LncRNA MTC-p65 Protein Mediates Skin Fibroblast Proliferation in Liaoning Cashmere Goat by Regulating the Nuclear Factor-Kappa B Signaling Pathway.

The aim of this study is to investigate the activation of NF-κB signaling pathway and the regulation of the expression of genes related to chorionic villus growth by the binding of LncRNA MTC (XLOC_005914) and p65 (transcription factor p65 [Capra hircus], XP_017898873.1). In addition, the regulation of LncRNA MTC and p65 binding on the proliferation of Liaoning Cashmere Goat skin fibroblasts is investigated. The upregulation of LncRNA MTC promoted the proliferation of skin fibroblasts, and the NF-κB signaling pathway played an important role in this process. Compared with the negative control (NC group), the expression of TNFα and NFKB2(NF-κB) genes was highly significantly up-regulated (P < 0.001), and NFKBIA(IκBɑ) genes were highly significantly down-regulated (P < 0.01) after LncRNA MTC overexpression (OE group). The expression levels of TNFα and NFκB-P-p65 proteins were upregulated in the OE group; NF-κB-p65 expression levels were upregulated in the nucleus, IκBα expression levels were downregulated in the cytoplasm, and P-IκBα expression levels were upregulated. LncRNA MTC and p65 proteins were co-localized in the cells. Meanwhile, LncRNA MTC and p65 protein showed significant nucleation in the OE group. RNA pull-down and LC-MS/MS verified that p65 protein was indeed an interacting protein of LncRNA MTC. LncRNA MTC binds to p65 protein, upregulates the expression of TNFα protein, nucleates p65 protein, and activates NF-κB signaling pathway to promote the proliferation of skin fibroblasts in Liaoning Cashmere Goat.

Effects of lncRNA MTC on protein expression in skin fibroblasts of Liaoning Cashmere goat based on iTRAQ technique.

Existing experiments have found a new intergenic lncRNA activated by melatonin, which is called lncRNA MTC. However, the regulatory mechanism of lncRNA MTC in Liaoning Cashmere goat skin fibroblasts has not been clarified. Specific knockdown of lncRNA MTC inhibits cell proliferation and increases apoptosis. iTRAQ reagent was used for relative and absolute quantification of proteins in lncRNA MTC-KD and NC groups to evaluate changes in protein expression during dermal fibroblast development following lncRNA MTC deletion. A total of 5931 proteins were found in Liaoning Cashmere goat skin fibroblasts, of which 123 were differentially expressed, including 32 up-regulated proteins and 91 down-regulated proteins. Of the 91 down-regulated proteins, 32 act mainly through related pathways (e.g., cell cycle, mitochondrial function, ribosomal structure, vesicular transport, cytoskeletal components and skin morphogenesis). LncRNA MTC facilitates the proliferation of Liaoning Cashmere goat skin fibroblasts by regulating ITGB5, TlN2, CTSS, POLG, RAP1B, CHAF1A, CDCA8 and other proteins involved in cell proliferation. The results of this study provide some candidate proteins for the in-depth investigation of the molecular mechanism of lncRNA MTC, which facilitates hair growth in cashmere goats and provides more insights into their regulatory networks and biochemical pathways.

Study on the roles of melatonin in regulating dermal fibroblast growth in Liaoning cashmere goats by transcriptome sequencing.

