Single nucleotide polymorphisms in the KRT82 promoter region modulate irregular thickening and patchiness in the dorsal skin of New Zealand rabbits.

BACKGROUND: While rabbits are used as models in skin irritation tests, the presence of irregular patches and thickening on the dorsal skin can affect precise evaluation. In this study, genes associated with patchiness or non-patchiness on the dorsal skin of New Zealand rabbits were investigated to identify potential regulators of the patchiness phenotype. RESULTS: The results showed that parameters associated with hair follicles (HFs), such as HF density, skin thickness, and HF depth, were augmented in rabbits with the patchiness phenotype relative to the non-patchiness phenotype. A total of 592 differentially expressed genes (DEGs) were identified between the two groups using RNA-sequencing. These included KRT72, KRT82, KRT85, FUT8, SOX9, and WNT5B. The functions of the DEGs were investigated by GO and KEGG enrichment analyses. A candidate gene, KRT82, was selected for further molecular function verification. There was a significant positive correlation between KRT82 expression and HF-related parameters, and KRT82 overexpression and knockdown experiments with rabbit dermal papilla cells (DPCs) showed that it regulated genes related to skin and HF growth and development. Investigation of single nucleotide polymorphisms (SNPs) in the exons and promoter region of KRT82 identified four SNPs in the promoter region but none in the exons. The G.-631G > T, T.-696T > C, G.-770G > T and A.-873 A > C alleles conformed to the Hardy - Weinberg equilibrium, and three identified haplotypes showed linkage disequilibrium. Luciferase reporter assays showed that the core promoter region of KRT82 was located in the - 600 to - 1200 segment, in which the four SNPs were located. CONCLUSIONS: The morphological characteristics of the patchiness phenotype were analyzed in New Zealand rabbits and DEGs associated with this phenotype were identified by RNA-sequencing. The biological functions of the gene KRT82 associated with this phenotype were analyzed, and four SNPs were identified in the promoter region of the gene. These findings suggest that KRT82 may be a potential biomarker for the breeding of experimental New Zealand rabbits.

4D label-free quantitative proteomic analysis identifies CRABP1 as a novel candidate gene for litter size in rabbits†.

In commercial rabbit breeding, litter size is a crucial reproductive trait. This trait directly determines the reproductive ability of female rabbits and is crucial for evaluating the production efficiency. We here compared differentially expressed proteins of in the ovary tissue from New Zealand female rabbits with high (H) and low (L) litter sizes by using 4D label-free quantitative proteomic technology and identified 92 differential proteins. The biological functions of these proteins were revealed through gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Most distributions of GO and KEGG were related to reproduction, growth development, and metabolism. Furthermore, a novel candidate gene cellular retinoic acid binding protein-1 (CRABP1), which was highly expressed in the L group, was selected for further biological function verification. The Cell Counting Kit-8 (CCK-8) assay and flow cytometry analysis revealed that CRABP1 can promote granulosa cell (GC) apoptosis and inhibit GC proliferation. Furthermore, qRT-PCR and western blotting analysis revealed that CRABP1 regulates the genes (HSD17B1, Wnt-10b, FSHR, TAF4B, BMP15, and BMP6) and protein (Wnt-10b) associated with steroid hormone synthesis and follicle development. The PCR product direct sequencing method revealed single nucleotide polymorphisms in the core promoter region of CRABP1. Luciferase activity assays revealed that the transcriptional activity of the GG genotype was significantly higher than that of the TT or TG genotype. Different genotypes are accompanied by changes in transcription factors, which indicates that T-359G polymorphism can regulate CRABP1 expression. In general, we identified litter size-related genes and revealed the mechanism underlying the effect of CRABP1 on litter size. CRABP1 serves as a key factor in the reproductive capacity of rabbits and can act as a molecular biomarker for the breeding of New Zealand rabbits.

NRAS promotes the proliferation of melanocytes to increase melanin deposition in Rex rabbits.

