Association between SPRY1 and TET3 in skin photoaging and natural aging mechanisms

SPRY1 is associated with the invasiveness and prognosis of various tumors, and TET3 affects aging by regulating gene expression.

include ultraviolet radiation, severe physical and mental stress, excessive alcohol intake, diet, environmental pollutants, and electromagnetic and ionizing radiations, with the effect of ultraviolet radiation being especially prominent. 2There is a vast market for anti-aging products and treatments, with research on skin aging gaining popularity.
Keratinocytes serve as the first line of defense against exogenous damage and pathogens and are affected by ultraviolet radiation. 3Sprouty 1 (SPRY1), first discovered in Drosophila, is a feedback inhibitor of the receptor tyrosine kinase (RTK) signaling pathway, which is involved in cell proliferation and migration as well as tissue growth and development. 4SPRY1 is associated with the invasiveness and prognosis of various tumors. 5Additionally, RTK functions as an activating regulator of fibroblast growth factor (FGF) and epidermal growth factor (EGF) signaling, thereby affecting skin activities and functions.During aging, methylation is an epigenetic modification involved in gene regulation and a reversible process associated with gene expression inhibition. 6TET3, a methylcytosine dioxygenase, affects the aging process by regulating gene expression through DNA demethylation. 7A has the longest wavelength among UV rays and can reach the subcutaneous area to trigger a series of biological effects in the dermis and connective tissue.The 340-400 nm wavelength is important in skin photoaging and DNA damage. 8To reveal the potential roles of TET3 and SPRY1 in natural aging and photoaging of the skin, we aimed to investigate the effects of these genes in an in vitro skin model, a replicative aging model, and a cell photoaging model, as well as the effect of UVA irradiation on overall DNA methylation of the genome.

| Quantitative PCR
The details of quantitative PCR are provided in the Data S1.Primer 6 was used to design primers for amplifying SPRY1, TET3, and GAPDH, and synthesized by Jiangsu Kaiyuan Biotechnology Co., Ltd.Relative expression levels were determined using the 2 −ΔΔ C T method.The primer sequences are listed in Table 1.

| HaCaT cell culture and replicative aging model
HaCaT

| qPCR verification following transfection of HaCaT with TET3 siRNA
HaCaT-P7 cells were cultured to a density of 70%-80%; siRNA was diluted in serum-free Opti-MEM.Opti-MEM was added to Lipo3000, and the mixture was incubated at 26°C for 5 min and added to the medium.After 4-6 h of incubation, the medium was replaced with fresh medium.
NC siRNA and TET3 siRNA were transfected into HaCaT cells; this process is repeated thrice.HaCaT-NC and HaCaT-siTET3 were synthesized by Jiangsu KeyGEN, and the RNA sequences are shown in Table 2.
RNA was extracted from HaCaT cells using TRIzol, and TET3 and SPRY1 levels were compared between the silenced (HaCaT-siTET3 group) and blank control groups (HaCaT-NC group) using real-time PCR, as described above.

| Real-time PCR
HaCaT cells with a fusion degree of over 75% were collected from each group and RNA was extracted using TRIzol, as described above.

| Western blotting
Immunoblotting was used to detect the expression of SPRY1 and TET3 in the skin of three age groups.Samples were freeze-thawed; then, 0.5 × 2 cm skin samples were minced to extract protein using a whole protein extraction kit (BCA method).The extracted protein samples were stored at −70°C to avoid repeated freeze-thaw.
Based on the absorbance of the samples, the corresponding protein content was obtained using standard curve.SDS-PAGE electrophoresis, membrane transfer, blocking, antigen-antibody reaction, color development, and G:BOX chemiXR5 imaging were performed; Gel-Pr32 software was used for grayscale analysis of the results.
Immunoblotting was used to detect the expression of the two proteins in the replicable aging, HaCaT photoaging, and HaCa-si TET3 photoaging models.HaCaT cells with a fusion degree of over 75% were collected to extract proteins as described above.

| ELISA to detect changes in DNA methylation
We used ELISA to detect changes in the overall DNA methylation level (5-mC content) of each group of HaCaT and HaCaT-siTET3 photoaging models.Wash buffer (1×) and standards were prepared (Table S1), where Slope is the slope of the curve and S is the amount of DNA added.The same method was used to detect 5-mC in each group of the photoaging model following cell transfection.

| TET3 and SPRY1 expression in the skin
The expression levels of SPRY1 in the skin of parts not exposed to radiation were different among the three groups, with the highest expression in the elderly group and the lowest expression in the chil-

| TET3 and SPRY1 expression in the replicative aging cell model
Western blotting was used to detect protein expression in the young and aging groups of HaCaT cells (Figures 4 and S1).As the number of passages increased, the expression of SPRY1 was upregulated (t = 23.67,p < 0.05), whereas that of TET3 was downregulated (t = 16.17,p < 0.05).

