LncRNA MSTRG.14227.1 regulates the morphogenesis of secondary hair follicles in Inner Mongolia cashmere goats via targeting ADAMTS3 by sponging chi-miR-433

Abstract

The cashmere goat is a type of livestock primarily known for its cashmere. Cashmere has a soft hand feel and good luster. It is a vital raw material in the textile industry, possessing significant economic value. Improving the yield and quality of cashmere can accelerate the development of the cashmere industry and increase the incomes of farmers and herdsmen. The embryonic stage is the main stage of the formation of hair follicle structure, which directly affects the yield and quality of cashmere. With the rapid advancements in modern molecular technology and high-throughput sequencing, many signaling molecules have been identified as playing critical roles in hair follicle development. Long non-coding RNA (lncRNA), which lacks protein-coding ability and exceeds 200 nucleotides in length, has been discovered to play a role in hair follicle development. In this study, the lncRNA MSTRG.14227.1, which is associated with the morphogenesis of secondary hair follicles, was screened and identified based on previously established lncRNA expression profiles derived from skin tissues of cashmere goats at different embryonic stages. This lncRNA has been shown to inhibit the proliferation and migration of dermal fibroblasts. Furthermore, we confirmed through bioinformatics analysis and dual-luciferase reporter assays that lncRNA MSTRG.14227.1 can function as a sponge for chi-miR-433, thereby alleviating the inhibitory effect of chi-miR-433 on its target gene ADAMTS3. In conclusion, the results of this study suggest that lncRNA MSTRG.14227.1 can inhibit the morphogenesis of secondary hair follicles through the chi-miR-433/ADAMTS3 signaling axis.

The results of this study will help further analyze molecular regulatory mechanisms during hair follicle development, which is crucial for improving the productive traits of fiber-producing livestock and accelerating the cultivation of new cashmere goat varieties.

Introduction

The cashmere goat represents a unique biological resource primarily utilized for cashmere production. Cashmere is not only one of the most significant raw materials in the textile industry but also possesses significant economic value. Hence, it is a highly sought-after commodity in the market. Worldwide, there are numerous varieties of cashmere goats, including the Liaoning cashmere goat, the Orenburg goat, and the Inner Mongolia cashmere goat, among others. Notably, the cashmere textile derived from the Inner Mongolia cashmere goat is renowned for its superior comfort and exceptional quality. The hair follicles of cashmere goats are categorized into 2 types: primary hair follicles and secondary hair follicles (Shang, 2023). Cashmere grows from secondary hair follicles. The embryonic period is pivotal for the formation of hair follicles ultimately influencing both the quality and yield of cashmere (Schneider et al., 2009). Therefore, studying the development of secondary hair follicles in cashmere goats is of great significance, not only for enhancing the quality and yield of cashmere but also for advancing the cashmere industry. Hair follicle formation relies on a series of signaling between the dermis and epidermis (Ma et al., 2024). These signals induce orderly proliferation and differentiation of dermal fibroblasts, forming a dermal papilla that regulates hair follicle growth and development (Kumamoto et al., 2003; Ma et al., 2024). It has been shown that, during the hair follicle development in cashmere goats, primary follicles develop earlier than secondary follicles (Shang, 2023). During the embryonic period of cashmere goats, by day 45, the fetal skin has developed a fully formed epidermal structure. By day 55 of embryonic development, the primary hair follicle begins to emerge, forming the characteristic hair bud structure. As development continues, by day 65, the hair bud structure of the primary hair follicle extends downward into the dermis of the skin. Subsequently, by day 75, the secondary hair follicle initiates its emergence (Zhang et al., 2007).

Hair follicle development is a highly complex process that is regulated by a series of signaling molecules. With the rapid advancements in modern molecular technology and high-throughput sequencing, an increasing number of genetic factors have been identified as playing crucial roles in vital life activities, including hair follicle development (Pan, 2023), muscle growth (Chengcheng et al., 2024), and adipogenesis (Abbas Raza et al., 2024). Among them, lncRNAs, which lack protein-coding ability and exceed 200 nucleotides in length, have been discovered to play a role in hair follicle development (Okazaki et al., 2002). For example, LncRNA-H19 can compete with chi-miR-214-3p for binding to β-catenin, a pivotal protein in Wnt signaling pathways. By regulating β-catenin, LncRNA-H19 indirectly controls the proliferation of dermal papilla cells, which are essential for hair follicle development (Zhang et al., 2022). LncRNA018392 can enhance the expression of CSF1R via the promoter SPI1. By increasing the activity of CSF1R, lncRNA018392 promotes the proliferation of dermal papilla cells (Jin et al., 2024). LncRNA 627.1 has been shown to regulate the expression of Edar, which subsequently affects the formation of the hair follicle matrix (Jiang et al., 2022). Additionally, LncRNA-XIST plays a pivotal role in promoting Shh gene expression by specifically targeting and binding to miR-424, effectively silencing its inhibitory effects. This interaction results in elevated levels of Shh, which activates signaling pathways essential for hair follicle regeneration (Lin et al., 2020). These findings indicate that lncRNA has a significant role in hair follicle development. However, the regulatory functions of novel lncRNAs in this process remain largely unexplored.

