Potential molecular characteristics in situ in response to repetitive UVB irradiation

Sunlight is essential for human life, but a previous study has revealed that it may induce 2 million new cases of non-melanoma skin cancer every year in the United States [1]. UV (ultraviolet) may impair the ability of basal keratinocytes to remove melanin and thus lead to hyperpigmentation [2]. Furthermore, UV radiation might be the major contributor to the risk of certain types of skin cancers in UV-radiated outdoor workers, such as basal cell carcinoma and squamous cell carcinoma [3]. UVB (ultraviolet-B) is a type of UV with wavelength ranging from 280 to 320 nm, and excessive UVB irradiation from sunlight may induce oxidative damage and inflammation in skin that further causes sunburn, photo-aging and various skin cancers [4].

Numerous researchers have been exploring the molecular characteristics in response to UVB exposure using animal models or cell culture systems. The activity of tyrosinase essential for melanogenic cascade can be stimulated by UVB exposure that further increases melanin synthesis required for UVB-induced delayed tanning [5]. The membrane-bound stem cell factor is up-regulated and then activates neighboring melanocytes through kit receptors in the course of UVB-caused pigmentation [6]. Nrf2 (nuclear factor, erythroid 2-like 2) may exert protective effects on an inflammation and a sunburn reaction that were induced by UVB exposure via inhibiting inflammation and extracellular matrix degradation [7]. More dangerously, UVB exposure can down-regulate the tumor suppressor TGFβ (Transforming growth factor β) signaling, suggesting its oncogenic effects [8]. Additionally, miRNAs (microRNAs) and TFs (transcription factors) are also implicated in the UVB irradiation-induced cellular response via regulating target genes [9, 10].

Although those previous studies have facilitated our understanding of molecular mechanisms in response to UVB exposure, they might not precisely reveal the molecular characteristics in situ where human skin is repetitively irradiated by UVB. Wonseon Choi et al. have demonstrated that UVB irradiation in situ can induce a large number of significant UVB-responsive genes implicated in regulating skin pigmentation, which has a different molecular characterization in response to UVA (ultraviolet-A) exposure [11]. However valuable, the above research focuses on the different molecular characteristics of human skin pigmentation under various types of UV irradiation. It is well known that different analysis procedures can obtain different findings. From a distinct perspective, this study was designed to explore the underlying molecular mechanisms in situ in response to UVB irradiation using various bioinformatics methods.

This study re-analyzed the published microarray data [11] to screen out DEGs (differentially expressed genes) between UVB-irradiated and non-irradiated skin biopsies, which were then subjected to enrichment analyses to identify biological metabolism and annotation analysis to identify tumor-associated DEGs. Then, PPIs (protein-protein interactions) among the DEGs were screened out to construct a PPI network, whilst TFs and miRNAs were screened out to construct regulatory networks. Accordingly, an integrated network among TFs, miRNAs and DEGs was constructed and further mined to identify densely connected modules.

Using expression profiling of UV radiation-induced melanocytes, Yang et al. revealed a set of p53 target genes in response to UV radiation through the Ingenuity® Pathway Analysis program [27]. In 2015, López et al. conducted a time-series analysis to compare the transcriptional profile of dark and light melanocyte lines under basal conditions and after UVB radiation, finding that ribosomal proteins can interact with the p53 signaling pathway in response to UVB [28]. This study identified a total of 151 up-regulated and 64 down-regulated genes in skin in response to UVB irradiation, which probably remained increased after repetitive exposure for approximately 2–3 weeks [11]. The up-regulated DEGs were significantly related to pigmentation, whereas the down-regulated DEGs were dramatically associated with immunity. The functional enrichment results were observed and were similar in the screened modules. As mentioned above, the up-regulated DEGs were significantly enriched in biological processes related to pigmentation. The obtained results are consistent with a previous study [11], implying the reliability of our research. However, the p53 pathway was not enriched in this study, indicating that in vitro and in situ studies have discrepancies and that an in situ study might be more credible. Importantly, this study further identified a number of TFs and miRNAs potentially targeting DEGs.

Several DEGs were enriched in pigmentation, such as DCT, SOX10, TYRP1, TYR, MLPH, KIT and GPR143, whose relationships with pigmentation have been revealed extensively in a previous study [11]. However, this study further revealed close interactions among DCT, SOX10, TYRP1, TYR and GPR143 in a densely connected module, suggesting a collaborative stimulating effect on the pigmentation process. Moreover, TYR had many interactions with other DEGs and was a hub node in the PPI network, implying predominant roles in the biological processes of pigmentation. Additionally, the up-regulated KIT, which was previously identified as a oncogenic drivers in sun-impaired skin, was annotated as a potential oncogene in this study [29].

