin treatment of Liaoning cashmere goat skin fibroblasts for 72 h significantly facilitates their proliferation¹¹. LncRNA018392 was screened based on the transcriptome sequencing data before and after the melatonin treatment of cells. LncRNA018392 is located between CSF1R and PDGFRB on chromosome 7 in the cashmere goats, and it is significantly expressed during the melatonin-promoted goat skin fibroblast proliferation. As revealed by this result, our lncRNA018392 plays a critical role in melatonin-promoted goat skin fibroblast proliferation.

Colony-stimulating factor 1 receptor (CSF1R) refers to M-CSFR (macrophage colony-stimulating factor). The transcriptional activation of CSF1R covers numerous transcription factors (e.g., the E26-specific transcription factor family, PU.1, ATF, and CEBP)¹². CSF1R can play a certain role in cytokine-cytokine receptor interactions, PI3K-AKT, Rap1 MAPK, and Ras signaling pathways, all of which are correlated with hair follicle development in goats¹³. The E26 transformation-specific (ETS) family of transcription factors, PU.1¹⁴, is also known as SPI1 in cashmeret goats. SPI1 takes on critical significance to hematopoietic differentiation, proliferation, and apoptosis¹⁵. Existing research has confirmed the transcription factor SPI1 as a vital transcription factor for the growth of fleece in Changthangi goats¹³. LINC00324 up-regulates FasL expression by recruiting SPI1, such that the proliferation, migration, and invasion of liver cancer stem cells (LCSCs) can be ultimately maintained¹⁶.

RNA immunoprecipitation (RIP) refers to an advanced technique that is capable of detecting the interaction between individual proteins and specific ncRNAs molecules in vivo¹⁷. Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) has been recognized as a technique for the genome-wide analysis of DNA-binding proteins¹⁸. In this study, the molecular regulatory mechanisms between lncRNA018392 and SPI1 and between SPI1 and CSF1R by RIP and ChIP-Seq will be explored.

2.1. Bioinformatics Analysis of lncRNA018392

As indicated by the results of The National Center for Biotechnology Information's Basic Local Alignment Search Tool (BLAST), lncRNA018392 was 1,304 bp long and located on chromosome 7. LncRNA018392 was located upstream of CSF1R, suggesting CSF1R as a potential cis-regulatory target gene for lncRNA018392 (Fig. 1A). ORF finder's prediction indicated that lncRNA018392 contains 13 ORFs, with the longest ORF being 282 nt. The longest ORF encoded 94 amino acids, less than the number of amino acids encoded by the coding gene. CPC2 result indicated that the coding capacity of lncRNA018392 was 0.2397, suggesting that lncRNA018392 exhibited a low coding potential. LncRNAs have been known to form complex secondary structures due to base pairing, and secondary structure prediction was performed for lncRNA018392 using the RNAfold web server. The minimum free energy (MFE) and center-of-mass algorithms were adopted for prediction, and the result indicated that lncRNA018392 would form several stem-loop structures (Figure S1). RNA was extracted from the nucleus and cytoplasm of goat skin fibroblasts before and after melatonin treatment using RNA nucleoplasm separation technique, followed by RT-qPCR. The above results suggested that lncRNA018392 accounted for 70 ± 0.17% in the nucleus and 30 ± 0.17% in the cytoplasm before melatonin treatment, and 70 ± 0.31% in the nucleus and cytoplasm by 30 ± 0.31% (Fig. 1B), thus revealing that lncRNA018392 can play a major role in the nucleus.

2.2. Melatonin Promotes Proliferation of Goat Skin Fibroblasts Through Up-regulation of lncRNA018392

To determine the role of lncRNA018392 in melatonin promotion of goat skin fibroblast proliferation, the expression of lncRNA018392 was down-regulated in goat skin fibroblasts by small interfering RNA transfection. siRNA-2 achieved a more efficient interference efficiency (Fig. 2A).CCK-8 results showed that lncRNA018392 knockdown reversed the proliferation-promoting effect of melatonin on goat skin fibroblasts (Fig. 2B). As indicated by the flow cytometry results, lncRNA018392 knockdown led to the increased proportion of goat skin fibroblasts at G0/G1 phase and the decreased proportion of cells in S phase (Fig. 2C); apoptosis was significantly higher (Fig. 2D). Hair follicle growth-associated genes, CDK4, EREG, FN1, PDGFB and TNF gene expression levels were significantly elevated after melatonin treatment, whereas lncRNA018392 knockdown reversed the above-mentioned phenomenon (Fig. 2E). The above-described results suggested that melatonin accelerated the goat skin fibroblast cycle, such that cell proliferation was facilitated, and apoptosis was inhibited through the up-regulation of lncRNA018392.