In this study, we performed a differential expression analysis of lncRNAs in the skin tissue of Inner Mongolia cashmere goat fetuses. We discovered that lncRNA MSTRG.14227.1 exhibited significant differential expression during secondary hair follicle morphogenesis. This lncRNA upregulated the expression of the ADAMTS3 gene by sponging chi-miR-433, subsequently inhibiting the proliferation and migration of dermal fibroblasts. Additionally, we found that this inhibitory effect on dermal fibroblasts may be mediated through reducing the proportion of cells in the S-phase. In conclusion, our findings indicate that lncRNA MSTRG.14227.1 plays a crucial role in the morphogenesis of secondary hair follicles in cashmere goats by regulating the chi-miR-433/ADAMTS3 signaling axis.

Materials and Methods

Ethics statement

All animal experiments followed guidelines and were approved by the Experimental Animal Ethics Committee of Inner Mongolia Agricultural University (Approval No. [2020] 056). Fetal skin samples were collected according to international principles.

Collection of tissue sample

The samples were obtained from Inner Mongolia Jinlai Animal Husbandry Technology Co., Ltd (Hohhot, China). According to the experimental records, skin tissue samples were collected from cashmere goat embryos at 45, 55, 65, and 75 d. Three samples were collected in each period to ensure the accuracy of the data. These samples were subsequently stored in liquid nitrogen to facilitate further studies (Ma et al., 2022).

Selection and validation of key lncRNAs during secondary hair follicle morphogenesis

In our previous research, we conducted transcriptome sequencing on skin tissues collected from 12 Inner Mongolia cashmere goats at 4 distinct embryonic stages: 45, 55, 65, and 75 d. As a result, we successfully obtained lncRNA expression profiles for these skin tissues (Ma et al., 2022). Initially, we utilized the Illumina HiSeq 4000 platform to perform paired-end sequencing, generating raw data. Subsequently, we employed Cutadapt to filter out low-quality data. To predict the coding potential of the RNA, we used CPC (Kang et al., 2017) and CNCI (L et al., 2013). Ultimately, we identified lncRNAs by selecting those with a transcript length exceeding 200 bp, a CPC score of ≤ 0.5, and a CNCI score of ≤ 0 for further investigation.

Based on this, the study screened out important lncRNAs that exhibited differential expression and were related to the morphogenesis and development of secondary hair follicles, taking into account the growth characteristics of primary and secondary hair follicles. The 3 comparison groups (d55 vs d45, d65 vs d45, and d65 vs d55) are designated as stage A, which pertains to the development of primary hair follicles. Subsequently, the comparison groups (d75 vs d45, d75 vs d55, and d75 vs d65) are designated as stage B, encompassing the development of both primary and secondary hair follicles. To isolate the lncRNAs specifically associated with the morphogenesis of secondary hair follicles, the common components shared between stage B and stage A were excluded from stage B. The remaining part of stage B was considered as lncRNA associated with the morphogenesis of secondary hair follicles. The screening criteria for differentially expressed lncRNAs were based on the FPKM. The differential expression analysis was performed using edgeR software, with |log2 (Foldchange) | ≥ 1 and P-value ≤ 0.05 as the threshold for screening.

Subcellular localization

We utilized LncLocator (Cao et al., 2018) to predict the subcellular localization of lncRNA MSTRG.14227.1 within cells. Additionally, we extracted and purified cytoplasmic and nuclear RNA from dermal fibroblasts using the Cytoplasmic & Nuclear RNA Purification Kit (Norgen Biotek, Thorold, ON, Canada), following the manufacturer’s guidelines. qRT-PCR was used to detect the expression of lncRNA MSTRG.14227.1 in the cytoplasm and nucleus (Supplementary material 2).

