Human umbilical cord was collected from puerperants who underwent childbirth at the Affiliated Hospital of Jining Medical University (Jining, China). HUCMSCs were isolated and characterized as described previously (32). The cells at passages 3 and 4 were used for experiments. HUCMSCs were cultured in low-glucose Dulbecco’s modified Eagle’s medium (DMEM, HyClone) supplemented with 10% fetal bovine serum (FBS, Gibco) under standard conditions (37℃ and 5% CO₂ in a humidified incubator).

The study conformed to the principles outlined in the Declaration of Helsinki and was conducted in accordance with approved guidelines. Written informed consent was obtained from each participant prior to the collection of umbilical cord. All samples were acquired in accordance with the regulations and approval of the Medical Ethics Committee of Jining Medical University.

The SOX9 shRNA sequence was synthesized and inserted into the shRNA expression vector pGPH1/Neo (GenePharma, Shanghai, China), and a scramble shRNA was used as a negative control (shNC). Cells were transfected with shSOX9 or shNC using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Stable cell lines were obtained after selection with 1 μg/ml puromycin (Invitrogen) for 15 days. The expression of shRNA was induced by the addition of 80 μg/ml doxycyc-line.

For the Cell Counting Kit-8 (CCK-8) assay, HUC-MSCs transfected with either sh-control or sh-Sox9 were incubated at 37℃. CCK-8 solution (Dojindo, Japan) was then added to each well and incubated for 2 h. Absorbance at 450 nm was measured at 12, 24, 36, and 48 h.

For cell counting, HUC-MSCs (5,000/well) transfected with either sh-control or sh-Sox9 were seeded in 24 plates and incubated in a 37℃ and 5% CO₂ incubator. HUC-MSCs were counted continuously for 5 days, and cell prolife-ration was evaluated.

HUC-MSCs transfected with either sh-control or sh-Sox9 were harvested and seeded in 35-mm plates (1,000 cells/well) overnight under standard conditions. Cells were incubated at 37℃ in a 5% CO₂ humidified incubator for 10 days. At the end of the incubation period, the cultures were fixed with 4% paraformaldehyde and stained with crystal violet.

HUC-MSCs transfected with either sh-control or sh-Sox9 (1×10⁵ cells/well) were added to the top chamber, and cells that migrated through the 8-μm pores and adhered to the lower surface of the membrane were fixed with 4% paraformaldehyde and then stained with crystal violet. Six random visual fields were observed and counted under a light microscope.

Total RNA was extracted from cells using RNA isolate (Vazyme Biotech Co., Ltd.), and equal amounts of RNA were used for real-time RT-PCR analyses. The cDNAs were synthesized by using a reverse transcription kit according to the manufacturer’s instructions (Vazyme Biotech Co., Ltd.). Real-time quantitative RT-PCR (qRT-PCR) was conducted with ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd). β-actin was used as an internal control. The sequences of the specific primers are listed in Table 1.

Adult female Sprague–Dawley rats (weighing 220±20 g) were purchased from Jinan Pengyue Experimental Animal Breeding. A rat model of skin deep second-degree burn wounds was established as described previously (21). Rats were anesthetized with sodium thiopental at a dose of 40 mg/kg body weight. After the hair on their upper back was shaved, the back skin of rats was injured with 80℃ water for 8 seconds to create a 16 mm diameter wound and then covered with gauze soaking saline for 6 minutes on the wound. Moreover, 1×10⁸ cells (HUC-MSCs transfected with either sh-control or sh-Sox9) suspended in 200 μl of PBS or 200 μl of PBS were injected subcutaneously at three sites. The animals were housed individually. The healing area of the skin damage scar was photographed at 12, 24, 36, and 48 h. At 15 days after treatment, the rats were sacrificed, and the wound area was cut for further analysis.

The wound skin (4 mm²) was fixed in 4% paraformaldehyde (pH 7.4), gradually dehydrated, embedded in paraffin, cut into 4-μm sections and stained with H&E for light microscopy. The thickness of the epidermis and dermis layers was measured. Immunofluorescence staining was performed as described previously (33). Primary antibodies were as follows: CK14 (Bioworld), CK18 (Bioworld), and Ki67 (Cell Signaling Technology). Fluorescent secondary antibodies were obtained from Cell Signaling Techno-logy. Images were sequentially acquired through light microscopy (Nikon, Tokyo, Japan) and confocal microscopy (GE, USA).

All data are shown as the mean±standard deviation (SD). The statistically significant differences between groups were assessed by analysis of variance (ANOVA) or t-test using Prism software (GraphPad, San Diego, USA). p values<0.05 were considered significant.