2.3 lncRNA018392 Promotes the Proliferation of Goat Skin Fibroblasts Through Up-regulation of CSF1R

LncRNA018392 interference was performed after melatonin treatment of goat skin fibroblasts to detect CSF1R expression level. Melatonin treatment significantly up-regulated CSF1R expression, whereas lncRNA018392 interference reversed this phenomenon (Fig. 3A). To determine the role of CSF1R in goat skin fibroblasts ,the expression of CSF1R was down-regulated in goat skin fibroblasts by small interfering RNA transfection, siRNA-2 achieved a more efficient interference efficiency (Fig. 3B).

Cell viability was decreased after interference with CSF1R (Fig. 3C), and interference with CSF1R reversed the cell proliferation caused by lncRNA018392 overexpression (Fig. 3D). The flow cytometry result reported that interfering with CSF1R led to the elevated ratio of goat skin fibroblasts at G0/G1 phase and the decreased ratio in S phase (Fig. 3E); the apoptosis rate was up-regulated significantly (Fig. 3F). When hair follicle growth-associated genes were detected, the expression levels of CDK4, EREG, FN1, PDGFB and TNF genes were significantly down-regulated after interference with CSF1R (Fig. 3G).

Besides, the addition of CSF1R inhibitor (PLX3397) reversed the cell proliferation induced by lncRNA018392 overexpression (Fig. 3H). The above-described observations suggested that lncRNA018392 facilitated the proliferation of goat skin fibroblasts by up-regulating CSF1R. The CSF1R-containing promoter sequence was inserted into the polyclonal site of the reporter gene vector pGL3-Basic, and the CSF1R promoter activity was measured using luciferase reporter gene to investigate the molecular mechanism of CSF1R regulation by lncRNA018392 in depth. The luciferase activity of the three recombinant plasmids was significantly increased after the transfection of cashmere skin fibroblasts for 24 h compared with the transfected internal reference plasmid and CSF1R promoter reporter vector set (Fig. 3I). The above-described results suggested that lncRNA018392 facilitated the proliferation of goat skin fibroblasts by positively regulating CSF1R expression through the region of the CSF1R promoter.

2.4. LncRNA018392 Promotes Proliferation of Goat Skin Fibroblasts Through Recruitment of SPI1

70 ± 0.31% of lncRNA018392 was detected in the nucleus (Fig. 1C), suggesting that lncRNA018392 was primarily involved in a role in transcriptional regulation in the nucleus. Prediction by an algorithm based on the individual interaction propensity of peptide and nucleotide sequence fragments catRAPID fragments indicated that the 1174–1227 and 1200–1253 nucleotide positions of lncRNA018392 sequence exhibited a high binding propensity to 126–177 amino acid residues of SPI1 protein (Fig. 4A). Next, RIP experiments confirmed that the RNA-binding protein SPI1 was significantly enriched in lncRNA018392, indicating the formation of the lncRNA018392-SPI1 complex in goat skin fibroblasts (Fig. 4B). To determine the role of SPI1 in lncRNA018392 proliferation of goat skin fibroblasts, down-regulation of SPI1 expression in goat skin fibroblasts by small interfering RNA transfection, siRNA-1 achieved a more efficient interference efficiency (Fig. 4C). pcDNA3.1-lncRNA018392 and si-SPI1 were cotransfected with cashmere skin fibroblasts. CCK-8 results showed that interfering with SPI1 reversed the proliferative effect of overexpressed lncRNA018392 on cells (Fig. 4D). The Flow cytometry results suggested that interfering with SPI1 after lncRNA018392 overexpression led to the increased ratio of goat skin fibroblasts at G0/G1 phase and the decreased ratio in S phase (Fig. 4E); the apoptosis rate was elevated (Fig. 4F). Detecting CSF1R and hair follicle growth-associated genes, interfering with SPI1 reversed the up-regulation of CSF1R, CDK4, EREG, FN1, PDGFB and TNF gene expression levels caused by overexpression of lncRNA018392 (Fig. 4G, 4H). In addition, interference with SPI1 reversed the up-regulation of gene expression levels of lncRNA018392 and SPI1 caused by overexpression of lncRNA018392 (Fig. 4I, 4J). The above results suggested that lncRNA018392 recruited SPI1 to form a complex that can facilitate cell proliferation and inhibit apoptosis by accelerating the goat skin fibroblast cell cycle;it up-regulated the expression of CSF1R and villi growth-associated genes.