Construction of the interference cell line

The cells used in this experiment were derived from a dermal fibroblast cell line established from the fetal skin tissue of a cashmere goat, which our research group had previously cultured (Pan, 2023). The plasmid was efficiently transfected into these dermal fibroblasts using lentiviral transfection techniques. The plasmid was prepared by Hanheng Biological Technology Co., Ltd (Hanheng, Shanghai, China). After transfection, puromycin was added to the culture medium for resistance-based cell selection.

Dual-luciferase report detection

Based on the results of bioinformatics prediction (Betel et al., 2008; Agarwal et al., 2015), we constructed a plasmid (wild-type/mutant) of the target gene (Ma et al., 2022). In 293T cells, chi-miR-433 mimics were co-transfected with the target gene plasmid (wild-type/mutant). After 48 h, we measured Firefly luciferase levels with the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA).

CCK8 assays

The proliferation of cells was assessed using the CCK8 assay (Solarbio, Beijing, China), following the manufacturer’s guidelines (Wang et al., 2024). Then, we measured the absorbance at a wavelength of 450 nm.

EdU assays

The EdU experiment process is as follows: The cells in 24-well plates were treated with 1 × EdU working solution for 2 h, followed by fixation with 4% paraformaldehyde for 15 min, permeabilization with 0.3% Triton X-100 for an additional 15 min, and staining with Hoechst stain for 10 to 15 min (Wang et al., 2024).

Cell cycle assays

The cell cycle was measured by utilizing the DNA content quantification method, according to the manufacturer’s instructions (Solarbio). The cells were collected and fixed for 24 h. Following fixation, RNase A solution was added, and the mixture was incubated for 30 min. Subsequently, PI staining solution was introduced, and the cells were incubated in the dark for an additional 30 min (Chengcheng et al., 2024).

Cell apoptosis

In accordance with the manufacturer’s directions, the Annexin V-APC/PI Cell Apoptosis Detection Kit was utilized to assess cell apoptosis (Elabscience Biotechnology, China). Cells were collected and 1x Buffer, 2.5 μl of APC Reagent, and 2.5 μl of PI Reagent were sequentially added to cells. The mixture was then incubated for 20 min (Wang et al., 2024). Finally, the apoptotic status of the cells was analyzed using flow cytometry.

Cell migration

The cell migration assay was conducted by using a sterile pipette tip to scratch the cell monolayer, followed by capturing images of the scratched area. Images of the cell scratch were acquired at both 0 and 24 h post-scratch (Wang et al., 2024).

Statistical analysis

Statistical analyses were primarily conducted using SPSS 26.0 (SPSS, USA) and GraphPad Prism 8.0 (GraphPad Software Inc., USA). All data are presented as the mean ± standard deviation derived from at least 3 independent repeated experiments. Differences between groups were analyzed using Student’s t-tests, with statistical significance indicated by P < 0.05 or P < 0.01.

Results

Selection of significant lncRNAs in the process of secondary hair follicle morphogenesis

Through analysis of skin transcriptome databases at various embryonic stages (Differential expression analysis was performed with |log2foldchange| ≥ 1 and P-value < 0.05 as screening conditions), we identified a total of 1209 differentially expressed lncRNAs (Figure 1B). Among them, 1,051 differentially expressed lncRNAs were identified in the d55 vs d45, d65 vs d45, and d65 vs d55 comparison groups. Additionally, we identified 903 differentially expressed lncRNAs in the d75 vs d45, d75 vs d55, and d75 vs d65 comparison groups. The embryonic period is a critical stage for the development of primary and secondary hair follicles. Secondary hair follicles develop later than primary hair follicles. Primary hair follicles initiate their development on embryonic day 55, whereas secondary hair follicles begin formation on embryonic day 75 (Zhang et al., 2007). Therefore, based on the characteristics of primary and secondary hair follicle morphogenesis and development during the embryonic period of the cashmere goat, We identified 1,051 lncRNAs (d55 vs d45, d65 vs d45, and d65 vs d55) as associated with primary hair follicle development (stage A). The 903 lncRNAs (d75 vs d45, d75 vs d55, and d75 vs d65) were considered to be associated with both primary and secondary hair follicle development (stage B). After excluding the common lncRNAs shared between stages B and A, we identified 158 lncRNAs in stage B that were significantly associated with secondary hair follicle morphogenesis (Figure 1A). The sequencing results indicated that the expression level of lncRNA MSTRG.14227.1 was extremely low in the samples collected on days 45, 55, and 65, whereas it was significantly upregulated in the samples from day 75 (Figure 1A). Furthermore, qRT-PCR was employed to validate these findings, and the results corroborated the sequencing data, demonstrating a significant upregulation of lncRNA MSTRG.14227.1 in the 75-d samples (Figure 1C). Consequently, lncRNA MSTRG.14227.1 was selected for subsequent study.

