The role of UVA-radiation-the major fraction in sunlight-in human skin carcinogenesis is still elusive. We here report that different UVA exposure regime (4 Â 5 J/cm 2 per week or 1 Â 20 J/cm 2 per week) caused tumorigenic conversion (tumors in nude mice) of the HaCaT skin keratinocytes. While tumorigenicity was not associated with general telomere shortening, we found new chromosomal changes characteristic for each recultivated tumor. Since this suggested a nontelomere-dependent relationship between UVA irradiation and chromosomal aberrations, we investigated for alternate mechanisms of UVA-dependent genomic instability. Using the alkaline and neutral comet assay as well as c-H2AX foci formation on irradiated HaCaT cells (20-60 J/cm 2 ), we show a dosedependent and long lasting induction of DNA single and double (ds) strand breaks. Extending this to normal human skin keratinocytes, we demonstrate a comparable damage response and, additionally, a significant induction and maintenance of micronuclei (MN) with more acentric fragments (indicative of ds breaks) than entire chromosomes particularly 5 days post irradiation. Thus, physiologically relevant UVA doses cause long-lasting DNA strand breaks, a prerequisite for chromosomal aberration that most likely contribute to tumorigenic conversion of the HaCaT cells. Since normal keratinocytes responded similarly, UVA may likewise contribute to the complex karyotype characteristic for human skin carcinomas.

MicroRNA (miRNA)-mediated regulation of the cellular transcriptome is an important epigenetic mechanism for fine-tuning regulatory pathways. These include processes related to skin cancer development, progression and metastasis. However, little is known about the role of microRNA as an intermediary in the carcinogenic processes following exposure to UV-radiation. We now show that UV irradiation of human primary keratinocytes modulates the expression of several cellular miRNAs. A common set of miRNAs was influenced by exposure to both UVA and UVB. However, each wavelength band also activated a distinct subset of miRNAs. Common sets of UVA- and UVB-regulated miRNAs harbor the regulatory elements GLYCA-nTRE, GATA-1-undefined-site-13 or Hox-2.3-undefined-site-2 in their promoters. In silico analysis indicates that the differentially expressed miRNAs responding to UV have potential functions in the cellular pathways of cell growth and proliferation. Interestingly, the expression of miR-23b, which is a differentiation marker of human keratinocytes, is remarkably up-regulated after UVA irradiation. Studying the interaction between miR-23b and its putative skin-relevant targets using a Luciferase reporter assay revealed that RRAS2 (related RAS viral oncogene homolog 2), which is strongly expressed in highly aggressive malignant skin cancer, to be a direct target of miR-23b. This study demonstrates for the first time a differential miRNA response to UVA and UVB in human primary keratinocytes. This suggests that selective regulation of signaling pathways occurs in response to different UV energies. This may shed new light on miRNA-regulated carcinogenic processes involved in UV-induced skin carcinogenesis.

UVA-radiation (315-400 nm) has been demonstrated to be capable of inducing DNA damage and is regarded as a carcinogen. While chromosomal aberrations found in UVA-irradiated cells and skin tumors provided evidence of the genetic involvement in UVA-carcinogenesis, its epigenetic participation is still illusive. We thus analysed the epigenetic patterns of 5 specific genes that are involved in stem cell fate (KLF4, NANOG), telomere maintenance (hTERT) and tumor suppression in cell cycle control (P16(INK4a), P21(WAFI/CIPI)) in chronically UVA-irradiated HaCaT human keratinocytes. A striking reduction of the permissive histone mark H3K4me3 has been detected in the promoter of P16(INK4a) (4-fold and 9-fold reduction for 10 and 15 weeks UVA-irradiated cells, respectively), which has often been found deregulated in skin cancers. This alteration in histone modification together with a severe promoter hypermethylation strongly impaired the transcription of P16(INK4a) (20-fold and 40-fold for 10 weeks and 15 weeks UVA-irradiation, respectively). Analysis of the skin tumor-derived cells revealed the same severe impairment of the P16(INK4a) transcription attributed to promoter hypermethylation and enrichment of the heterochromatin histone mark H3K9me3 and the repressive mark H3K27me3. Less pronounced UVA-induced epigenetic alterations were also detected for the other genes, demonstrating for the first time that UVA is able to modify transcription of skin cancer associated genes by means of epigenetic DNA and histone alterations.

No abstract

Cellular microRNAs (miRNA) have proven to be critical regulators of inflammatory gene expression across many pathways within autoimmunity. Circulating miRNAs serve as a new class of disease biomarkers. Nevertheless, the functional roles of miRNAs, particularly extracellular miRNAs, in systemic lupus erythematosus (SLE) remain poorly understood. Therefore, we aimed to link changes in extracellular miRNAs to lymphocyte gene regulation and the pathophysiology of SLE. Here, we demonstrate that circulating miR-22-3p levels are associated with SLE, and miR-22-3p regulates T and B cell function and SLE-associated kidney disease. Based on high-throughput small RNA sequencing and real-time PCR, extracellular miR-22-3p levels were found to be significantly increased in whole plasma in human SLE subjects. To determine the functional impact of miR-22-3p in SLE, miR-22-3p loss-of-function studies were performed in a mouse model of SLE (B6.SLE1.2.3). We found that in vivo administration of locked-nucleic acid inhibitors of miR-22-3p (LNA-22) reduced lymphocyte accumulation in both the spleen and lymph nodes compared to LNA scramble (LNA-Scr) control-treated mice. Strikingly, LNA-22-3p treatments reduced kidney disease pathology and glomerular IgG deposition compared to LNA-Scr treatments in SLE mice. Moreover, miR-22-3pinhibition reduced the proportion of T effector memory IFN-g producing CD4 + T cells, suggesting that miR-22-3p regulates Th1 T cell differentiation. We also found that miR-22 inhibition in mice reduced STAT1 phosphorylation in the kidney which was correlated with loss of IFNg production by splenic CD4 + T cells. In conclusion, our findings suggest that miR-22-3p is a critical regulator of SLE-associated CD4 + T cell immunity and kidney disease. These results provide therapeutic potential for limiting splenic Th1 signaling and preventing the progression of lupus nephritis. Key Findings:• Extracellular miR-22-3p levels are significantly increased in plasma from human SLE subjects.• Inhibition of miR-22-3p in vivo significantly reduced lymphocyte accumulation in both the spleen and lymph nodes in a mouse model of SLE, thus reducing splenomegaly and lymphadenopathy.• miR-22-3p inhibition significantly reduced IFN-g expression and secretion from splenic T cell subsets.• Inhibition of miR-22-3p in vivo resulted in decreased IgG deposition in the kidney, decreased STAT1 phosphorylation, and decreased kidney disease in a mouse model of SLE.