In this study, the genes related to the Downy growth of Liaoning cashmere goats were screened for their expression with simultaneous melatonin administration, so as to investigate the effects of target genes on the proliferation of skin fibroblasts in this animal species. Genes related to the villus growth of skin fibroblasts were screened by in vitro transcriptome sequencing and verified by qPCR. In addition, gene overexpression and interference were used to study the effects of target genes on the proliferation of skin fibroblasts. Groups treated with M1_24H, M2_24H and M2_72H exhibited significant differences compared with the control group. Among them, the differentially expressed transcripts in the M2_72H group were significantly enriched in the TNF and NOD-like receptor signaling pathways, which are associated with the villus. In addition, eight differentially expressed genes were screened from the TNF and the NOD-like receptor signaling pathways. Verification by qPCR showed that the expression of TNF-α, IL-6, TNFAIP3, PYCARD and NFKBIA genes were significantly upregulated, which was consistent with the sequencing results. Melatonin treatments can significantly lead to an increase in the expression of IL-6 and TNF-α genes. Besides, melatonin treatments can affect cashmere growth in Liaoning cashmere goats by regulating several signaling pathways, including TNF, NOD-like receptor and NF-κB.

Effects of sodium arsenite and dimethyl arsenic acid on Liaoning cashmere goat skin fibroblasts.

The morphology and oxidation state of arsenic in its compounds affects the skin cell toxicity. Accordingly, the present study was conducted to explore the effects of two different arsenic compounds on the proliferation and survival of Liaoning cashmere goat skin fibroblasts. Based on MTT assay results, at 24 h, the proliferation concentration, critical concentration, and half inhibitory concentration (IC50) of sodium arsenite were 0.50, 5.00, and 45.66 µmol/L, respectively. The corresponding values for dimethyl arsenic acid were 0.85, 1.00, and 38.68 mmol/L. Immunofluorescence, transmission electron microscopy, and mitochondria membrane potential (MMP) assays showed that sodium arsenite promotes microtubule polymerization and increases MMP, while cells treated with dimethyl arsenic acid exhibited cytoskeletal collapse and decreased MMP. In the IC50 groups for both arsenic agents, the cytoskeletons collapsed, microtubules were gathered into bundles, and MMP was significantly decreased. Dimethyl arsenic acid had a stronger effect on MMP than sodium arsenite. Flow cytometry revealed a slightly lower occurrence of apoptosis in the sodium arsenite proliferation group, while it was slightly increased in the dimethyl arsenic acid proliferation group. Apoptosis was increased more significantly in the sodium arsenite IC50 group than in the dimethyl arsenic acid IC50 group. These results indicate that the differences in cell proliferation and cytotoxicity induced by inorganic and organic arsenic are related to their effects on cellular structures.

Expression of lipid-protein gene PLP2 in Liaoning cashmere goat.

The relationship between PLP2 gene and cashmere fiber quality of Liaoning cashmere goat was investigated. The sheep fibroblast cells were treated with exogenous cytokines and melatonin, independently, and RNA interference, RT-PCR and in situ hybridization were utilized for investigating the PLP2 gene regulation mechanism underlying the Liaoning cashmere growth. The results showed that the expression of PLP2 gene in the prosperous and degenerative stage is higher than that of the primary follicle, indicating that the PLP2 gene promotes the secondary follicle, wherein the gene is expressed only in the inner root sheath, suggesting its correlation to hair loss. The results of RT-PCR showed that the trend of FGF5 expression in PLP2 gene was positively regulated. The influence of MT on the expression of PLP2 gene was negatively regulated, and the inhibition was gradually enhanced with the passage of time. Studies have confirmed that the Noggin gene is an inhibitor of the BMP signaling pathway. After the noggin gene interferes with the lentivirus infection, the expression of the PLP2 gene is downregulated. Therefore, the PLP2 gene, along with the other suppressor genes including the noggin gene, might affect the development of hair follicles by inhibiting the BMP（Bone morphogenetic proteins）pathway.

Gene Expression and Localization of LAMTOR3 in the Skin Cells of Liaoning Cashmere Goats.

