The NHDF cells were purchased from Lonza (Basel, Switzerland) and grown in DMEM media (Gibco-Invitrogen Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA) with penicillin/streptomycin in a humidified chamber at 37°C under 5% CO₂. Cells (4×10³) were seeded into 96-well plates a day before UVB exposure and treatment with the C. asiatica extract. For RNA purification, 7×10⁵ cells were seeded into 60-mm dishes.

Prior to UVB irradiation, cells were pre-treated with control dimethylsulfoxide (DMSO; Sigma-Aldrich) or TECA (Bayer HealthCare, Berlin, Germany) for 4 h. Cells were washed with PBS twice and then exposed to 100 mJ/cm² UVB without covering the 96-well plates or 60-mm dishes, so that the UVB light was not filtered. Following irradiation, the cells were cultured for 24 h in DMEM media containing 10% FBS with DMSO or TECA.

NHDF cells exposed to UVB with or without TECA were collected, and then total-RNA, including mRNAs, small RNAs and miRNAs, was extracted and purified from each cell pellet using TRIzol reagent (Invitrogen Life Technologies) according to the manufacturer’s protocol. The integrity of each RNA sample was verified using an Agilent 2100 Bioanalyzer^® (Agilent Technologies, Santa Clara, CA, USA). The quality and concentration of each RNA sample were determined using MaestroNano^®, a micro-volume spectrophotometer (Maestrogen, Las Vegas, NV, USA). RNA quality parameters for the miRNA microarray analysis were: A260/280 and A260/A230 values >1.8 and an RNA integrity number (RIN) >8.0.

The miRNA profiling analysis was performed using the SurePrint G3 Human v16 miRNA 8×60K (Agilent Technologies) that contained probes for 1,205 and 144 human viral miRNAs. The qualified RNA samples (100 ng) were first dephosphorylated using calf intestinal alkaline phosphatase (CIP) at 37°C for 30 min. Next, DMSO was added to each sample, and the samples were incubated at 100°C for 10 min and immediately transferred to an ice-water bath. The dephosphorylated RNA samples were labeled with cyanine 3-pCp using T4 RNA ligase by incubation at 16°C for 2 h. After the labeling reaction, the samples were completely dried using a vacuum concentrator at 55°C for 4 h. The dried samples were treated with GE Blocking Agent (Agilent Technologies) and hybridized to the probes on the microarray at 55°C with a constant rotation at 20 rpm in the Agilent Microarray Hybridization Chamber (Agilent Technologies) for 20 h. The microarray slide was washed and scanned using the Agilent scanner to obtain the microarray image. The numerical data for the miRNA profiles were extracted from the image using the Feature Extraction program (Agilent Technologies). These data were analyzed with the aid of the GeneSpring GX software version 7.3 (Agilent Technologies).

Among the total miRNAs probed on the microarray, 866 human miRNAs were selected for further analysis. The miRNAs whose flags were present in at least one sample were filtered and applied to the fold-change analysis. The fold-change analysis was conducted to select miRNAs whose expression changed by a factor of 1.2-fold or more between the following two groups: UVB-exposed and DMSO-treated NHDF control cells and UVB-exposed and 50 μg/ml TECA-treated NHDFs.

Changes in miRNA expression of 1.2-fold and more between the two groups were selected, and their putative cellular target genes were determined using MicroCosm Target version 5 (www.ebi.ac.uk/enright-srv/microcosm/thdoc/targets/v5/). Using a gene ontology (GO) analysis tool, AmiGO (amigo.geneontology.org/cgi-bin/amigo/browse.cgi), the target genes were categorized into the following four groups: aging, apoptosis, cell proliferation and skin development. Further GO analysis for miRNA target genes that were also identified by cross-linking and Argonaute (Ago) immunoprecipitation coupled with high-throughput sequencing (CLIP-Seq) data was performed using starBase web-based bioinformatics tools (starBase.sysu.edu.cn) (21).

We first screened for the dose range of TECA that is cytotoxic to NHDF cells. NHDF cells were treated with a series of four concentrations of TECA (25, 50, 100 and 200 μg/ml) for 24 h, and the WST-1-based cellular toxicity assay was used to determine the level of cell viability. As shown in Fig. 1A, low doses (up to 50 μg/ml) of TECA increased cell viability slightly; however, relatively high doses (100 and 200 μg/ml) of TECA decreased cell viability. In particular, although 100 μg/ml of TECA exhibited a low toxicity on NHDF cells, 200 μg/ml of TECA largely decreased cell viability. Therefore, TECA concentrations of 25, 50 and 100 μg/ml were chosen for further experiments. Next, we investigated the protective effect of TECA treatment against UVB-mediated damage of NHDFs. A day before UVB irradiation, NHDF cells were seeded and incubated in 96-well plates. The cells were then pre-treated with TECA, at the indicated concentration, for 4 h. The cells were then washed with PBS and exposed to 100 mJ/cm² of UVB without putting any protective covers on the microplates. Following UVB irradiation, the cells were incubated with TECA, at the indicated concentration, for 24 h. Cell viability was determined using the WST-1 assay, which revealed that treatment with TECA, in the range of 25 and 50 μg/ml, markedly restored the UVB-mediated loss of cell viability in NHDFs to the normal status in a dose-dependent manner (Fig. 2B). Treatment with 100 μg/ml of TECA did not increase cell viability more than the 50 μg/ml of TECA, which can be attributed to the cytotoxic effect of 100 μg/ml TECA on NHDF cells. Therefore, TECA displayed a protective effect against UVB-mediated loss of cell survival observed in NHDF cells.