Melanocytes play a major role in the formation of mammalian fur color and are regulated by several genes. Despite playing the pivotal role in the study of melanoma, the mechanistic role of NRAS (neuroblastoma RAS viral oncogene homolog) in the formation of mammalian epidermal color is still elusive. First of all, the expression levels of NRAS mRNA and protein in the dorsal skin of different colored Rex rabbits were detected by qRT-PCR and Western blot. Then, the subcellular localization of NRAS was identified in melanocytes by indirect immunofluorescence. Next, the expression of NRAS was overexpressed and knocked down in melanocytes, and its efficiency was verified by qRT-PCR and Western blot. Subsequently, NaOH, CCK-8, and Annexin V-FITC were used to verify the changes in melanin content, proliferation, and apoptosis in melanocytes. Finally, we analyzed the regulation of NRAS on other genes (MITF, TYR, DCT, PMEL, and CREB) that affect melanin production. In silico studies showed NRAS as a stable and hydrophilic protein, and it is localized in the cytoplasm and nucleus of melanocytes. The mRNA and protein expression levels of NRAS were significantly different in skin of different colored Rex rabbits, and the highest level was found in black skin (P < 0.01). Moreover, the NRAS demonstrated impact on the proliferation, apoptosis, and melanin production of melanocytes (P < 0.05), and the strong correlation of NRAS with melanin-related genes was evidently observed (P < 0.05). Our results suggested that NRAS can be used as a gene that regulates melanin production and controls melanocyte proliferation and apoptosis, providing a new theoretical basis for studying the mechanism of mammalian fur color formation.

Establishment and functional characterization of immortalized rabbit dermal papilla cell lines.

Hair follicle (HF) undergo periodic growth and development in mammals, which regulated by dermal papilla cells (DPCs) are reported to play an important role in HF morphogenesis and development. However, primary DPCs have low proliferative activity, age quickly, and fresh cell isolation is both time-consuming and laborious. In this study, we introduced the SV40 large T antigen (SV40T) into dissociated early passage rabbit vibrissae DPCs with lentiviral vectors and established seven immortalized DPC lines (R-1, R-2, R-3, R-4, R-5, R-6 and R-7). These cell lines displayed early passage morphology and high alkaline phosphatase activity. RT-PCR and immunofluorescence staining showed that all the immortalized cell lines expressed the DPC markers (α-SMA, IGF1, ALPL, FGF2, BMP2 and TGFß2), but α-SMA was only expressed well in R-3, R-4, and R-7. Furthermore, it was found that R-7 was the only line to survive beyond 50 passages. Compared to melanoma cells, R-7 did not undergo malignant transformation. Karyotyping and cell growth viability analysis illustrated that the R-7 cell line preserved the basic characteristics of primary DPCs. The R-7 DPCs established have potential application for future hair research. The study provides the theoretical basis in the cell research of HF growth and development.

Dermal PapillaCell-Derived Exosomes Regulate Hair Follicle Stem Cell Proliferation via LEF1.

Hair follicle (HF) growth and development are controlled by various cell types, including hair follicle stem cells (HFSCs) and dermal papilla cells (DPCs). Exosomes are nanostructures that participate in many biological processes. Accumulating evidence indicates that DPC-derived exosomes (DPC-Exos) mediate HFSC proliferation and differentiation during the cyclical growth of hair follicles. In this study, we found that DPC-Exos increase ki67 expression and CCK8 cell viability readouts in HFSCs but reduce annexin staining of apoptotic cells. RNA sequencing of DPC-Exos-treated HFSCs identified 3702 significantly differentially expressed genes (DEGs), including BMP4, LEF1, IGF1R, TGFß3, TGFα, and KRT17. These DEGs were enriched in HF growth- and development-related pathways. We further verified the function of LEF1 and showed that overexpression of LEF1 increased the expression of HF development-related genes and proteins, enhanced HFSC proliferation, and reduced HFSC apoptosis, while knockdown of LEF1 reversed these effects. DPC-Exos could also rescue the siRNA-LEF1 effect in HFSCs. In conclusion, this study demonstrates that DPC-Exos mediated cell-to-cell communication can regulate HFSCs proliferation by stimulating LEF1 and provide novel insights into HF growth and development regulatory mechanisms.