2.4. ChIP-Seq to Verify the Binding of SPI1 to CSF1R

To reveal the regulatory mechanism of SPI1 on CSF1R, we used chromatin immunoprecipitation combined gene sequencing (ChIP-Seq) technology to detect the binding sites of SPI1 to CSF1R, and the percentage of reads that passed internal quality filtering was 93.68% after sequencing was completed. A total of 19096224 reads were mapped to the reference genome and we identified 5,314 ChIP-Seq-based Spi1 target genes (Table S1). The distribution of SPI1 binding sites on chromosomes (Fig. 5A) indicated that SPI1 was correlated with all chromosomes. As depicted in Fig. 5B, the distribution of SPI1 binding sites peaked at nearly 1000 pb of TSS. To examine whether SPI1 has different interaction preferences for a wide variety of chromatin regions, their presence in specific chromatin regions was compared, i.e., promoters (1.61%), exons (4.40%), introns (47.71%), transcription start sites (TSS) (1.61%), transcription termination sites (TTSs) (1.34%), and intergenic regions ( 44.48%) (Fig. 5C). The above-described results suggested that the correlation of SPI1 with chromatin may play the critical role in distal intergenic domains and intron regions. GO functional annotation and enrichment analysis of the KEGG metabolic pathway were performed on the ChIP-Seq results of high-throughput SPI1. GO annotation indicated that these target genes were primarily enriched in transcriptional activator activity, RNA polymerase II transcription, calcium binding, positive regulation of gene expression, positive regulation of RNA polymerase II promoter transcription, and so forth. CSF1R played a role in certain biological functions (e.g., protein autophosphorylation and protein phosphatase binding) (Fig. 5D ,Table S2). As indicated by the result of KEGG pathway annotation, these target genes played a role in 20 generative metabolic pathways, and the pathways associated with hair follicle growth and development comprised Wnt signaling pathway, Hippo signaling pathway, calcium signaling pathway, TGF-β signaling pathway, PI3K-Akt signaling pathway, and so forth. CSF1R played a role in PI3K -Akt signaling pathway and MAPK signaling pathway, and so forth. (Fig. 5E Table S3). The characteristics of SPI1 protein binding motif sequences were investigated using the MEME-ChIP program, and 18 motifs were obtained based on all targets (Table S4). Two motif sequences were identified (i.e., GTTTTC and AAGGAAAT) (Fig. 5F) in the sixth intron of CSF1R after sequence alignment, suggesting thatSPI1 in Liaoning cashmere goat skin fibroblasts can regulate CSF1R expression by binding to GTTTTC and AAGGAAAT on the sixth intron of CSF1R.

Fibroblasts are the main resident cell type in the skin dermis, mediating embryonic hair follicle morphogenesis, and hedgehog signaling in fibroblasts is critical to the induction of new hair follicle formation within healing wounds¹⁹. Fibroblasts play a role in fibroblast-epidermal interactions during hair development and in the interfollicular region of the skin²⁰. During the hair follicle cycle, the skin accumulation of fibroblasts is the "energy source" for the hair follicle at the respective stage of growth²¹. Increasing evidence has reported that lncRNAs are of great importance in the development of the hair follicle cycle in the cashmere goat^22,23. Extensively research has been conducted on the lncRNAs and their regulatory networks during secondary hair follicle development in the cashmere goat²⁴. LncRNAs, a type of ncRNA, play a role in the dynamic processes of skin and hair follicle morphogenesis and hair follicle growth and development^25,26. LncRNAs are capable of regulating gene expression by interacting with proteins, RNA, and DNA. lncRNAs' function is significantly correlated with its subcellular localization. In the nucleus, lncRNAs regulate gene expression at the epigenetic and transcriptional levels, whereas in the cytoplasm, they regulate gene expression at the post-transcriptional and translational levels²⁷. Therefore, their location in the cell needs to be determined before elucidating the pro-proliferative role of lncRNA018392 in cashmere skin fibroblasts. The result of nucleoplasmic separation experiments in this study indicated that lncRNA018392 was primarily localized in the nucleus.

On that basis, it was suggested that lncRNA018392 can up-regulate CDK4, EREG, PDGFRB, and TNF expression during the acceleration of the goat skin fibroblast cycle, promotion of cell proliferation, and inhibition of apoptosis. Hair follicle morphogenesis is affected by multiple genes. Cell cycle protein-dependent kinase 4, i.e., cytokinin kinase 4, takes on great significance to the G1/S phase transition of the cell cycle²⁸. Existing research has suggested that up-regulated lncRNA-LET expression promotes human skin fibroblast proliferation while inhibiting apoptosis via the cyclin D1-CDK4 and Bax/Bcl-2/cystatin-3 signaling pathways²⁹. EREG (extra-epidermal regulatory protein) refers to a member of the epidermal growth factor (EGF) family, playing a certain role in inflammation and wound healing. EREG knockdown can inhibit hair growth, and EREG activate EGFR and ErbB4 on epidermal and hair papilla cells, respectively, in the hair cycle³⁰. Fibronectin 1 (FN1) plays a role in cell adhesion and migration processes (e.g., embryogenesis and wound healing) as well as in a wide variety of biochemical processes. Fibronectin 1 (FN1) inhibits apoptosis in the human trophectoderm by activating the PI3K/Akt signaling pathway³¹. Platelet-derived growth factor (PDGF) is a potential mitogen and chemoattractant correlated with a variety of biological processes (e.g., cell survival, proliferation, and migration)³². Both platelet-derived growth factor subunits PDGF-AA and PDGF-BB play a role in the induction and maintenance of the anagen phase of the mouse hair cycle³³. TNF is a pleiotropic cytokine that serves as a vital mediator and regulator of the immune response in healthy organisms. TNF can control the development of the immune system, cell survival signaling pathways, and proliferation while regulating metabolic processes³⁴. TNF knockdown can attenuate hair follicle neogenesis in wounds³⁵.