Figure 1.

Screening and identification of lncRNA related to secondary hair follicle morphogenesis: (A) Screening of lncRNAs related to secondary hair follicle morphogenesis and development. (B) Expression of lncRNA in different treatment groups. (C) Expression of lncRNA MSTRG.14227.1 in skin tissues at different embryonic periods. (D) lncLocator software predicts the distribution of lncRNA MSTRG.14227.1 in cells. (E) Detection of lncRNA MSTRG.14227.1 expression in the nucleus and cytoplasm of dermal fibroblasts. **** indicates P < 0.0001.

LncRNA MSTRG.14227.1 identification, characterization, and expression profiling

Meanwhile, we found that lncRNA MSTRG.14227.1 is an RNA molecule with a full length of 43,497 base pairs, located within the intronic region of the gene TTC28. Subsequently, we analyzed the coding potential of lncRNA MSTRG.14227.1 using CPC and CNCI software. The results indicated that the CPC score for lncRNA MSTRG.14227.1 was 0.483, while the CNCI score was −0.023, indicating that this lncRNA does not possess the ability to encode proteins. Furthermore, through the application of the lncLocator software and nucleoplasmic separation experiments, we discovered that lncRNA MSTRG.14227.1 is predominantly expressed in the cytoplasm (Figure 1D and E). Therefore, we hypothesize that it may function through competitive endogenous RNA (ceRNA) regulatory mechanisms.

LncRNA MSTRG.14227.1 inhibits the proliferation and migration of dermal fibroblasts

Furthermore, we studied the effects of lncRNA MSTRG.14227.1 on hair follicle cells. Specifically, we focused on the embryonic stage, where dermal fibroblasts undergo a series of transformations and eventually develop into the dermal papilla, which plays a crucial role in hair follicle development (Ma et al., 2024). Consequently, dermal fibroblasts were selected as the primary target for subsequent experiments. Firstly, we designed 3 small interfering RNAs of lncRNA MSTRG.14227.1 (Supplementary material 1), and transfected dermal fibroblasts with lncRNA MSTRG.14227.1-sh1, sh2, and sh3, respectively, using lentiviral vectors (Figure 2A). The results of qRT-PCR showed that lncRNA MSTRG.14227.1-sh1 exhibited the highest interference efficiency (Figure 2B). Consequently, lncRNA MSTRG.14227.1-sh1 was chosen for subsequent experiments.

Figure 2.

Functional analysis of lncRNA MSTRG.14227.1 in dermal fibroblasts: (A) Construction of lncRNA MSTRG.14227.1-sh cell line. (B) Screening of cell lines with lncRNA MSTRG.14227.1 interference. (C) Flow cytometry was used to detect the effect of lncRNA MSTRG.14227.1 interference on the cycle distribution of dermal fibroblasts. (D) Using the EdU method to detect the proliferation of dermal fibroblasts following interference with lncRNA MSTRG.14227.1. (E) The effect of lncRNA MSTRG.14227.1 interference on the apoptosis rate of dermal fibroblasts was detected using Annexin V-APC/PI double staining. (F) The CCK8 method was used to detect the proliferation of dermal fibroblasts after interference with lncRNA MSTRG.14227.1. (G) Expression levels of genes related to proliferation/apoptosis in dermal fibroblasts were detected after lncRNA MSTRG.14227.1 interference. (H) The migration of dermal fibroblasts was detected after interference with lncRNA MSTRG.14227.1. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001.

Subsequently, we carried out a series of cell phenotype experiments in lncRNA MSTRG.14227.1-sh cell line. After the interference, there was a significant increase in the number of EdU-positive dermal fibroblasts and a marked enhancement in their proliferative capacity (Figure 2D and F). Additionally, the results from Annexin V-APC/PI double staining showed a significant reduction in the apoptotic rate (Figure 2E). In addition, the expression of proliferation-related genes (PCNA, CCND2, and CCND1) and apoptosis-related genes (Bax, Bcl2, and Casp9) was detected using qRT-PCR. The results demonstrated a significant increase in the expression of proliferation-related genes, while the expression of apoptosis-related genes showed an opposite trend, indicating that this interference promotes cell proliferation in dermal fibroblasts (Figure 2G). Meanwhile, the results of flow cytometry showed that lncRNA MSTRG.14227.1-sh could increase the proportion of S-phase cells (Figure 2C). During the development of mammalian hair follicles, dermal fibroblasts migrate and aggregate downward to form mature dermal papilla. The cell migration assay data revealed that interference with lncRNA MSTRG.14227.1 significantly enhanced migration (Figure 2H). Taken together, these results indicated that lncRNA MSTRG.14227.1-sh has the potential to enhance both cell proliferation and migration in dermal fibroblasts.