Characterization and functional analysis of lncRNA2690 in regulating the growth cycle of the hair follicle in rabbits.

Hair follicles (HFs) achieve hair growth and renewal by periodic regeneration. Therefore, exploring the key factors affecting hair growth in rabbits is of great significance for precisely breeding Angora rabbits and improving the competitiveness of the rabbit industry. Based on the results of our previous studies, lncRNA2690 was differentially expressed in the HF cycle using lncRNA-Seq, and the full-length sequence was annotated by bioinformatics analysis. The lncRNA2690 is 363 nt long and is found on chromosome 14 from 163 321 514 to 163 321 872. The lncRNA2690 was predicted to not have the coding ability through open reading frame and CPC2, and the nuclear-cytoplasmic separation experiment showed the lncRNA2690 to be highly expressed in the nucleus (p < 0.01). The expression pattern of lncRNA2690 was further analyzed in the different HF development stages of Angora rabbits using quantitative real-time PCR. The results showed that lncRNA2690 was periodically expressed in HF development, and the expression level was found to be high in the HF resting phases. The overexpression and knockdown of lncRNA2690 were found to significantly upregulate and downregulate the expression of the genes WNT2, CCND1, BMP2, LEF1, and SIAH1 in the rabbit dermal papilla cells (p < 0.01), promoting cell apoptosis and inhibiting cell proliferation (p < 0.01). This indicated that lncRNA2690 negatively regulates the periodic regeneration of the HFs in rabbits. These results provide a basis for the further study of lncRNA2690 in the HF growth cycle of Angora rabbits.

Effects of feeding restriction on skeletal muscle development and functional analysis of TNNI1 in New Zealand white rabbits.

While restricting nutrition can improve diseases related to the digestive tract, excessive restriction of food intake can also lead to malnutrition and delayed physical growth. Therefore, this brings the demand to study the effect and potential mechanism of restricted feeding on skeletal muscle development in rabbits. This study utilized hematoxylin-eosin (HE) staining to detect muscle fiber area which depicted significant reduction in skeletal muscle fiber upon 30% feed restriction (p < 0.05). The control group and 30% feed restricted group showed 615 deferentially expressed genes (DEGs). Through the GO and KEGG functional enrichment analysis demonstrated 28 DEGs related to muscle development. KEGG analysis showed enrichment of pathways including PI3K/Akt signaling pathway, MAPK signaling pathway, and Hedgehog signaling pathway. Further, the full length of troponin I1, slow skeletal type (TNNI1) was cloned. We studied the expression of skeletal muscle differentiation-related genes such as MyoD, Myf5 gene and Desmin. Specifically, the TNNI1 gene overexpression and knockdown studies were conducted. The over-expression of TNNI1 significantly enhanced the expression of the skeletal muscle development-related genes. Contrastingly, the silencing of TNNI1 gene reduced the expression significantly. These findings showed that TNNI1 may be a regulator for regulating the expression of muscle development-related genes.

Simulation Experiment and Analysis of GNSS/INS/LEO/5G Integrated Navigation Based on Federated Filtering Algorithm.

This article examines the positioning effect of integrated navigation after adding an LEO constellation signal source and a 5G ranging signal source in the context of China's new infrastructure construction. The tightly coupled Kalman federal filters are used as the algorithm framework. Each signal source required for integrated navigation is simulated in this article. At the same time, by limiting the range of the azimuth angle and visible height angle, different experimental scenes are simulated to verify the contribution of the new signal source to the traditional satellite navigation, and the positioning results are analyzed. Finally, the article compares the distribution of different federal filtering information factors and reveals the method of assigning information factors when combining navigation with sensors with different precision. The experimental results show that the addition of LEO constellation and 5G ranging signals improves the positioning accuracy of the original INS/GNSS by an order of magnitude and ensures a high degree of positioning continuity. Moreover, the experiment shows that the federated filtering algorithm can adapt to the combined navigation mode in different scenarios by combining different precision sensors for navigation positioning.