The colony-stimulating factor 1 receptor refers to a receptor tyrosine kinase36. CSF1R has been widely considered a regulator of cell proliferation³⁷. CSF1R-mediated signaling is essential for the survival, function, proliferation, and differentiation of Langerhans cells in the skin¹². Platelet-rich plasma is effective in androgenetic alopecia, and the growth factor CSF1R is contained in the supernatant of platelet lysate³⁸. Hair growth is affected by various growth factors, among which CSF1R plays a certain role in wound-induced hair growth³⁹. It has been shown that the transcriptional activation of CSF1R is affected by SPI1⁴⁰. SPI1, a key transcription factor, plays a crucial role in proliferation and differentiation, apoptosis, and hematopoiesis^41,42,43. The finding of this study confirmed that the binding motifs of SPI1 and CSF1R are GTTTTC and AAGGAAAT further supports the regulatory role of SPI1 on CSF1R. Unlike previous studies that reported SPI1 binding to the CSF1R promoter region⁴⁴, SPI1 bound to a sixth intron in this study, probably due to the unconserved nature of the CSF1R promoter. Extensive research on mammalian liver-specific enhancers has reported that the promoter is much more conserved than the enhancer⁴⁵, in contrast to CSF1R. The promoter of CSF1R is weakly conserved in mammals, retaining only extensive purine-rich structures⁴⁰.

KEGG analysis of genes targeted by SPI1 showed that targets interacting with SPI1 play a role in hair follicle growth and a wide variety of signaling pathway processes (e.g., the TGF-β signaling pathway, PI3K-Akt signaling pathway, and Rap1 signaling pathway). Hydrangea has been reported to promote hair regrowth and repair in mouse models of hair loss through activation of TGF-β signaling and mitochondrial signaling pathways⁴⁶. The PI3K-AKT signaling pathway is critical to human hair follicle regeneration⁴⁷. In addition, PI3K-AKT, MAPK, Ras and Rap1 signaling pathways play a certain role in the growth of in vitro cultured Inner Mongolian cashmere goat hair follicle stem cells⁴⁸. GO analysis of ChIP-Seq data of SPI1 indicated that SPI1 primarily regulated transcriptional activator activity, RNA polymerase II transcription, calcium binding, and positive regulation of gene expression. As revealed by the above-mentioned result, multiple physiological processes and signaling pathways are involved in the proliferation of Liaoning cashmere goat skin fibroblasts promoted by SPI1, and subsequent research directions will place stress on the role of specific components in the binding sites of SPI1 and target genes.

On that basis, a conclusion was drawn that lncRNA018392 can facilitate CSF1R transcription by recruiting transcription factor SPI1 and accelerate the cashmere skin fibroblast cycle, such that cell proliferation can be promoted, and apoptosis can be inhibited. This study can provide novel perspectives and ideas for the melatonin-mediated regulation of lncRNA-lncRNA018392, i.e., a novel lncRNA capable of regulating the growth and development of cashmere fleece and hair follicle cycle regeneration.

4.1. Cell culture and Treatment

Liaoning cashmere goat skin fibroblasts preserved in the laboratory were cultured in DMEM supplemented with 20% FBS (solarbio, China), 1% glutamine at 37°C and 5% CO2 in an incubator. At the time of cell passaging, cells were treated with 100 nM of CSF1R inhibitor PLX3397 (Macklin, China) for 24 h.

4.2. RNA Isolation and Quantitative RT-PCR Analysis

Nuclear and cytoplasmic RNA extraction was performed using the PARIS™ kit (Invitrogen, China) according to the manufacturer's instructions. Total RNA (1 µg) was reverse transcribed using BeyoRT II M-MLV reverse transcriptase (Beyotime, China), and 1 µl cDNA was employed for quantitative mRNA analysis. The detection was performed using a fluorescent quantitative PCR instrument (Exicycler 96, Korea). Relative expression levels were determined using the 2^-ΔΔCt method⁴⁹, with GAPDH and β-actin as reference genes. GAPDH was assigned to a positive control for cytoplasmic gene expression, and U6 RNA was assigned to a positive control for cytosolic gene expression. The specific primers are shown in the supplementary table 5.

4.3. Cell Counting Kit-8

Cell counting kits (wanleibio, China) were employed for cell proliferation analysis in accordance with the CCK-8 reagent manufacturer's instructions. Cells were inoculated in 96-well plates (1×104 cells/well), and OD450 nm was examined at 24 h, 48 h and 72 h using an enzyme marker (BIOTEK, USA).

4.4. Cell Cycle Analysis and Apoptosis Assay

For cell cycle assay, pre-cooled 70% ethanol was introduced to the treated cells and then fixed overnight at 4 ℃. The fixed cells were centrifuged, the supernatant was discarded, washed twice with PBS. Subsequently, the cells were incubated with 500 µl of PI staining solution at 4 ℃ for 30 min in the dark, collected and detected on a flow cytometer (Agilent, USA). Next, positive cells were detected.