LncRNA MSTRG.14227.1 directly binds chi-miR-433 and inhibits its activity

Next, we explored the regulatory mechanism of lncRNA MSTRG.14227.1. Given its primary localization in the cytoplasm, we hypothesized that lncRNA MSTRG.14227.1 might serve as a ceRNA to influence the morphogenesis of secondary hair follicles. Firstly, we utilized the TargetScan (Agarwal et al., 2015) and miRanda (Betel et al., 2008) databases to predict the target miRNAs of lncRNA MSTRG.14227.1. The results indicated that this lncRNA had potential binding sites for 24 miRNAs. Subsequently, we further screened some of the miRNAs with superior overall scores based on the evaluations from TargetScan and miRanda. qRT-PCR results showed that the expression of the targeted miRNAs was upregulated in this cell line, with chi-miR-433 exhibiting the highest expression level. Therefore, chi-miR-433 was chosen for further verification in subsequent experiments (Figure 3A). Further, the sequence information regarding the binding site between lncRNA MSTRG.14227.1 and chi-miR-433 was obtained through RNAhybrid software. The prediction is shown in Figure 3B. Subsequently, qRT-PCR results showed that chi-miR-433 was significantly under-expressed at the 75th day (Figure 3C). In summary of the results, we predicted that lncRNA MSTRG.14227.1 has a target-binding site for chi-miR-433. Subsequently, we constructed a reporter plasmid (wild-type/mutant) containing the sequence of lncRNA MSTRG.14227.1 (Figure 3D). The results showed that chi-miR-433 mimics significantly downregulated the luciferase activity in the lncRNA MSTRG.14227.1-WT reporter gene, whereas no significant change was observed in the lncRNA MSTRG.14227.1-Mut reporter gene. This suggests the existence of a specific target-binding relationship between lncRNA MSTRG.14227.1 and chi-miR-433 (Figure 3E).

Figure 3.

LncRNA MSTRG.14227.1 acts as a sponge for chi-miR-433 in cashmere goat: (A) Relative expression of target miRNAs detected after transfection of dermal fibroblasts with lncRNA MSTRG.14227.1-sh. (B) RNAhybrid software predicts the sequence of lncRNA MSTRG.14227.1 binding site to chi-miR-433. (C) Expression of chi-miR-433 in skin tissues of different embryonic periods. (D) Schematic diagram of wild-type/mutant lncRNA MSTRG.14227.1 luciferase reporter vector construction. (E) Dual-luciferase reporter gene system to detect target binding of lncRNA MSTRG.14227.1 to chi-miR-433. * indicates P < 0.05, *** indicates P < 0.001, **** indicates P < 0.0001, and ns indicates that the difference is not significant.

Chi-miR-433 reverses the inhibitory effect of lncRNA MSTRG.14227.1 on dermal fibroblast proliferation and migration

Based on the above results, we introduced a chi-miR-433 inhibitor into the lncRNA MSTRG.14227.1-sh cell line to further investigate the targeting relationship between these through rescue experiments. Firstly, we observed that the interference of chi-miR-433 led to an upregulation of lncRNA MSTRG.14227.1 expression in the cells. However, in contrast, the expression pattern of lncRNA MSTRG.14227.1 in the cell line overexpressing chi-miR-433 was reversed compared to that in the cell line with chi-miR-433 inhibitor (Figure 4A). Subsequently, EdU experiments and cell cycle analysis showed that knocking down lncRNA MSTRG.14227.1 significantly increased the percentage of EdU-positive cells and the number of S-phase cells in dermal fibroblasts. However, in the co-transfected cell lines, both the number of EdU-positive cells and S-phase cells decreased markedly. Additionally, the expression of genes that promote cell proliferation was significantly reduced (Figure 4B, D, and E). At the apoptosis level, the chi-miR-433 inhibitor reversed the inhibitory effect of lncRNA MSTRG.14227.1-sh on apoptosis (Figure 4C). Regarding cell migration, lncRNA MSTRG.14227.1-sh accelerated dermal fibroblast migration, but this effect was negated by co-transfection with the chi-miR-433 inhibitor (Figure 4F). In summary, our findings suggest that the chi-miR-433 inhibitor can reverse the proliferative and migratory effects of lncRNA MSTRG.14227.1-sh.

