LncRNA ST6GALNAC3 inhibits dermal fibroblast proliferation and migration in cashmere goat hair follicles via the chi-miR-24-3p/ID4 axis

Abstract

Objective: Dermal papilla is developed from the continuous proliferation and differentiation of dermal fibroblasts, which is the key to the normal development of hair follicles. This study aims to elucidate the role of lncRNA ST6GALNAC3, which is significantly differentially expressed during the secondary hair follicle development stage in cashmere goats, on dermal fibroblasts, and to analyze the regulatory mechanism of this lncRNA thoroughly.

Methods: We conducted a screening process and characterization for lncRNAs associated with the development of secondary hair follicles. The effects of lncRNA ST6GALNAC3 on cell proliferation and migration were assessed using CCK8, EdU, and flow cytometry. Subsequently, we employed bioinformatics analysis to identify the target miRNAs of lncRNA ST6GALNAC3 and the corresponding target genes of these miRNAs, respectively, and initially constructed the regulatory axis of lncRNA ST6GALNAC3-chi-miR-24-3p-ID4. Luciferase reporter assays and rescue experiments were performed to confirm the regulatory axis at both molecular and cellular levels, thus elucidating the mechanism by which lncRNA ST6GALNAC3 regulates dermal fibroblasts.

Results: One hundred fifty-eight lncRNAs related to secondary hair follicle morphogenesis were identified. Among them, lncRNA ST6GALNAC3 was significantly differentially expressed on embryonic day 75 and significantly inhibited the proliferation and migration of dermal fibroblasts. The results showed that lncRNA ST6GALNAC3 can target chi-miR-24-3p, which in turn can target the ID4 gene. The results of the luciferase reporter assay and rescue assay showed that chi-miR-24-3p binds to both lncRNA ST6GALNAC3 and ID4. Furthermore, lncRNA ST6GALNAC3 can indirectly regulate the proliferation and migration of dermal fibroblasts through chi-miR-24-3p/ID4 axis.

Conclusion: LncRNA ST6GALNAC3 inhibits the proliferation and migration of dermal fibroblasts through the chi-miR-24-3p/ID4 axis, thus suppressing the formation of dermal papilla structures and influencing the morphogenesis of secondary hair follicles during embryonic development.

Keywords: Cashmere Goats; Dermal Fibroblast; Hair Follicle; LncRNA; Morphogenesis.

Figure 1

Screening and identification of lncRNAs related to secondary hair follicle morphogenesis. (A) Screening of lncRNAs associated with secondary hair follicle morphogenesis. (B) lncLocator software predicts the distribution of lncRNA ST6GALNAC3 in cells. (C) Detection of lncRNA ST6GALNAC3 expression in the nucleus and cytoplasm of dermal fibroblasts. (D) Expression of lncRNA ST6GALNAC3 in skin tissues at different embryonic periods. (E) The distribution of lncRNA ST6GALNAC3 in cells was detected by FISH assay.

Figure 2

Functional analysis of lncRNA ST6GALNAC3 in dermal fibroblasts. (A) Screening of lncRNA ST6GALNAC3 interference vector. (B) CCK8 was used to detect the proliferation of the lncRNA ST6GALNAC3-sh cell line. (C) The EdU was used to detect the proliferation of the lncRNA ST6GALNAC3-sh cell line. (D) The expression of cell proliferation/apoptosis marker genes was detected by qRT-PCR. (E) The apoptosis of dermal fibroblasts was detected after lncRNA ST6GALNAC3 interference. (F) DNA staining was used to detect the cell cycle of the lncRNA ST6GALNAC3-sh cell line. (G) The cell scratch assay was used to detect the migration of the lncRNA ST6GALNAC3-sh cell line. qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction.

Figure 3

Construction and verification of lncRNA ST6GALNAC3-chi-miR-24-3p-ID4 regulatory network. (A) Construction of a regulatory network. (B) GO enrichment analysis of chi-miR-24-3p target genes. (C) RNAhybrid software predicts the sequence of lncRNA ST6GALNAC3 binding site to chi-miR-24-3p. (D) RNAhybrid software predicts the sequence of the ID4 binding site to chi-miR-24-3p. (E) KEGG enrichment analysis of chi-miR-24-3p target genes. (F) chi-miR-24-3p interference/overexpression of dermal fibroblast cell lines to detect lncRNA ST6GALNAC3/ID4 expression. (G) Dual-luciferase reporter gene system to detect target binding of lncRNA ST6GALNAC3 to chi-miR-24-3p. (H) Dual-luciferase reporter gene system to detect target binding of ID4 to chi-miR-24-3p.

Figure 4

Functional analysis of ID4 in dermal fibroblasts. (A) Screening of ID4 interference vector. (B) CCK8 was used to detect the proliferation of the ID4-sh cell line. (C) The apoptosis of dermal fibroblasts was detected after ID4 interference. (D) The EdU was used to detect the proliferation of the ID4-sh cell line. (E) The cell scratch assay was used to detect the migration of the ID4-sh cell line. (F) The expression of cell proliferation/apoptosis marker genes was detected by qRT-PCR. (G) DNA staining was used to detect the cell cycle of the lncRNA ID4-sh cell line. qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction.

Figure 5

Chi-miR-24-3p can reverse the effect of lncRNA ST6GALNAC3-sh/ID4-sh on the cell phenotype of dermal fibroblasts. (A) The EdU assay was used to detect cell proliferation. (B) The expression of cell proliferation/apoptosis marker genes was detected by qRT-PCR. (C) The cell scratch assay was used to detect cell migration. qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction.

Figure 6

Chi-miR-24-3p can reverse the effect of lncRNA ST6GALNAC3-sh/ID4-sh on the cell phenotype of dermal fibroblasts. (A) Annexin V-APC/PI double staining was used to detect cell apoptosis. (B) DNA staining was used to detect the cell cycle.

Figure 7

Diagram of the lncRNA ST6GALNAC3/chi-miR-24-3p/ID4 regulatory mechanism.