Liaoning cashmere goats are the most precious genetic resources in China. The function of LAMTOR3 [late endosomal/lysosomal adaptor, mitogen-activated protein kinase (MAPK), and mammalian target of rapamycin activator 3/MAPK scaffold protein 1] gene is expressed in the skin of Liaoning cashmere goats. In situ hybridization (ISH) found that LAMTOR3 is expressed in the inner root sheath (IRS) of hair follicles. During the anagen or catagen phase, the expression of LAMTOR3 is higher in secondary hair follicles than in primary hair follicles. Expression of LAMTOR3 in skin cells treated with melatonin or insulin-like growth factor-1 (IGF-1) is lower than in untreated cells. In addition, the simultaneous treatment of fibroblast growth factor 5 and melatonin decrease the expression of LAMTOR3 in skin cells. The simultaneous treatment with melatonin and 10-5 g/L IGF-1 or 10-4 g/L IGF-1 increases the expression of LAMTOR3 gene in skin cells. If Noggin expression is decreased, then LAMTOR3 expression is increased. This hypothesis suggested that LAMTOR3 influences the character of cashmere fiber, and it may regulate the development of hair follicle and cashmere growth by inducing the MAPK signaling pathway.

Long noncoding RNA and gene expression analysis of melatonin-exposed Liaoning cashmere goat fibroblasts indicating cashmere growth.

Cashmere produced from Liaoning cashmere goat is highly valuable. Melatonin is an important factor affecting cashmere growth and can regulate the growth cycle via effects on gene expression. Long noncoding RNAs (lncRNAs) regulate gene expression, but detailed studies of their effect on hair growth are lacking. To explore how lncRNA mediates the effects of melatonin on cashmere growth, we used RNA-Seq including a control condition (C) and three melatonin treatments (1.0 g/L 24 h (M1_24H), 0.2 g/L 24 h (M2_24H), 0.2 g/L 72 h (M2_72H)). M1_24H, M2_24H, and M2_72H had 32, 10, and 113 differentially expressed lncRNAs, respectively. Gene ontology (GO) and pathway analyses results showed that melatonin was most beneficial to cashmere growth at 0.2 g/L 72 h, and nuclear factor (NF)-κB signaling corresponding to an effect of LncRNA MTC was involved in hair follicle development. We found that melatonin upregulated XLOC_005914 lncRNA (LncRNA MTC). Proliferation increased in the 0.2 g/L 72 h condition and cells with high LncRNA MTC expression, but it was reduced in fibroblasts with knocked down LncRNA MTC expression. This is the first report that LncRNA MTC promotes fibroblast proliferation and regulates hair follicle development and cashmere growth by activating NF-κB signaling.

Cashmere growth control in Liaoning cashmere goat by ovarian carcinoma immunoreactive antigen-like protein 2 and decorin genes.

OBJECTIVE: The study investigated the biological functions and mechanisms for controlling cashmere growth of Liaoning cashmere goat by ovarian carcinoma immunoreactive antigen-like protein 2 (OCIAD2) and decorin (DCN) genes. METHODS: cDNA library of Liaoning cashmere goat was constructed in early stages. OCIAD2 and DCN genes related to cashmere growth were identified by homology analysis comparison. The expression location of OCIAD2 and DCN genes in primary and secondary hair follicles (SF) was performed using in situ hybridization. The expression of OCIAD2 and DCN genes in primary and SF was performed using real-time polymerase chain reaction (PCR). RESULTS: In situ hybridization revealed that OCIAD2 and DCN were expressed in the inner root sheath of Liaoning cashmere goat hair follicles. Real-time quantitative PCR showed that these genes were highly expressed in SF during anagen, while these genes were highly expressed in primary hair follicle in catagen phase. Melatonin (MT) inhibited the expression of OCIAD2 and promoted the expression of DCN. Insulin-like growth factors-1 (IGF-1) inhibited the expression of OCIAD2 and DCN, while fibroblast growth factors 5 (FGF5) promoted the expression of these genes. MT and IGF-1 promoted OCIAD2 synergistically, while MT and FGF5 inhibited the genes simultaneously. MT+IGF-1/MT+FGF5 inhibited DCN gene. RNAi technology showed that OCIAD2 expression was promoted, while that of DCN was inhibited. CONCLUSION: Activation of bone morphogenetic protein (BMP) signaling pathway up-regulated OCIAD2 expression and stimulated SF to control cell proliferation. DCN gene affected hair follicle morphogenesis and periodic changes by promoting transforming growth factor-ß (TGF-ß) and BMP signaling pathways. OCIAD2 and DCN genes have opposite effects on TGF-ß signaling pathway and inhibit each other to affect the hair growth.