Studies have revealed SOX9 regulates cancer stem-like properties and SLUG is required for SOX9 stabilization and functions to promote cancer stem cells (27, 28). And, SOX9 also regulates adipogenic, chondrogenic, osteogenic differentiation of mesenchymal stromal cells (29-31). So SOX9 is likely to be participated in regulating the regeneration roles of MSCs. To explore the influences of SOX9 on HUC-MSCs, we first isolated HUC-MSCs from the umbilical cord. Fig. 1 shows HUC-MSC growth from the tissue mass of the umbilical cord.

Wang et al. (34) have demonstrated that knockdown of SOX9 in triple‑negative breast cancer cells increased apoptosis and the cell cycle was arrested at the G0/G1 phase, RNA sequencing identified numerous genes regulated by SOX9. Gao et al. (35) also revealed SOX9 downregulation decreased cyclin D1, CDK4 expression and Rb phosphorylation, which correlated with a reduced population of cells in the S phase and suppressed growth of glioma cells. These reports revealed SOX9 could regulate the cell proliferation by influencing on cell cycle. We evaluated the effects of SOX9 on the proliferation of HUC-MSCs by Cell Counting Kit-8 assays and continuous cell counting for 5 days and found that knockdown of SOX9 expression significantly decreased the proliferation of HUC-MSCs (Fig. 2A and 2B). A colony formation assay further confirmed this result; knockdown of SOX9 in HUC-MSCs significantly inhibited colony formation (Fig. 2C and 2D). MSCs possess a homing ability, ensuring that they can migrate to injured sites (36). Therefore, exploring the influences of SOX9 on the migration of HUC-MSCs is important for its application in regenerative medicine. We found that knockdown of SOX9 expression significantly attenuated the migration of HUC-MSCs (Fig. 2E and 2F). These results indicated that SOX9 plays critical roles in regulating the proliferation and migration of HUC-MSCs.

Interleukin (IL)-6 and interleukin (IL)-8, which are the most abundant cytokines secreted by MSCs, play critical roles in regulating tumor cell proliferation, tumor cell apoptosis, and tumorigenesis (37, 38). We detected the IL-6 and IL-8 mRNA expression in HUC-MSCs transfected with sh-control or shSOX9 by qRT-PCR and found that knockdown of SOX9 decreased IL-8 mRNA expression significantly and promoted IL-6 mRNA expression with no significant effect (Fig. 3A and 3B). Liu and Hwang (39) found that mesenchymal stem cells from cord blood secreted growth factors, including GM-CSF and VEGF, by a cytokine protein array. We also measured the GM-CSF and VEGF mRNA expression in HUC-MSCs transfected with sh-control or shSOX9 by qRT-PCR and found that knockdown of SOX9 significantly enhanced VEGF mRNA expression and promoted GM-CSF mRNA expression with no significance (Fig. 3C and 3D). SOX9 interacted with Janus kinase 1 (JAK1) and regulated IL-6-JAK1-STAT3 signaling activation (40), and STAT3 signaling activation promoted the secretion of IL-6 cytokines and enhanced the stemness abilities of cells (41). Detailed molecular mechanisms involved in the secretion of cytokines of HUC-MSCs regulated by SOX9 need to further explore in the future. Knockdown of SOX9 also significantly inhibited the mRNA levels of the stemness-related genes OCT4 and SALL4 (Fig. 3E and 3F). These results indicated that SOX9 regulates the expression of cytokines and stemness-related genes in HUC-MSCs.

To explore the effects of SOX9 on the pro-healing ability of HUC-MSCs, we established a skin wound model in rats. As shown in our previous report (21), HUC-MSCs obviously promoted the skin repair of burn injury in rats (Fig. 4A). Knockdown of SOX9 expression in HUC-MSCs obviously decreased the pro-healing ability of HUC-MSCs in a skin wound model (Fig. 4A and 4B). We further evaluated the effects of SOX9 knockdown in HUC-MSCs on the repair of histological structure in the burn injury area. The results of histological evaluation of wounds post-infusion showed that the thickness of epidermal layer obviously increased in wounds treated with the HUC-MSCs transfected with sh-control (Fig. 5A and 5B), while epidermis repair of the wounds after treatment with PBS or the HUC-MSCs transfected with shSox9 was worse than that after treatment with the HUC-MSCs transfected with sh-control (Fig. 5A and 5B). The thickness of the dermal layer was not obviously different in these three groups (Fig. 5A and 5B). The results of Ki67 immunofluorescence staining showed that the HUC-MSCs transfected with sh-control had more Ki67-positive cells in the wound area than the HUC-MSCs transfected with shSox9 (Fig. 5C). We also measured the expression of epidermal markers, including CK14 and CK18, by immunofluorescence. The results suggested that epithelial repair in the wound injury of rats treated with the HUC-MSCs transfected with sh-control was more effective than that in the rats treated with PBS or the HUC-MSCs transfected with shSox9, and the accessory structures of skin, including hair follicles and glands, were also greater in the wound region of the rats treated with the HUC-MSCs transfected with sh-control (Fig. 5C). These results revealed that SOX9 influences the pro-healing ability of HUC-MSCs.