Promoter Methylation Changes in KRT17: A Novel Epigenetic Marker for Wool Production in Angora Rabbit.

Wool production is an important economic trait of Angora rabbits. Exploring molecular markers related to wool production is one of the essentials of Angora rabbits' breeding. KRT17 (Keratin 17) is an important gene of hair follicle development, which must be explored for genetic/epigenetic variation to assess its effect on wool production. Based on the effective wool production data of 217 Angora rabbits, the high and low yield groups were screened with 1.5 standard deviations of the population mean. The full-length sequence of KRT17 was obtained by rapid amplification of cDNA ends technology, and the polymorphism was analyzed in the promoter, exon, and intron regions by direct sequencing. KRT17, SP1 over-expression plasmids, and siRNA were constructed and transfected into dermal papilla cells. The mRNA expressions of relevant genes were analyzed by RT-qPCR. The methylation level of the KRT17 promoter was determined by Bisulfite Sequencing PCR. Dual-luciferase system, site-directed mutagenesis, and electrophoretic mobility shift assays were used to analyze the binding relationship between SP1 and the promoter of KRT17. The structure map of KRT17 was drawn, and no SNPs were found in the promoter, exon, and intron, indicating a relatively conserved structure of KRT17. Expression of KRT17 was significantly higher in cutaneous tissues than in other tissues and was significantly upregulated in the high-yield group compared to the low-yield group (p < 0.05). Furthermore, the overall high methylation levels of KRT17 CpG I and CpG III showed significant association with low wool yield; the methylation levels of 5 CpG locus (CpG I site 4 and CpG III site 2−5) were significantly different between the high and low yield groups (p < 0.05). The methylation levels of 3 CpG locus (CpG I site 4 and CpG III site 4, 14) showed a significant correlation with KRT17 expression (p < 0.05). Overall, CpG III site 4 significantly affects wool production and KRT17 expressions (p < 0.05). This site promotes SP1 binding to the KRT17 promoter region (CGCTACGCCC) to positively regulate the KRT17 expression. KRT17 CpG III site 4 can be used as candidate epigenetic markers for the breeding of high wool-producing Angora rabbits.

DNA Methylation Mediates lncRNA2919 Regulation of Hair Follicle Regeneration.

Hair follicles (HFs) are organs that periodically regenerate during the growth and development of mammals. Long non-coding RNAs (lncRNAs) are non-coding RNAs with crucial roles in many biological processes. Our previous study identified that lncRNA2919 is highly expressed in catagen during the HF cycle. In this study, the in vivo rabbit model was established using intradermal injection of adenovirus-mediated lncRNA2919. The results showed that lncRNA2919 decreased HF depth and density and contributed to HF regrowth, thereby indicating that lncRNA2919 plays a negative role in HF regeneration. Moreover, methylation levels of the lncRNA2919 promoter at different HF cycle stages were detected through bisulfite sequencing. The key CpG site that negatively correlates with lncRNA2919 expression during the HF cycle was identified. 5-Aza-dc-induced demethylation upregulated lncRNA2919 expression, and the core promoter region of lncRNA2919 was verified on the basis of luciferase activity. Furthermore, we found that DNA methylation could prevent the binding of EGR1 to the lncRNA2919 promoter region, thereby affecting the transcriptional expression of lncRNA2919. Collectively, DNA methylation inhibits the transcriptional expression of lncRNA2919, which plays a vital role in the HF cycle and HF regrowth. These findings contribute to the basic theory of epigenetics in HF biology and provide references for further research in HF disease treatment and animal wool production.

Characterization of HTATIP2 and its role during hair follicle cycles in Angora rabbit.