Figure 4.

Chi-miR-433 can reverse the effect of lncRNA MSTRG.14227.1 on the cell phenotype of dermal fibroblasts: (A) Chi-miR-433 interference/overexpression of dermal fibroblast cell lines to detect lncRNA MSTRG.14227.1 expression. (B) The expression of cell proliferation and apoptosis marker genes was detected after co-transfection of lncRNA MSTRG.14227.1-sh and chi-miR-433 inhibitor. (C) The apoptosis of cells was detected after co-transfection of lncRNA MSTRG.14227.1-sh and chi-miR-433 inhibitor. (D) The cell cycle was detected after co-transfection of lncRNA MSTRG.14227.1-sh and chi-miR-433 inhibitor. (E) The cell proliferation was detected after co-transfection of lncRNA MSTRG.14227.1-sh and chi-miR-433 inhibitor. (F) The cellular migration was detected after co-transfection of lncRNA MSTRG.14227.1-sh and chi-miR-433 inhibitor. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001.

ADAMTS3 can participate in secondary hair follicle morphogenesis by binding to chi-miR-433

To identify potential regulatory mechanisms, we conducted bioinformatics analyses using the TargetScan and miRanda databases. We first constructed the chi-miR-433-mRNA regulatory network (Figure 5A) and then performed enrichment analysis on target genes (Figure 5B and C). The results indicated enrichment in hair follicle development pathways, including TGF-β, PI3K-AKT, and Wnt. ADAMTS3 is an important member of the ADAMTS proteinase family (Jones and Riley, 2005). Previous research has demonstrated that ADAMTS3 plays a crucial role in the development of hair follicles (Ma et al., 2022). Consequently, we decided to further investigate ADAMTS3, a molecule prominently found in the TGF-β signaling pathway, due to its potential importance in our research.

Figure 5.

Target gene prediction and functional enrichment analysis of chi-miR-433: (A) Construction of the chi-miR-433-mRNA regulatory network. (B) GO enrichment analysis of chi-miR-433 target genes. (C) KEGG enrichment analysis of chi-miR-433 target genes.

Firstly, we examined the expression of ADAMTS3 in the chi-miR-433 inhibitor cell line and the chi-miR-433 mimic cell line. The results indicated that interference with chi-miR-433 enhanced the expression of ADAMTS3, whereas overexpression of chi-miR-433 inhibited its expression (Figure 6A). Based on these findings, we speculated that ADAMTS3 has a target-binding site for chi-miR-433. To verify this speculation, we constructed a reporter plasmid (wild-type/mutant) containing the sequence of the ADAMTS3-3’UTR (Figure 6B). The results showed that chi-miR-433 mimics significantly downregulated the luciferase activity in ADAMTS3-3’UTR-WT reporter gene, but no significant change was observed in ADAMTS3-3’UTR-Mut reporter gene (Figure 6C). This suggests the existence of a specific target-binding relationship between ADAMTS3 and chi-miR-433.

Figure 6.

Chi-miR-433 can regulate the phenotype of dermal fibroblasts by targeting ADAMTS3: (A) chi-miR-433 interference/overexpression of dermal fibroblast cell lines to detect ADAMTS3 expression. (B) Schematic diagram of wild-type/mutant ADAMTS3-3’UTR luciferase reporter vector construction. (C) Dual-luciferase reporter gene system to detect target binding of chi-miR-433 to ADAMTS3-3’UTR. (D) Screening of cell lines with ADAMTS3 interference. (E) Detection of gene expression levels related to cell proliferation and apoptosis. (F) EdU detection of cell proliferation. (G) Cell cycle detection by flow cytometry. (H) Annexin V-APC/PI detection of apoptotic cells. (I) Cell migration detected by cell scratching. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001.

Inhibition of dermal fibroblast proliferation and migration by lncRNA MSTRG.14227.1 via chi-miR-433/ADAMTS3 axis