For the apoptosis assay, pre-cooled 70% ethanol was added to the treated cells and fixed overnight at 4 ℃. The groups of cells were collected through centrifugation at 150 g for 5 min, and the mixture was mixed with 10 µl of PI staining solution and 5 µl of membrane-linked protein V-FITC solution (wanleibio, China) at room temperature for 15 min in the dark. Positive cells were subsequently detected through flow cytometry (Agilent, USA).

4.5. Plasmid Preparation

Goat CSF1R promoter 2000bp was ligated into vector pGL3-Basic, and the enzyme cut site was: NheI /XhoI.

The CDS region of goat SPI1 was ligated to vector PECMV-3flag with enzyme cut sites BamHI and XhoI.

The full-length sequence of goat lncRNA018392 obtained through pre-sequencing was ligated to pcDNA3.1 vector with enzyme cut site: NheI / XhoI at a concentration of 156.9 ng/µl. Goat skin fibroblasts were transfected with expressed lncRNA018392 vector or empty control vector and after 48 h, the cells were employed for further experiments.

4.6. Small Interfering RNA

Small interfering RNAs (siRNA-CSF1R, siRNA-SPI1, and siRNA-lnRNA01839)and goat skin fibroblasts were infected with liposomes of the above gene interference targets, and the cells were adopted for further experiments. The siRNAs sequence for RNA interference are shown in the supplementary table 6.

4.7. Luciferase Reporter Gene Assay

The constructed plasmids pGL3-CSF1R and pcDNA3.1-lncRNA018392 and the internal reference plasmid (pRL-TK) were cotransfected with goat skin fibroblasts, while the empty plasmid pGL3-Basic, PcDNA and internal reference plasmid cotransfection group was set as the control, and goat skin fibroblasts were collected after 24 h. The luciferase assay kit (KeyGEN Bio, China) was employed to determine dual luciferase activity (Briefly enzyme and Renilla enzyme).

4.8. RNA Immunoprecipitation Assay

The constructed PECMV-3flag-SPI1 plasmid was sequenced, and the sequencing results were analyzed through the comparison with GeneBank, showing that the plasmid was successfully constructed. A pull-down assay was performed using the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, China). The cells were collected and then lysed in complete RIPA buffer supplemented with a protease inhibitor mixture and RNase inhibitor. Next, the cell lysates were incubated with RIP buffer supplemented with magnetic beads coupled with Flag-tagged antibodies or control normal IgG. Samples are digested with proteinase K to isolate immunoprecipitated RNA. Finally, real-time fluorescent quantitative PCR is performed on purified RNA to demonstrate the presence of bound targets.

4.9. Chromatin Immunoprecipitation Sequencing (ChIP-Seq)

For ChIP-Seq, cashmere skin fibroblasts were transfected with PECMV-3flag-SPI1. Immunoprecipitation ChIP kit (wanleibio, China) was employed. Real-time quantitative PCR assays were performed to examine SPI1 overexpression efficiency to confirm successful transfection. Flag antibody (Proteintech, China) was the antibody applied for ChIP sequencing. After immunoprecipitation, DNA fragments were sequenced and then characterized at Shenyang Wan Class Biotechnology .

4.10. Bioinformatics Analysis

Gene information was acquired from UniProt (https://www.UniProt.org/), and promoter information originated from the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/). R language tools were used for KEGG pathway and GO analysis. Motif searches of DNA sequences were performed using MEME-ChIP⁵⁰(http://meme-suite.org/tools/meme-chip). Peaks in the samples were annotated to the nearest gene based on the latest UCSC RefSeq database. The enriched regions of ChIP-seq were obtained using MACS2 software, and the peaks were visualized using IGV. Potential open reading frames (ORFs) were predicted using ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder51. The potential coding capacity was estimated using the Coding Potential Calculator 2 (CPC2, http://cpc2.cbi.pku.edu.cn)⁵². The secondary structure of lncRNA018392 was predicted using the RNAfold web server (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi?PAGE=3&ID=UhzKa0Peie). The interactions between lncRNA018392 and SPI1 were predicted using catRAPID (http://service.tartaglialab.com/page/catrapid_group).

4.11. Statistical Analysis

All experiments were performed at least three times, and all results are expressed as mean ± standard deviation. Statistical analysis was conducted using one-way ANOVA and two-way ANOVA in GraphPad Prism 9.4 software for correlation analysis. p-value < 0.05 indicated that the difference that achieved statistical significance.

Author contributions: Mei Jin guided the experiment , designed experiments and applied the fund . Suhe Lv executed experiments, literature review, and manuscript. Xinyang Liu performed experiments and collected data. Lixin Cui conducted some of the experiments.Weiyu Fan reviewed the literature, interpreted the data, and reviewed the manuscript.