Hair follicle (HF) growth and cycling is a complex biological process that occurs in most mammals. As HF growth and cycling directly impacts rabbit wool yield, it is important to better understand the potential regulation pattern of HF development. Our previous study demonstrated that HTATIP2 may participate in regulating rabbit HF cycles, but the molecular mechanism of HTATIP2 remained unclear. In this study, the coding sequence of the HTATIP2 gene in Angora rabbit was cloned. The length of the coding region sequence was 840 bp, which could code 279 amino acids, and exhibited high homology in different mammals. Bioinformatics analyses indicated that the HTATIP2 protein is stable, hydrophilic, located around the cytoplasm, and has a putative signal peptide. Moreover, we verified that HTATIP2 is highly expressed during catagen and telogen of the HF cycle. The overexpression vector was constructed and siRNAs were designed. Overexpression and knockdown of HTATIP2 appeared to regulate JAK-STAT pathway genes, such as BCL2, CCND1, c-Myc, and STAT2. It is therefore likely that HTATIP2 promotes cell apoptosis and inhibits cell proliferation. Our results indicate that HTATIP2 is highly expressed during catagen and telogen and may play an important role in JAK-STAT signaling. This study provides a theoretical foundation for investigating HTATIP2 in biological processes.

Characterization and functional analysis of SIAH1 during skin and hair follicle development in the angora rabbit (Oryctolagus cuniculus).

BACKGROUND: Seven in absentia homolog 1 (SIAH1) is an E3 ubiquitin ligase containing a RING-finger domain and a key regulator of normal development. Skin and hair follicle development is a complex and special process of morphogenesis involving multiple signaling pathways. SIAH1 is enriched in the Wnt signaling pathway and potentially related to hair follicle cycle and skin development. This study aims to provide evidence for the role of SIAH1 in skin and hair development. RESULTS: Full-length cloning and analysis of SIAH1 was conducted to better understand its function. Phylogenetically, the sequence of SIAH1 in the rabbit shares the greatest homology with Home sapiens, Pongo abelii and Mus mulatta. Based on the rabbit hair follicle synchronization model, we found that the expression level of SIAH1 in the regressive period of the rabbit hair cycle is significantly lower than in the active growth and rest periods. In addition, the mRNA expression levels of skin and hair follicle development-related genes changed significantly when SIAH1 was overexpressed and silenced. After SIAH1 overexpression, the expression levels of WNT2, LEF1 and FGF2 decreased, and those of SFRP2 and DKK1 increased (P < 0.05). After interference of SIAH1, the expression levels of WNT2, LEF1 and FGF2 increased (P < 0.05), and SFRP2 and DKK1 decreased. CONCLUSIONS: SIAH1 can affect skin and hair follicle development and exert an inhibitory effect. These results could provide foundamental insights into the role of SIAH1 as a target gene in rabbit skin and hair follicle development.

miR-218-5p regulates skin and hair follicle development through Wnt/ß-catenin signaling pathway by targeting SFRP2.

The hair follicle is a complex biological system involved in a dynamic process governed by gene regulation. MicroRNAs play a critical role in the regulation of gene expression. We demonstrated that the expression of miR-218-5p and SFRP2 showed the opposite relationship in catagen and telogen phases and that miR-218-5p promoted the growth of hair shafts. The luciferase reporter assays confirmed that SFRP2 is the direct target of miR-218-5p. The expression of miR-218-5p may decrease the expression of SFRP2, which activates the Wnt signaling pathway, including the regulation of downstream genes and ß-catenin/T-cell-specific factor transcriptional activity. Moreover, miR-218-5p enhanced apoptosis, but inhibition of miR-218-5p decreased apoptosis and inhibited RAB-9 cell proliferation. In this study, we show that miR-218-5p positively regulates the Wnt signaling pathway by targeting SFRP2 and acts as a dynamic governor during skin and hair follicle development.

Correction to: Morphological Characterization and Gene Expression Patterns for Melanin Pigmentation in Rex Rabbit.

The original article has been published with an incorrect grant number in the Funding section.

Morphological Characterization and Gene Expression Patterns for Melanin Pigmentation in Rex Rabbit.