Notably, some TFs and miRNAs were screened from the up-regulated DEGs. KIT is a potential target of miR-206 and miR-496, and DCT may be targeted by miR-184. As previously reported, miR-206 acts as a tumor suppressor in melanoma via inhibiting cancer cell growth and migration [30], miR-496 is involved in silencing gene expression in breast cancer, and miR-184 antagonizes miR-205 to protect against stratified squamous epithelia [31]. In consideration of the involvement of KIT and DCT in pigmentation and carcinogenesis, miRNAs might also be implicated in pigmentation in response to UVB irradiation. Moreover, miR-337, which can have a tumor suppressive effect on pancreatic ductal adenocarcinoma [32], was most significantly enriched, implying its involvement in the cellular response to UVB exposure. On the other hand, PAX5 and HNF4A were identified as two TFs targeting MLPH and GPR143, respectively. MLPH has been demonstrated as a putative pigmentation genes in East Asia [33], and GPR143 is targeted to melanosomes as a G protein-coupled receptor in pigment cells [34]. Thus, PAX5 and HNF4A might be involved in the regulation of pigmentation under UVB exposure. The up-regulation of DEGs may be associated with the screened the miRNAs and TFs under UVB irradiation.

On the other hand, the down-regulated DEGs were related to the immune system, suggesting an inactive immune regulation, e.g., GPR183, INHBA, PTPRC, PLEK, CD8A and IKZF1. UVB irradiation is responsible for inducing systemic immune suppression, which is an important contributor to skin cancer [35]. The relevance of those DEGs to the immune system or cancer has been revealed. Constitutively activated PTPRC modulates neutrophil recruitment and integrin activation during inflammation [36]. GPR183, which is primarily expressed in lymphoid cells, is important for the adaptive immune response [37]; as well as PLEK and CD8A are also important in this response [38, 39]. In addition, IKZF1 encodes a TF regulating lymphocyte differentiation [40], and INHBA, which belongs to the TGF-β superfamily, is associated with several cancers [41]. In the screened module, we further identified close interactions among GPR183, PTPRC and PLEK and observed their enrichment in the immune system. Notably, PTPRC and PLEK were hub nodes in the PPI network. Therefore, these down-regulated DEGs might jointly contribute to the suppressive immune response to UVB exposure, which might be a cause of skin cancer development. Additionally, the down-regulated DNAJB4, which is implicated in human gastric carcinomas [42], and SLIT2, which is involved in pancreatic cancer [43], are also annotated as tumor suppressors, suggesting their potential roles in UVB-induced skin cancers.

Additionally, some miRNAs were identified for the down-regulated DEGs, among which miR-21 and miR-16 were the most significantly enriched. The expression of miR-21 could be decreased by UVB irradiation in NIH3T3 cells [44]. MiR-16 targets the thyroid hormone receptor and nuclear factor-kappaB, which are vital for initiating epithelial immune responses [45]. Thus, miR-21 and miR-16 may play a role in the UVB-induced immunosuppressive response via down-regulating immune-related genes. In addition, GPR183, PTPRC and PLEK are all targeted by TFs BATF, SPI1 and EP300, which have been demonstrated to be involved in the immune system [46‐48]. Notably, we also observed interactions of EP300 with other down-regulated DEGs in the screened module. The p53/p300 complex can be recruited to the promoters of p53 target genes that are related to cell growth and apoptosis under UVB irradiation [49]. Therefore, the TFs and miRNAs may be involved in the cellular immune response to UVB exposure.

In conclusion, this study identified 151 up-regulated and 64 down-regulated genes implicated in the pigmentation and immune system response to UVB exposure. UVB-induced pigmentation has been demonstrated in previous studies, but the down-regulation of respectively by UVB exposure involved in the immune system have not been revealed. Furthermore, this study screened some TFs and miRNAs for the DEGs, which might play important roles in UVB irradiation-induced pigmentation and the immunosuppressive response. However, although we identified many DEGs, TFs and miRNAs with potential critical roles in UVB irradiation-induced cellular response, the obtained results relied on bioinformatics methods and need further validation.