Since miRNA is an important small non-coding RNA molecule that regulates development, differentiation, proliferation and apoptosis (22–25), we determined the protective effect of TECA against UVB-induced cell damage through a miRNA expression profiling analysis. Total-RNAs were purified from UVB-irradiated NHDF control cells and from 50 μg/ml of TECA-stimulated and UVB-irradiated NHDF cells, after which miRNA microarrays were performed using the Agilent SurePrint G3 Human v16 miRNA 8×60K, as described in Materials and methods. A total of 1,205 human miRNAs, including 144 human viral miRNAs, were selected to analyze the miRNA profiles. The human miRNAs whose flags were present in at least one sample were continuously filtered to obtain more defined data using the Agilent GeneSpring software. As shown in Fig. 2A, a total of 40 human miRNAs were differentially expressed following stimulation with TECA in UVB-irradiated NHDF cells compared to cells exposed to UVB alone. Upregulated miRNAs are shown in the left panel and downregulated miRNAs are shown in the right panel of Fig. 2A. The color bar displaying altered fluorescence intensity corresponds to miRNAs that were either upregulated (red colors) or downregulated (blue color) by TECA stimulation. The full list of the 40 miRNAs whose expression was altered by TECA is listed in Table I. The asterisk following the name indicates non-functional miRNA or passenger strand that is released from the miRNA duplex (26). Recent studies suggest that miRNA^* may offer potential opportunities for contributing to the regulation network (27). As shown in Fig. 2B, 19 miRNAs were upregulated and 21 miRNAs were downregulated under the experimental conditions. Although the majority of miRNAs did not show significant changes in expression, treatment with TECA did affect certain miRNA expression levels in NHDFs. These differentially expressed miRNAs may be involved in specific mechanisms of TECA-mediated cellular responses during the inhibition of UVB-induced cell damage in NHDFs.

miRNA expression profiling suggested a protective role of TECA against UVB-induced damage that may be dependent on the regulation of TECA-specific miRNA expression. These results further highlight the significance of the altered miRNA expression in light of the photoprotective property of TECA in UVB-induced damage of NHDFs. Since the cellular functions of miRNAs are mediated by controlling their target gene expression (28), we analyzed the cellular meaning of the TECA-dependent miRNA expression changes by sorting them according to three independent criteria (Fig. 3): i) the putative target genes of the differentially expressed miRNAs; ii) the cellular functions of the target genes; and iii) the target genes involved in TECA-mediated protective properties. First, using the miRBase Target Database tool, Microcosm, we identified approximately 2,758 potential targets for all miRNA, excluding human viral miRNAs. A total of 1,479 genes were identified as potential target genes for the upregulated miRNAs and 1,279 genes were identified as potential target genes for the downregulated miRNAs. Next, we identified the potential target genes involved in TECA-mediated protective properties against UVB damage, such as aging, apoptosis, cell proliferation and skin development. Using a GO web-based tool, AmiGO, we arranged the gene information in four classes: a total of 388 genes in aging, 3,824 genes in apoptosis, 3,148 genes in cell proliferation and 399 genes in skin development were identified (data not shown). We then compared these genes with the putative target genes (1,479 genes corresponding to upregulated miRNAs and 1,279 genes corresponding to downregulated miRNAs), and the overlapping genes in the two groups are listed in Tables II and III. Some of the miRNAs were potentially targeted by more than one miRNA, since a single miRNA may target a number of mRNAs, and, conversely, a single mRNA target may be modulated by several miRNAs (29).

The GO terms in Tables II and III cover a relatively wide range of cellular processes. For example, the GO term of apoptosis encompasses all the genes involved in apoptosis-promoting and apoptosis-inhibiting processes. Therefore, for a more accurate analysis, we rearranged the results shown in Tables II and III into a subset of GO terms, such as positive or negative regulation of the cell cycle, cell division, cell proliferation, cell growth and apoptosis, GTPase-, Ras-, MAPKK-mediated signal transduction and DNA-dependent transcription. As shown in Fig. 4, the target genes of the upregulated miRNAs are involved in promoting processes of cell proliferation; however, the target genes of the downregulated miRNAs are involved in inhibiting processes of apoptosis. Therefore, these findings suggest that TECA-mediated protective effects against UVB-induced damage in NHDFs is related to the changes in expression of specific miRNAs involved in cell proliferation and apoptosis.