Animal melanin has an important role in the formation of animal fur and skin, which is determined by its quantities, character, and distribution. To identify the effect of melanin on the formation of multi-colored Rex rabbits (Black, Chinchilla, Beaver, Protein cyan, Protein yellow, White), the structure of hair follicles and melanin content in multi-colored Rex rabbit skins were observed by Hematoxylin and Eosin (H&E) staining and melanin staining, respectively. The melanin granules were primarily found in the epidermis and hair follicle roots. The melanin content of skin was measured by extracting melanin from skin tissue. The results demonstrated that the melanin content was the highest in the skin of black Rex rabbit. Additionally, we measured the mRNA and protein expression levels of melanin-related key genes (MITF and TYR) in the skin of different hair color by quantitative real-time PCR and Wes assay, respectively. The results revealed that the mRNA expression levels in the skin of black Rex rabbit was highly expressed when as compared with other Rex rabbit skin (P < 0.01), and they were the lowest in the skin of white Rex rabbit. Finally, correlation analysis was conducted between melanin content and the expression levels of mRNA and protein. The results indicated a significant correlation between melanin content and the mRNA expression of MITF (P < 0.05), but it was not correlated with the mRNA expression of TYR (P > 0.05). In summary, melanin deposition has important economic value, and the coat color of fur-bearing animals is partly determined by the melanin-related genes.

Slc7a11 Modulated by POU2F1 is Involved in Pigmentation in Rabbit.

Solute carrier family 7 member 11 (Slc7a11) is a cystine/glutamate xCT transporter that controls the production of pheomelanin pigment to change fur and skin color in animals. Previous studies have found that skin expression levels of Slc7a11 varied significantly with fur color in Rex rabbits. However, the molecular regulation mechanism of Slc7a11 in pigmentation is unknown. Here, rabbit melanocytes were first isolated and identified. The distribution and expression pattern of Slc7a11 was confirmed in skin from rabbits with different fur colors. Slc7a11 affected the expression of pigmentation related genes and thus affected melanogenesis. Meanwhile, Slc7a11 decreased melanocyte apoptosis, but inhibition of Slc7a11 enhanced apoptosis. Furthermore, the POU2F1 protein was found to bind to the -713 to -703 bp region of Slc7a11 promoter to inhibit its activity in a dual-luciferase reporter and site-directed mutagenesis assay. This study reveals the function of the Slc7a11 in melanogenesis and provides in-depth analysis of the mechanism of fur pigmentation.

Characterization and Establishment of an Immortalized Rabbit Melanocyte Cell Line Using the SV40 Large T Antigen.

Melanocytes (MCs) are specialized cells that synthesize melanin within the melanosome. Cultured MCs are useful in order to study their role in relation to pigmentation. However, MC isolation is laborious and the obtained cells have a limited culture time. In this study, we transformed lentivirus-mediated simian virus 40 Large T (SV40-LT) into primary rabbit melanocytes (Pri RMCs) to establish an immortalized cell line. Morphologically, the immortalized RMCs (Im RMC) were indistinguishable from the Pri RMCs, and dendrites were visible following Dopa staining. No significant differences in cell proliferation or growth between immortalized and primary RMCs were observed. Based on melanocyte-specific markers, the expression of MITF, TYR, and TYRP1 were detected by PCR, immunofluorescence staining, and western blot analysis. Through karyotype, soft agar, and tumorigenesis assays, the immortalized RMCs did not undergo malignant transformation. Our results show that Im RMCs can be used as a tool cell for future MC studies on the pigmentation mechanisms of fur animals.

Gene expression profiling analysis reveals fur development in rex rabbits (Oryctolagus cuniculus).