Molecular characterization and expression pattern of a novel Keratin-associated protein 11.1 gene in the Liaoning cashmere goat (Capra hircus).

OBJECTIVE: An experiment was conducted to determine the relationship between the KAP11.1 and the regulation wool fineness. METHODS: In previous work, we constructed a skin cDNA library and isolated a full-length cDNA clone termed KAP11.1. On this basis, we conducted a series of bioinformatics analysis. Tissue distribution of KAP11.1 mRNA was performed using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. The expression of KAP11.1 mRNA in primary and secondary hair follicles was performed using real-time PCR (real-time polymerase chain reaction) analysis. The expression location of KAP11.1 mRNA in primary and secondary hair follicles was performed using in situ hybridization. RESULTS: Bioinformatics analysis showed that KAP11.1 gene encodes a putative 158 amino acid protein that exhibited a high content of cysteine, serine, threonine, and valine and has a pubertal mammary gland) structural domain. Secondary structure prediction revealed a high proportion of random coils (76.73%). Semi-quantitative RT-PCR showed that KAP11.1 gene was expressed in heart, skin, and liver, but not expressed in spleen, lung and kidney. Real time PCR results showed that the expression of KAP11.1 has a higher expression in catagen than in anagen in the primary hair follicles. However, in the secondary hair follicles, KAP11.1 has a significantly higher expression in anagen than in catagen. Moreover, KAP11.1 gene has a strong expression in inner root sheath, hair matrix, and a lower expression in hair bulb. CONCLUSION: We conclude that KAP11.1 gene may play an important role in regulating the fiber diameter.

The Study on Biological Function of Keratin 26, a Novel Member of Liaoning Cashmere Goat Keratin Gene Family.

In our research, we explored the relationship between Keratin 26 and the regulation of fine hair, BMP signaling pathway, MT, FGF5, and IGF-I. The result of hybridization in situ revealed that Keratin 26 was specially expressed in cortex of skin hair follicles; the result of immunohistochemistry indicated that Keratin 26 was expressed in internal root sheath, external root sheath. Then, Real-time quantitative PCR results showed that relative expressive quantity of Keratin 26 was 1.08 or 3.3 × greater in secondary follicle than primary follicle during anagen or catagen; the difference during anagen was not remarkable (p>0.05), however, that of catagen was extremely significant (p<0.01). Relative expressive quantity of Keratin 26 increased during telogen; the difference was extremely significant (p<0.01). Moreover, after Noggin expression interference using RNAi technology, we found that relative expressive quantity of Keratin 26 extremely remarkably declined (p<0.01); after K26 overexpression, we found that relative expressive quantity of Noggin extremely remarkably increased (p<0.01). We detected expressive quantity change of Keratin 26 and Keratin 26 using Real-time quantitative PCR and immunofluorescence technologies after fibroblasts were treated with MT, FGF5 or IGF-I; the results indicated that MT and FGF5 played a positive role in Keratin 26 and Keratin 26 expression, IGF-I played a negative role in Keratin 26 expression, positive role in Keratin 26 expression. The results above showed that Keratin 26 could inhibit cashmere growth, and was related to entering to catagen and telogen of hair follicles; Keratin 26 and BMP signaling pathway were two antagonistic pathways each other which could inhibit growth and development of cashmere; MT, FGF5 and IGF-I could affect expression of Keratin 26 and Keratin 26, and Keratin 26 was one of the important pathways that MT induced cashmere production in advance, FGF5 regulated cashmere growth and IGF-I promoted cashmere growth and development.