Funding:This study was supported by National Natural ScienceFoundation of China [grant number 31772557]; DalianScience and Technology Innovation Fund Project [grant number 2019J12SN65].

Informed Consent Statement：Not applicable.

Data Availability Statement: The data presented in this study are available in the article and Supplementary Materials. The datasets generated during the current study are available in the Short Read Archive (SRA) of NCBI repository, https://dataview.ncbi.nlm.nih.gov/object/PRJNA951442?reviewer=ub5k5m3kkoe3ggjcj2udr52aet.

Conflicts of Interest：The authors declare no conflict of interest.

Millar, S. E. Molecular mechanisms regulating hair follicle development. J Invest Dermatol118, 216-225, doi:10.1046/j.0022-202x.2001.01670.x (2002).
Zhao, Y. H. et al. Expression of the RORalpha gene in Inner Mongolian cashmere goat hair follicles. Genet Mol Res14, 380-388, doi:10.4238/2015.January.23.11 (2015).
Jin, M. et al. Long noncoding RNA and gene expression analysis of melatonin-exposed Liaoning cashmere goat fibroblasts indicating cashmere growth. Naturwissenschaften105, 60, doi:10.1007/s00114-018-1585-6 (2018).
Ge, W. et al. Melatonin promotes Cashmere goat (Capra hircus) secondary hair follicle growth: a view from integrated analysis of long non-coding and coding RNAs. Cell Cycle17, 1255-1267, doi:10.1080/15384101.2018.1471318 (2018).
Liu, Z. et al. Identification of key pathways and genes that regulate cashmere development in cashmere goats mediated by exogenous melatonin. Front Vet Sci9, 993773, doi:10.3389/fvets.2022.993773 (2022).
Li, B. et al. LncRNA RAD51-AS1 Regulates Human Bone Marrow Mesenchymal Stem Cells via Interaction with YBX1 to Ameliorate Osteoporosis. Stem Cell Rev Rep19, 170-187, doi:10.1007/s12015-022-10408-x (2023).
Zheng, Y. et al. LncRNA-000133 from secondary hair follicle of Cashmere goat: identification, regulatory network and its effects on inductive property of dermal papilla cells. Anim Biotechnol31, 122-134, doi:10.1080/10495398.2018.1553788 (2020).
Jiao, Q. et al. Identification and molecular analysis of a lncRNA-HOTAIR transcript from secondary hair follicle of cashmere goat reveal integrated regulatory network with the expression regulated potentially by its promoter methylation. Gene688, 182-192, doi:10.1016/j.gene.2018.11.084 (2019).
Zhang, Y., Li, F., Shi, Y., Zhang, T. & Wang, X. Comprehensive Transcriptome Analysis of Hair Follicle Morphogenesis Reveals That lncRNA-H19 Promotes Dermal Papilla Cell Proliferation through the Chi-miR-214-3p/beta-Catenin Axis in Cashmere Goats. Int J Mol Sci23, doi:10.3390/ijms231710006 (2022).
Int J Mol Sci16, 3251-3266, doi:10.3390/ijms16023251 (2015).
Jin, M., Fan, W., Piao, J., Zhao, F. & Piao, J. Effects of lncRNA MTC on protein expression in skin fibroblasts of Liaoning Cashmere goat based on iTRAQ technique. Anim Biotechnol, 1-10, doi:10.1080/10495398.2022.2119406 (2022).
Mun, S. H., Park, P. S. U. & Park-Min, K. H. The M-CSF receptor in osteoclasts and beyond. Exp Mol Med52, 1239-1254, doi:10.1038/s12276-020-0484-z (2020).
Bhat, B., Yaseen, M., Singh, A., Ahmad, S. M. & Ganai, N. A. Identification of potential key genes and pathways associated with the Pashmina fiber initiation using RNA-Seq and integrated bioinformatics analysis. Sci Rep11, 1766, doi:10.1038/s41598-021-81471-6 (2021).
Li, G., Hao, W. & Hu, W. Transcription factor PU.1 and immune cell differentiation (Review). Int J Mol Med46, 1943-1950, doi:10.3892/ijmm.2020.4763 (2020).
Oikawa, T. et al. The role of Ets family transcription factor PU.1 in hematopoietic cell differentiation, proliferation and apoptosis. Cell Death Differ6, 599-608, doi:10.1038/sj.cdd.4400534 (1999).
Gao, J., Dai, C., Yu, X., Yin, X. B. & Zhou, F. Long noncoding RNA LINC00324 exerts protumorigenic effects on liver cancer stem cells by upregulating fas ligand via PU box binding protein. FASEB J34, 5800-5817, doi:10.1096/fj.201902705RR (2020).
Selth, L. A., Gilbert, C. & Svejstrup, J. Q. RNA immunoprecipitation to determine RNA-protein associations in vivo. Cold Spring Harb Protoc2009, pdb prot5234, doi:10.1101/pdb.prot5234 (2009).
Wan, M., Li, Y. T., Zheng, L. W. & Zhou, X. D. [Epigenetic regulation mechanism: roles in enamel formation and developmental defects of enamel]. Zhonghua Kou Qiang Yi Xue Za Zhi58, 68-74, doi:10.3760/cma.j.cn112144-20221019-00542 (2023).
Liu, Y. et al. Hedgehog signaling reprograms hair follicle niche fibroblasts to a hyper-activated state. Dev Cell57, 1758-1775 e1757, doi:10.1016/j.devcel.2022.06.005 (2022).
Sorrell, J. M. & Caplan, A. I. Fibroblast heterogeneity: more than skin deep. J Cell Sci117, 667-675, doi:10.1242/jcs.01005 (2004).
Marsh, E., Gonzalez, D. G., Lathrop, E. A., Boucher, J. & Greco, V. Positional Stability and Membrane Occupancy Define Skin Fibroblast Homeostasis In Vivo. Cell175, 1620-1633 e1613, doi:10.1016/j.cell.2018.10.013 (2018).
Yin, R. H. et al. LncRNA-599547 contributes the inductive property of dermal papilla cells in cashmere goat through miR-15b-5p/Wnt10b axis. Anim Biotechnol33, 493-507, doi:10.1080/10495398.2020.1806860 (2022).
Bai, W. L. et al. LncRNAs in Secondary Hair Follicle of Cashmere Goat: Identification, Expression, and Their Regulatory Network in Wnt Signaling Pathway. Anim Biotechnol29, 199-211, doi:10.1080/10495398.2017.1356731 (2018).
Jiao, Q. et al. Identification and molecular analysis of cashmere goat lncRNAs reveal their integrated regulatory network and potential roles in secondary hair follicle. Anim Biotechnol32, 719-732, doi:10.1080/10495398.2020.1747477 (2021).
