Foreskin tissue was donated (with signed informed consent) by a healthy young male following circumcision. The donor's health status was established through physical examinations and laboratory investigations which revealed no obvious abnormalities. The sample was immersed in an iodine complex for 15 min and washed three times with phosphate buffered saline (PBS). Following removal of subcutaneous tissue, the sample was cut into pieces and digested with trypsin to isolate fibroblasts. Fibroblasts were collected, washed, and then incubated with high-glucose Dulbecco's Modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (HyClone; GE Healthcare Life Sciences, Logan, UT, USA). Cells in logarithmic phase from the third to eighth passage were used for experiments.

Cells that had reached ~70% confluence were irradiated with UVB using a SS-07 light therapy device (36 W power; time of irradiation automatically adjusted to dose of irradiation; cat. no. 1,047,469; Shanghai Sigma Hi-Tech Co., Ltd., Shanghai, China). Prior to irradiation, culture medium was removed from cells, which were then washed twice with PBS. Fibroblasts, coated with a thin layer of PBS, were vertically irradiated with UVB immediately following opening the culture dish cover. The distance of irradiation was 20 cm. Complete medium was added to the culture dish immediately following irradiation and the culture dish was then incubated in the original culture environment.

MALAT1 siRNA (5′-CACAGGGAAAGCGAGTGGTTGGTAA-3′) and the negative control non-targeting siRNA were designed and synthesized by GeneChem Co., Ltd. (Shanghai, China). When cells reached 30–50% confluence, siRNAs (50 nM) were transfected into cells using Lipofectamine^® 3000 (Thermo Fisher Scientific, Inc.,) in serum-free DMEM according to the manufacturer's protocol. Following 6 h of incubation, cells were returned to normal medium in the incubator. When cells reached 70% confluence, they were exposed to irradiation. Cells were pretreated with 10 mM N-acetyl-L-cysteine (NAC; Beyotime Institute of Biotechnology, Haimen, China) 1 h prior to irradiation. Following this, cells were supplemented with 10 mM NAC for 24 h.

Cells were seeded into a 96-well plate (10⁴ cells/well). Confluent cells were treated with serum-free medium for 24 h and irradiated with UVB. Following irradiation, cells were incubated with normal medium for 24 h and then treated with 10 µl Cell Counting kit (CCK)-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) for 4 h. Absorbance values were measured at a wavelength of 450 nm using a microplate reader (GEN5 CHS v.2.00, BioTek Instruments, Inc., Winooski, VT, USA).

Total RNA was extracted from cells using TRIzol^® reagent (Thermo Fisher Scientific, Inc.). Extracted total RNA, following purification and quality assessments, was reverse-transcribed to cDNA. RT-qPCR was performed to detect MALAT1 expression using the SYBR-Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) with a ABI PRISM 7700 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: Initial denaturation at 95°C for 2 min, then denaturation at 95°C for 15 sec, annealing at 56°C for 15 sec and extension at 68°C for 20 sec, for a total of 40 cycles. β-actin served as an internal reference. The expression level of MALAT1 was calculated using the 2^−∆∆Cq method (13) method. The primer sequences used were as follows: Forward, 5′-AAAGCAAGGTCTCCCCACAAG-3′ and reverse, 5′-GGTCTGTGCTAGATCAAAAGGCA-3′ for MALAT1. Experiments were performed in triplicate.

Following irradiation, cells were incubated with serum-free medium for 24 h. Cell culture medium was collected and centrifuged at 1,000 × g/min for 10 min at room temperature, and then supernatants were harvested. MMP-1 concentration was detected in supernatants using the MMP-1 kit (cat. no. EK0458; Boster Systems, Inc., Pleasanton, CA, USA) according to the manufacturer's protocol.

Following 24 h of UVB irradiation, cells were stained using an SA-β-Gal staining kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Senescent cells presented with a blue color around the nuclei and the percentage of senescent cells was equal to number of cells stained blue/total number of cells ×100%.

Following 24 h of UVB irradiation, cells were treated with serum-free medium containing 10 µM dichloro-dihydro-fluorescein diacetate (DCFH-DA; cat. no. KGT010-1; Nanjing Keygen Biotech Co., Ltd., Nanjing, China) for 30 min at 37°C, according to the manufacturer's protocol, and then washed three times with DMEM. Images were captured using a fluorescence microscope. Cells were collected and the level of green fluorescence was detected to evaluate the levels of intracellular ROS using flow cytometry (FACSCalibur, BD Biosciences, Franklin Lakes, NJ, USA) with BD CellQuest Pro software version 6.0 (BD Biosciences) according to the manufacturer's protocol.

