Genetic and cell studies point to miR-539-3p–SOX9 pattern in keloids

An exploratory study combining genetic analysis, scar-tissue profiling, computational biology and laboratory experiments has identified a miR-539-3p/SOX9-associated expression pattern in keloids, the raised scars that grow beyond the boundaries of an original wound. The researchers report that miR-539-3p was the only microRNA meeting both a nominal genetic-association criterion and a disease-state differential-expression criterion. In follow-up tests, miR-539-3p and SOX9 were elevated in keloid-derived fibroblasts, while stronger SOX9 protein staining was observed in keloid tissue. The results point to a candidate pathway for further investigation, but they do not establish that either molecule causes keloids or that miR-539-3p directly regulates SOX9.

Keloids are chronic fibroproliferative lesions marked by persistent inflammation, altered wound healing and excessive extracellular matrix deposition. Unlike ordinary scars, they continue to enlarge beyond the original wound margins and may cause itching, pain and substantial cosmetic distress. Surgery, intralesional corticosteroids, radiotherapy, laser therapy and cryotherapy can reduce lesion burden in selected patients, yet recurrence is common. The study’s focus was therefore not a clinical treatment trial, but an attempt to connect inherited variation in circulating microRNAs with molecular changes measured in established keloid tissue and primary cells.

For the genetic component, the investigators used two-sample Mendelian randomization. Genetic variants associated with plasma microRNA levels were treated as instrumental variables for the corresponding circulating microRNAs, while summary association data from the Million Veteran Program were used to assess keloid susceptibility. The miRNA quantitative-trait-locus data came from a genome-wide association study of 2,178 participants in which 2,083 plasma microRNAs were quantified. The keloid outcome analysis included 447,008 analyzable individuals. Linkage-disequilibrium clumping, removal of palindromic variants and instrument-strength checks were used to prepare the analyses, with instruments for most microRNAs represented by a single genetic variant.

Nine microRNAs showed nominal associations with keloid susceptibility, but none remained significant after correction for multiple testing. miR-539-3p had a nominal inverse genetic association, with an odds ratio of 0.68, a 95% confidence interval of 0.47 to 0.97 and a P value of 0.035; its false-discovery-rate-adjusted P value was 0.986. Because that estimate came from one SNP, the investigators could not perform candidate-specific sensitivity analyses such as MR-Egger regression, weighted median estimation or leave-one-out analysis. The result is therefore an exploratory selection signal, not evidence that higher circulating miR-539-3p protects against keloids.

The researchers next analyzed the public GEO dataset GSE237753, which contains bulk microRNA sequencing from three keloid tissues and three normal skin controls. After quality control and filtering of low-abundance microRNAs, the DESeq2 analysis identified 44 differentially expressed microRNAs. miR-539-3p was the only candidate to overlap with the nominal Mendelian-randomization results. It was strongly upregulated in keloid tissue, with a log2 fold change of 3.29 and an adjusted P value of 4.71 × 10−6. This overlap was used to prioritize the molecule for biological follow-up rather than to confirm a genetic causal effect.

Potential downstream targets were assembled from miRDB, TargetScan and miRTarBase. Nine genes appeared in all three resources: SOX9, CTBP1, NRF1, ARID1A, SRSF2, PCGF3, HS3ST3B1, KPNA2 and MTF2. Their combined annotation was associated with developmental processes, regulation of cell number, stem-cell population maintenance and extracellular matrix organization, along with transcriptional and PI3K–Akt-related functions. SOX9 was selected for further examination because it was a shared predicted target, had a curated human miR-539-3p/SOX9 record in miRTarBase, and was considered biologically relevant to mesenchymal remodeling and fibrosis. The selection combined computational prediction, curated evidence and literature-guided biological judgment.

Experimental measurements in primary cells and tissue provided further support for the expression pattern. The researchers tested eight matched pairs of normal skin-derived and keloid-derived fibroblasts from eight patients. Both miR-539-3p and SOX9 messenger RNA levels were higher in the keloid-derived cells, with P values of 3.69 × 10−5 and 1.09 × 10−5, respectively. In an independent set of 20 specimens—10 normal skin samples and 10 keloids—semiquantitative immunohistochemistry showed stronger SOX9 staining in the dermal or lesional compartment of keloids. Mean immunoreactivity scores were 4.20 ± 1.38 for keloids and 1.55 ± 0.64 for normal skin, with P = 5.18 × 10−5.

SOX9-centered networks placed the candidate within several processes relevant to scar remodeling. GeneMANIA analysis connected SOX9 with genes involved in connective-tissue development, extracellular matrix organization and mesenchyme development, with enrichment for Notch signaling, cytoskeletal processes and PI3K–Akt signaling. A high-confidence STRING network was enriched for connective-tissue and mesenchyme development, epithelial-to-mesenchymal transition, collagen-containing extracellular matrix, Wnt signaling, TGF-β signaling, Hippo signaling, Hedgehog signaling and Notch signaling. These computational associations describe the biological context around SOX9; they do not demonstrate that SOX9 controls keloid formation or directly governs matrix deposition.

The investigators also transfected primary fibroblasts with a miR-539-3p inhibitor or a negative-control oligonucleotide. Across three independent experiments per assay, SOX9 messenger RNA was higher after inhibitor treatment than after control treatment, with P = 0.0007. Western blotting likewise showed higher SOX9-to-GAPDH protein abundance after inhibition, with a nominal P value of 0.0296. The result is compatible with the possibility that miR-539-3p can influence SOX9, but direct targeting was not tested. The study did not examine whether miR-539-3p binds the SOX9 3′ untranslated region, and it did not manipulate SOX9 to determine whether the protein changes fibroblast activation or matrix production.

Several limitations constrain the conclusions. The genetic signal was nominal, based on one SNP and unable to support robust candidate-specific sensitivity testing. The microRNA-QTL exposure cohort was of European ancestry, while the multi-ethnic keloid GWAS was predominantly European, limiting generalizability. The discovery transcriptomic dataset contained only three keloid and three control tissues, and the paired-cell and tissue experiments, although independent of that dataset, assessed established lesions rather than the process of scar initiation. The differing directions of the genetic and expression findings also reflect different biological contexts: genetically predicted circulating microRNA levels were compared with molecular measurements from established lesions and lesion-derived cells. The study has therefore narrowed a broad candidate space to an experimentally tractable miR-539-3p/SOX9 association. Direct binding assays, targeted perturbation of both molecules and rescue experiments in keloid-derived cells will be needed to determine whether the relationship is direct, indirect or simply part of a broader remodeling program.

Subject of Research: Exploratory genetic, transcriptomic and experimental analysis of miR-539-3p and SOX9 expression in keloid tissue and fibroblasts

Article Title: Integrated genetic and transcriptomic analyses suggest a miR-539-3p/SOX9-associated expression pattern in keloids

Article References: Luo, Z., Ge, L., Chen, L., Lin, S., Wei, T., Lu, B., & Jin, Z. (2026). Integrated genetic and transcriptomic analyses suggest a miR-539-3p/SOX9-associated expression pattern in keloids. Frontiers in Cell and Developmental Biology, 14, Article 1931861. https://doi.org/10.3389/fcell.2026.1931861

Image Credits: AI Generated

DOI: 10.3389/fcell.2026.1931861

Keywords: keloids, fibrosis, miR-539-3p, SOX9, microRNA, Mendelian randomization, transcriptomics, extracellular matrix, primary fibroblasts, Integrated, genetic, transcriptomic