Si, Y. et al. LncRNA PlncRNA‑1 regulates proliferation and differentiation of hair follicle stem cells through TGF‑beta1‑mediated Wnt/beta‑catenin signal pathway. Mol Med Rep17, 1191-1197, doi:10.3892/mmr.2017.7944 (2018).
Cai, B. et al. Long non‑coding RNA regulates hair follicle stem cell proliferation and differentiation through PI3K/AKT signal pathway. Mol Med Rep17, 5477-5483, doi:10.3892/mmr.2018.8546 (2018).
Xing, C., Sun, S. G., Yue, Z. Q. & Bai, F. Role of lncRNA LUCAT1 in cancer. Biomed Pharmacother134, 111158, doi:10.1016/j.biopha.2020.111158 (2021).
Arsenijevic, T. et al. CDK4/6 Inhibitors in Pancreatobiliary Cancers: Opportunities and Challenges. Cancers (Basel)15, doi:10.3390/cancers15030968 (2023).
Zheng, W. & Yu, A. EZH2-mediated suppression of lncRNA-LET promotes cell apoptosis and inhibits the proliferation of post-burn skin fibroblasts. Int J Mol Med41, 1949-1957, doi:10.3892/ijmm.2018.3425 (2018).
Choi, N., Kim, W. S., Oh, S. H. & Sung, J. H. Epiregulin promotes hair growth via EGFR-medicated epidermal and ErbB4-mediated dermal stimulation. Cell Prolif53, e12881, doi:10.1111/cpr.12881 (2020).
Ji, J. et al. Fibronectin 1 inhibits the apoptosis of human trophoblasts by activating the PI3K/Akt signaling pathway. Int J Mol Med46, 1908-1922, doi:10.3892/ijmm.2020.4735 (2020).
Xing, A. P. et al. Cigarette smoke extract stimulates rat pulmonary artery smooth muscle cell proliferation via PKC-PDGFB signaling. J Biomed Biotechnol2012, 534384, doi:10.1155/2012/534384 (2012).
Tomita, Y., Akiyama, M. & Shimizu, H. PDGF isoforms induce and maintain anagen phase of murine hair follicles. J Dermatol Sci43, 105-115, doi:10.1016/j.jdermsci.2006.03.012 (2006).
Varfolomeev, E. & Vucic, D. Intracellular regulation of TNF activity in health and disease. Cytokine101, 26-32, doi:10.1016/j.cyto.2016.08.035 (2018).
Wang, X. et al. Macrophages induce AKT/beta-catenin-dependent Lgr5(+) stem cell activation and hair follicle regeneration through TNF. Nat Commun8, 14091, doi:10.1038/ncomms14091 (2017).
Xiang, C., Li, H. & Tang, W. Targeting CSF-1R represents an effective strategy in modulating inflammatory diseases. Pharmacol Res187, 106566, doi:10.1016/j.phrs.2022.106566 (2023).
Fujiwara, T. et al. CSF1/CSF1R Signaling Inhibitor Pexidartinib (PLX3397) Reprograms Tumor-Associated Macrophages and Stimulates T-cell Infiltration in the Sarcoma Microenvironment. Mol Cancer Ther20, 1388-1399, doi:10.1158/1535-7163.MCT-20-0591 (2021).
Zhu, M. et al. Platelet sonicates activate hair follicle stem cells and mediate enhanced hair follicle regeneration. J Cell Mol Med24, 1786-1794, doi:10.1111/jcmm.14873 (2020).
Gentile, P. et al. Platelet-Rich Plasma and Micrografts Enriched with Autologous Human Follicle Mesenchymal Stem Cells Improve Hair Re-Growth in Androgenetic Alopecia. Biomolecular Pathway Analysis and Clinical Evaluation. Biomedicines7, doi:10.3390/biomedicines7020027 (2019).
Rojo, R., Pridans, C., Langlais, D. & Hume, D. A. Transcriptional mechanisms that control expression of the macrophage colony-stimulating factor receptor locus. Clin Sci (Lond)131, 2161-2182, doi:10.1042/CS20170238 (2017).
Hegde, S. et al. Stat3 promotes the development of erythroleukemia by inducing Pu.1 expression and inhibiting erythroid differentiation. Oncogene28, 3349-3359, doi:10.1038/onc.2009.202 (2009).
Goto, H. et al. Restoring PU.1 induces apoptosis and modulates viral transactivation via interferon-stimulated genes in primary effusion lymphoma. Oncogene36, 5252-5262, doi:10.1038/onc.2017.138 (2017).
Gupta, P. et al. PU.1: An ETS family transcription factor that regulates leukemogenesis besides normal hematopoiesis. Stem Cells Dev15, 609-617, doi:10.1089/scd.2006.15.609 (2006).
Ross, I. L., Yue, X., Ostrowski, M. C. & Hume, D. A. Interaction between PU.1 and another Ets family transcription factor promotes macrophage-specific Basal transcription initiation. J Biol Chem273, 6662-6669, doi:10.1074/jbc.273.12.6662 (1998).
Villar, D. et al. Enhancer evolution across 20 mammalian species. Cell160, 554-566, doi:10.1016/j.cell.2015.01.006 (2015).
Zhu, H. L., Gao, Y. H., Yang, J. Q., Li, J. B. & Gao, J. Serenoa repens extracts promote hair regeneration and repair of hair loss mouse models by activating TGF-beta and mitochondrial signaling pathway. Eur Rev Med Pharmacol Sci22, 4000-4008, doi:10.26355/eurrev_201806_15285 (2018).
Chen, Y. et al. PI3K/Akt signaling pathway is essential for de novo hair follicle regeneration. Stem Cell Res Ther11, 144, doi:10.1186/s13287-020-01650-6 (2020).
He, N., Su, R., Wang, Z., Zhang, Y. & Li, J. Exploring differentially expressed genes between anagen and telogen secondary hair follicle stem cells from the Cashmere goat (Capra hircus) by RNA-Seq. PLoS One15, e0231376, doi:10.1371/journal.pone.0231376 (2020).
Schmittgen, T. D. & Livak, K. J. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc3, 1101-1108, doi:10.1038/nprot.2008.73 (2008).
Machanick, P. & Bailey, T. L. MEME-ChIP: motif analysis of large DNA datasets. Bioinformatics27, 1696-1697, doi:10.1093/bioinformatics/btr189 (2011).
Rombel, I. T., Sykes, K. F., Rayner, S. & Johnston, S. A. ORF-FINDER: a vector for high-throughput gene identification. Gene282, 33-41, doi:10.1016/s0378-1119(01)00819-8 (2002).
Kang, Y. J. et al. CPC2: a fast and accurate coding potential calculator based on sequence intrinsic features. Nucleic Acids Res45, W12-W16, doi:10.1093/nar/gkx428 (2017).