| SPRY1 and TET3 expression in the replicative aging cell model
As the number of passages increased, SPRY1 expression was upregulated (t = 10.81,p < 0.05), whereas TET3 expression was downregulated (t = 6.824, p < 0.05).Statistical analysis results are shown in Figure S2.

| HaCaT DNA damage induced by UVA irradiation
Through immunofluorescence staining (Figure S3), it was found that the optical density value of the HaCaT irradiated group was higher than that of the HaCaT non-irradiated group (t = 5.449, p < 0.05).
Furthermore, the optical density value of the HaCaT-siTET3 irradiated group was higher than that of the HaCaT-NC irradiated group

2. 3 |
Overall experimental structure SPRY1 and TET3 expression in skin was measured using immunoblotting and real-time PCR.Thereafter, senescence and DNA damage were analyzed using staining techniques.Real-time PCR, western blotting, and enzyme-linked immunosorbent assay (ELISA) were used to further analyze the samples.The study was conducted from July 2020 to December 2022.

2. 3 . 4 | 2 . 3 . 5 |TA B L E 1
Establishing HaCaT and HaCaT-siTET3 photoaging models HaCaT and HaCaT-siTET3 cells were irradiated with UVA after transfection for 48 h.The UVA irradiation conditions were as follows: UVA wavelength, 365 nm; LED light, 10 J/cm 2 UVA irradiation, 1000 s each time, at an interval of 40 h, four times.Cell samples were collected 1 day after non-irradiation/irradiation four times, and each group of sample was subcultured for 15 generations for the subsequent experiments.HaCaT photoaging model grouping: HaCaT non-irradiated and HaCaT irradiated groups.HaCaT-siTET3 photoaging model grouping: HaCaT-NC irradiated, HaCaT-NC non-irradiated, HaCaT-siTET3 irradiated, and HaCaT-siTeT non-irradiated groups.Detection of γH2AX via immunofluorescence staining The details of immunofluorescence staining are provided in the Data S1.Protein expression in the cells was observed under a microscope and photos of the high expression areas were captured.Optical density value was determined using Image-Pro Plus 6.0 (Media Cybernetics, USA).Primer sequences and product lengths of SPRY1, TET3, and GAPDH.

2. 3 . 6 |
β-Galactosidase staining Each group of cells was fixed in β-galactosidase staining fixative for 15 min; the cell fixative was then removed.The cells were washed with PBS thrice, and then 1 mL of staining working solution was added.The cells were incubated overnight at 37°C, sealed, observed, and photographed under a regular optical microscope.
and then, DNA binding was stimulated.The 5-mC Detection Complex Solution turned blue after a few minutes, indicating the presence of sufficient methylated DNA; the color of the NC wells remained unchanged.When 5% of the positive control wells turned dark blue, a stop solution was added to terminate the enzyme reaction, and the wells were left undisturbed for 1-2 min to allow the color reaction to stop.The absorbance of the samples at 450 nm was measured using a microplate reader within 2-15 min, and the following was calculated:

GraphPad Prism 9 . 5 3 | RE SULTS 3 . 1 |
was used to conduct statistical analyses of the results, which are expressed as mean ± standard deviation.A univariate analysis of variance and Tukey's test were used for comparisons among groups, and t-tests were used for comparisons between groups.p < 0.05 was used to indicate statistically significant differences; ns p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and **p ≤ 0.0001.TET3 and SPRY1 expression in the skin SPRY1 expression was the highest in the elderly group and lowest in the children's group (F = 615.4,p < 0.05).TET3 expression was the lowest in the elderly group and highest in the children's group (F = 174.6,p < 0.05).The expression levels and statistical analysis results are shown in Figure 1.

F I G U R E 1
TET3 and SPRY1 expression in the skin not exposed to radiation among the indicated age groups.F I G U R E 2 TET3 and SPRY1 expression levels in the skin not exposed to radiation among the indicated age groups.F I G U R E 3 Comparison of TET3 and SPRY1 levels in the skin among the indicated age groups.F I G U R E 4 TET3 and SPRY1 levels in the cell replicative aging model.

.2 | Overall experimental structure
posed to light were collected from healthy people belonging to three age groups [children (<18 years), middle-aged individuals (46-60 years), and older adults (>60 years)], with 20 cases per group.The samples were immediately rinsed with 0.9% NaCl and stored in liquid nitrogen.