Drawing from prior research, we found that chi-miR-433 inhibitors could counteract the stimulatory effects on proliferation and migration, as well as the inhibitory effect on apoptosis, that is caused by the low expression of lncRNA MSTRG.14227.1. In addition, dual-luciferase reporter results revealed a targeting relationship between ADAMTS3 and chi-miR-433. Therefore, we speculated that chi-miR-433 inhibitors might function as inhibitors of ADAMTS3-sh. First, we constructed 3 interference plasmids for ADAMTS3 (Supplementary material 1). The qRT-PCR results showed that ADAMTS3-sh2 exhibited the highest interference efficiency (Figure 6D). The results of cell proliferation and cell migration experiments showed that the proliferation and migration abilities of dermal fibroblasts were significantly enhanced following interference ADAMTS3. However, when chi-miR-433 inhibitors were added to the cell, the enhancement of cell proliferation and migration induced by the interference with ADAMTS3 was alleviated (Figure 6F and I). Cell cycle experiment results indicated that interference with ADAMTS3 led to a significant increase in the proportion of dermal fibroblasts in the S-phase. When chi-miR-433 inhibitors were introduced into the cells, the alterations in the cell cycle induced by ADAMTS3-sh were rescued. It is suggested that the proliferation phenomenon observed in dermal fibroblasts, which is caused by the interference with ADAMTS3-sh, may be attributed to an increase in the proportion of S-phase cells (Figure 6G). In apoptosis experiments, we found that ADAMTS3-sh inhibited the apoptosis of dermal fibroblasts. However, this inhibitory effect was reversed when the chi-miR-433 inhibitor was added. (Figure 6H). qRT-PCR experiments showed that ADAMTS3-sh promoted proliferation-related marker genes and inhibited apoptosis-related genes. Interestingly, when co-transferred with chi-miR-433 inhibitors, the proliferation-related marker gene expression was significantly inhibited, while the expression of apoptosis-related genes was upregulated, compared to the ADAMTS3-sh group (Figure 6E). The above results indicated that ADAMTS3, as a target gene of chi-miR-433, could promote apoptosis and inhibit the proliferation and migration of dermal fibroblasts.

In summary, lncRNA MSTRG.14227.1 can act as a sponge for chi-miR-433 to regulate the expression of the downstream target gene ADAMTS3, which in turn affects the proliferation and migration ability of dermal fibroblasts, thereby regulating secondary hair follicle morphogenesis and development (Figure 7).

Figure 7.

Diagram of the lncRNA MSTRG.14227.1/chi-miR-433/ADAMTS3 regulatory mechanism.

Discussion

The cashmere produced by cashmere goats is a significant raw material in the textile industry possessing high economic value. With the rapid advancements in high-throughput sequencing and modern molecular technology, more and more lncRNAs have been identified as playing crucial roles in vital life activities. For instance, lncRNA018392 can enhance the expression of CSF1R via the promoter SPI1. By increasing the activity of CSF1R, lncRNA018392 promotes the proliferation of dermal papilla (Jin et al., 2024). LncRNA MSTRG.20890.1 regulates hair follicle development through competitive binding with miR-24-3p for ADAMTS3 (Wang et al., 2024). LncRNA-XIST competes with Shh to bind miR-424, thereby promoting hair follicle regeneration (Lin et al., 2020). In this study, we screened a total of 158 lncRNAs that are associated with secondary hair follicle morphogenesis, utilizing a previously established transcriptome database of skin samples derived from different embryonic stages of cashmere goats (Ma et al., 2022). Among them, lncRNA MSTRG.14227.1 stood out as being significantly overexpressed during the process of secondary hair follicle morphogenesis. Through comprehensive functional analysis, we discovered that lncRNA MSTRG.14227.1 functions as a sponge for chi-miR-433, thereby alleviating the inhibitory effect of chi-miR-433 on ADAMTS3 expression in dermal fibroblasts (Figure 7).