Fur is an important economic trait in rabbits. The identification of genes that influence fur development and knowledge regarding the actions of these genes provides useful tools for improving fur quality. However, the mechanism of fur development is unclear. To obtain candidate genes related to fur development, the transcriptomes of tissues from backs and bellies of Chinchilla rex rabbits were compared. Of the genes analyzed, 336 showed altered expression in the two groups (285 upregulated and 51 downregulated, P ≤ 0.05, fold-change ≥2 or ≤0.5). Using GO and KEGG to obtain gene classes that were differentially enriched, we found several genes to be involved in many important biological processes. In addition, we identified several signaling pathways involved in fur development, including the Wnt and MAPK signaling pathways, revealing mechanisms of skin and hair follicle development, and epidermal cell and keratinocytes differentiation. The obtained rabbit transcriptome and differentially expressed gene profiling data provided comprehensive gene expression information for SFRP2, FRZB, CACNG1, SLC25A4, and SLC16A3. To validate the RNA-seq data, the expression levels of eight differentially expressed genes involved in fur development were confirmed by qRT-PCR. The results of rabbit transcriptomic profiling provide a basis for understanding the molecular mechanisms of fur development.

ChIP-Seq analysis reveals PRKACB as a target gene of HOXC13 involved in rabbit hair follicle development.

Dermal papilla cells (DPCs) are key regulators of hair follicle (HF) development and growth, which not only regulate HF growth and cycling but play a role in the pathogenesis of hair loss. The transcription factor Homeobox C13 (HOXC13) can modulate the growth and development of HFs. Nevertheless, the specific genes and pathways regulated by HOXC13 in DPCs have yet to be determined. Thus, to gain a better understanding of genomic binding sites involved in HOXC13-regulated HF development, chromatin immunoprecipitation followed by high throughput sequencing (ChIP-Seq) was performed on rabbit DPCs with pcDNA3.1-3 × Flag-HOXC13 overexpression. A complete set of 9670 enrichment peaks was acquired by applying HOXC13-Flag ChIP. Subsequently, the peak sequence was annotated to the rabbit genome, revealing that 6.1 % of the peaks were identified within in the promoter region. Thereafter, five annotated genes were verified using RT-qPCR. The peak-associated genes were mainly enriched in signaling pathways related to HF development, such as MAPK and PI3K-Akt. Furthermore, by using a dual-luciferase reporter assay, we found that HOXC13 can target the protein kinase cAMP­dependent catalytic ß (PRKACB) promoter region (-1596 âˆ¼ -1107 bp) and inhibit its transcription, which was consistent with data obtained from ChIP-seq analysis. Overexpression of PRKACB gene significantly modulated the expression of BCL2, WNT2, LEF1, and SFRP2 genes related to HF development as determined by RT-qPCR (P < 0.01, P < 0.05). The CCK-8 and flow cytometry assays showed that PRKACB significantly inhibited the proliferation of DPCs and promoted apoptosis (P < 0.01). In conclusion, our research revealed that PRKACB has the potential to serve as a novel target gene of HOXC13, contributing to the regulation of the proliferation and apoptosis of DPCs. The process of identifying global target genes can contribute to the understanding of the intricate pathways that HOXC13 regulates in the growth of HFs.

Characterization and establishment of an immortalized rabbit ovary granulosa cell line for reproductive biology experiments.

Granulosa cells (GCs) are the key components of ovarian follicles and regulate the maturation, communication, growth, and development of oocytes. GCs have great potential as human therapeutic models and in livestock breeding. In this study, we established an immortalized cell line (Im-RGCs) by transforming primary rabbit granulosa cells (Pri-RGCs) with lentivirus-mediated simian virus 40 Large T (SV40LT). Morphologically, Im-RGCs were indistinguishable from Pri-RGCs and maintained intact cell structure as observed by H&E staining. Also, Im-RGCs exhibited no significant change in cell proliferation, viability, and growth. Furthermore, GC-specific markers, such as FSHR, StAR, CYP11A1, and CYP19A1, were examined by PCR, immunofluorescence, and Western blotting. ELISA and karyotype analysis showed that Im-RGCs can synthesize steroid hormones and maintain the normal number of chromosomes during the infinite passage. Furthermore, soft-agar cloning and nude mice tumorigenic experiments indicated the absence of any malignancy transformation in Im-RGCs. In conclusion, we successfully established the immortalized granulosa cell line of rabbit follicles that can be used for biological, animal husbandry, and female reproductive research.