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LncRNA018392 promotes proliferation of Liaoning cashmere goat skin fibroblasts through up-regulation of CSF1R by binding to SPI1

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Abstract

Figures

1. Introduction

2. Results

2.1. Bioinformatics Analysis of lncRNA018392

2.2. Melatonin Promotes Proliferation of Goat Skin Fibroblasts Through Up-regulation of lncRNA018392

2.3 lncRNA018392 Promotes the Proliferation of Goat Skin Fibroblasts Through Up-regulation of CSF1R

2.4. LncRNA018392 Promotes Proliferation of Goat Skin Fibroblasts Through Recruitment of SPI1

2.4. ChIP-Seq to Verify the Binding of SPI1 to CSF1R

3. Discussion

4. Materials And Methods

4.1. Cell culture and Treatment

4.2. RNA Isolation and Quantitative RT-PCR Analysis

4.3. Cell Counting Kit-8

4.4. Cell Cycle Analysis and Apoptosis Assay

4.5. Plasmid Preparation

4.6. Small Interfering RNA

4.7. Luciferase Reporter Gene Assay

4.8. RNA Immunoprecipitation Assay

4.9. Chromatin Immunoprecipitation Sequencing (ChIP-Seq)

4.10. Bioinformatics Analysis

Declarations

References

Additional Declarations

Supplementary Files

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