Hair follicles are the unique organs in mammals that undergo cyclic growth throughout their entire life cycle. They are composed of various types of cells and possess a highly complex structure. These organs play a significant role in maintaining skin homeostasis, regulating body temperature, and facilitating metabolism. Dermal fibroblasts are a crucial cell type in the formation of hair follicle structures (Osawa et al., 2005). They can eventually differentiate into the dermal papilla, which serves as the signaling center that ensures the normal development of hair follicles, and directly determines the fate of hair follicles (Ma et al., 2024). In this study, we initially investigated the impact of lncRNA MSTRG.14227.1 on the phenotypic characteristics of dermal fibroblasts. Our findings revealed that lncRNA MSTRG.14227.1 was predominantly localized within the cytoplasm of these cells. Upon interference with lncRNA MSTRG.14227.1, we observed a significant enhancement in dermal fibroblasts’ proliferation and migratory capacities. In the past few years, growing evidence has shown that lncRNAs can play a regulatory role as sponge molecules for miRNAs. LncRNA indirectly regulates hair follicle development through interactions with numerous key factors and signaling pathways. Additionally, lncRNA can indirectly influence the formation of the dermal papilla, as well as the proliferation and differentiation of hair follicle cells, by regulating the expression of hair-related genes. Furthermore, it plays a crucial role in the formation of both the dermal papilla and the hair follicle placode. For example, lncRNA 627.1 has been demonstrated to competitively bind to miR-29a-5p, thereby relieving its inhibitory effect on Edar and promoting the proliferation of hair follicle stem cells (Jiang et al., 2022). Furthermore, lncRNA H19 is implicated in promoting the proliferation of dermal papilla cells and modulating hair follicle formation and growth (Zhang et al., 2022). In this study, we employed predictive analysis to identify 24 miRNAs that may interact with lncRNA MSTRG.14227.1. Notably, chi-miR-433 showed the least expression levels in dermal fibroblast cell lines with silenced lncRNA MSTRG.14227.1. Dual-luciferase reporter assays definitively confirmed the presence of specific binding sites between lncRNA MSTRG.14227.1 and chi-miR-433, validating our hypothesis regarding their interaction. Similarly, we predicted the target mRNA for chi-miR-433. The results demonstrated that chi-miR-433 could specifically bind to the 3ʹUTR region of ADAMTS3, thereby inhibiting its expression. The ADAMTS protease family, initially identified in mice by Kuno (Kuno et al., 1997), belongs to the MA clan within the metalloproteinase and exhibits a structural similarity to the ADAM enzymes (Jones and Riley, 2005). The ADAMTS proteinase family can be broadly divided into 4 subdivisions. Among them, ADAMTS3, ADAMTS2, and ADAMTS14 belong to the second subdivision (Jones and Riley, 2005). Previous research indicated that ADAMTS3 plays a pivotal role in the morphological development of cashmere goat hair follicles during the embryonic stage(Ma et al., 2022). In this study, we further analyzed the regulatory network of lncRNA MSTRG.14227.1/chi-miR-433/ADAMTS3 at the cellular level. Experimental results demonstrated that silencing of either ADAMTS3 or lncRNA MSTRG.14227.1 significantly promoted the proliferation and migration of dermal fibroblast. However, following the introduction of the chi-miR-433 inhibitor into the lncRNA MSTRG.14227.1-sh and ADAMTS3-sh cell lines, respectively, the stimulatory effects on dermal fibroblast proliferation and migration observed under both knockdown conditions were progressively eliminated.

In summary, we have discovered that the lncRNA MSTRG.14227.1, which is associated with secondary hair follicle morphogenesis, can regulate the proliferation and migration of dermal fibroblasts. Dermal fibroblasts, through their continuous proliferation and differentiation, ultimately give rise to the dermal papilla, which serves as the “signaling center” of the hair follicle and regulates its normal growth and development. In this study, we identified lncRNA MSTRG.14227.1 as a ceRNA of chi-miR-433, thereby enhancing ADAMTS3 expression. Furthermore, we observed that ADAMTS3 influences secondary hair follicle development via the TGF-β pathway. This study establishes a theoretical foundation for accelerating the improvement of cashmere goat breeding. It also provides a theoretical reference for improving the quality of cashmere produced by these goats.

Conclusions

In summary, our results suggest that lncRNA MSTRG.14227.1 regulates the proliferation and migration of dermal fibroblasts through competitive binding with chi-miR-433 to ADAMTS3, ultimately inhibiting the formation of the dermal papilla structure. This study will help to understand the molecular regulatory mechanisms of hair follicle development, which is crucial for improving the productive traits of fiber-producing livestock and accelerating the cultivation of new cashmere goat varieties.

Glossary

Abbreviations

lncRNA

long non-coding RNA

ADAMTS

ADAM metallopeptidase with thrombospondin type-1 motif

ceRNA

competing endogenous RNA

qRT-PCR

real-time fluorescence quantitative PCR

Conflict of interest statement

The authors declare no conflicts of interest.

Data availability statement

The raw transcriptome sequencing data of Inner Mongolia cashmere goat skin have been deposited in the SRA database with accession numbers SRR13306938-SRR13306949.

Author contributions

Rong Ma (Conceptualization, Data curation, Formal analysis, Methodology, Validation, Writing—original draft, Writing—review & editing), Min Wang (Conceptualization, Formal analysis, Methodology), Qing Ma (Conceptualization, Data curation, Methodology, Resources, Validation, Visualization), Yiming Zhang (Data curation, Methodology, Resources, Validation, Visualization), Fangzheng Shang (Conceptualization, Data curation, Methodology, Resources, Writing—review & editing), Ruijun Wang (Formal analysis, Methodology, Resources, Software, Writing—review & editing), and Yanjun Zhang (Conceptualization, Funding acquisition, Methodology, Project administration, Writing—review & editing)