Following fibroblast irradiation with UVB for 48 h, total protein was collected. Expression levels of ERK, phosphorylated ERK (p-ERK), p38, phosphorylated p38 (p-p38), JNK and phosphorylated JNK (p-JNK) were measured using western blot analysis. The process was conducted as previously described (14). Briefly, the cells were rinsed with PBS and lysed in radioimmunoprecipitation lysis buffer (Beyotime Institute of Biotechnology) with a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Inc.) on ice for 30 min. Cell lysates were centrifuged at 13,000 × g at 4°C for 20 min. The supernatants were collected and the proteins were quantified using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). Protein samples (20 µg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gels were transferred to nitrocellulose membranes, blocked with 5% non-fat-milk and then probed with anti-ERK (dilution, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA; cat. no. 9926), anti-p-ERK (dilution, 1:1,000; Cell Signaling Technology, Inc.; cat. no. 9910), anti-p38 (dilution, 1:1,000; Cell Signaling Technology, Inc.; cat. no. 9926), anti-p-p38 (dilution, 1:1000; Cell Signaling Technology, Inc.; cat. no. 9910), anti-JNK (dilution, 1:1,000; Cell Signaling Technology, Inc.; cat. no. 9926), or anti-p-JNK (dilution, 1:1,000; Cell Signaling Technology, Inc.; cat. no. 9910) antibody at 4°C overnight. Following incubation with a rabbit horseradish peroxidase-conjugated secondary antibody (dilution, 1:2,000; Abcam, Cambridge, UK; cat. no. ab6721) at room temperature for 2 h, immunoreactive bands were visualized using a electrochemiluminescence detection reagent (Yeasen; Shanghai Yi Sheng Biological Technology Co., Ltd., Shanghai, China; cat. no. 36208ES60) according to the manufacturer's instructions. Integrated optical density (IOD) was calculated using ImageJ version 1.46 software (http://rsb.info.nih.gov/ij). The relative intensity of the level of the protein of interest was calculated using the following formula: IOD_{phosphorylated protein band of interest}/IOD_{corresponding total protein band}. Representative blots of at least three independent experiments are presented.

Data were analyzed using SPSS software, version 22.0 (IBM SPSS, Armonk, NY, USA) and expressed as the mean ± standard deviation. All experiments were performed at least in triplicate. Significance tests were conducted on the data groups using analysis of variance followed by a comparison between the specific groups using a Student-Newman-Keuls test and analysis of differences between only two groups was performed using an unpaired Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

To investigate the phototoxicity of UVB on fibroblasts, cell viability was detected using the CCK-8 method, on cells irradiated with various doses of UVB for 24 h. The results demonstrated UVB suppressed melanocyte viability, an inhibitory effect enhanced by increasing doses of UVB. The inhibitory rate of 60 mJ/cm² UVB was ~25% (P<0.001; Fig. 1A) and results further demonstrated that 60 mJ/cm² UVB increased MALAT1 expression (P<0.01; Fig. 1B).

To investigate the effects of MALAT1 on photo-aging, MALAT1 expression in fibroblasts was silenced and effects on MMP-1 secretion observed. The results demonstrated that MALAT1 siRNA suppressed MALAT1 expression (P<0.01 and P<0.0001; Fig. 2A) and inhibited 60 mJ/cm² UVB-induced MMP-1 secretion (P<0.01 and P<0.001; Fig. 2B).

The ratio of senescent cells was markedly greater following UVB irradiation (74.4%) compared with cells that had not been exposed to irradiation (15.7%). In addition, the ratio of senescent cells was significantly lower following intervention with MALAT1 siRNA (52.1%) compared with the UVB irradiation group (P<0.01 and P<0.0001; Fig. 3).

Activation of MAPK signaling is important in photo-aging. The present study investigated the effect of the silencing of MALAT1 on activation of MAPK signaling pathway elements, induced by UVB. The results demonstrated that 60 mJ/cm² UVB upregulated ERK, JNK and p38 phosphorylation levels, however MALAT1 siRNA inhibited UVB-induced ERK phosphorylation (P<0.01), with no significant influence on JNK and p38 phosphorylation levels (P>0.05; Fig. 4).

ROS alters the expression levels of numerous molecules. The present study examined whether ROS underlies UVB-induced MALAT1 alteration using NAC, which is a ROS scavenger. The effects on MALAT1 expression in NAC-pretreated cells were investigated. The results indicated an increase in ROS content following fibroblast irradiation with UVB, which was then inhibited by NAC. However, NAC did not have a significant effect on MALAT1 expression (P=0.2307). These findings indicated that UVB-induced MALAT1 expression is not dependent on ROS generation (P<0.01, P<0.001 and P<0.0001 respectively; Fig. 5).