UVB suppresses PTEN expression by upregulating miR-141 in HaCaT cells

MicroRNAs (miRNAs) are 21 to 24 nucleotide, non-coding RNA molecules that post-transcriptionally regulate the expression of target genes. Ultraviolet B (UVB) radiation has been shown to inhibit phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression in HaCaT cells through an unknown mechanism. In this study, we investigated whether miR-141 can regulate UVB exposure-mediated inhibition of PTEN expression. Real-time RT-PCR, annexin V/fluorescein isothiocyanate staining, Western blotting and anti-miRNA oligonucleotide transfection were employed in this study. We found that upregulation of miR-141 expression after UVB irradiation was inversely correlated with PTEN expression levels in HaCaT cells. Furthermore, miR-141 expression increased apoptosis, while anti-miR-141 partly restored PTEN expression and reversed the pro-apoptosis effect of UVB. UVB suppresses the expression of PTEN by upregulating miR-141 in HaCaT cells. Therefore, miR-141 is a potential gene therapy target for UVB-induced photodamage.


Introduction
Ultraviolet (UV) radiation from the sun, particularly the UVB component (290-320 nm), is the major cause of skin cancer. In addition, UV radiation elicits a variety of adverse effects, such as erythema, sunburn, inflammation, hyperplasia, hyper-pigmentation, immunosuppression, premature skin aging, and photocarcinogenesis [1,2] . These cellular events are mediated by the activation or suppression of a variety of genes after UVB exposure [3] . However, the precise molecular events involved in UVB-regulated gene expression remain largely unknown.
MicroRNAs (miRNAs) are short, noncoding RNAs of approximately 22 nucleotides, which regulate gene expression through sequence-specific base pairing with the 3'-untranslated region (3'-UTR) of target mRNAs [4,5] . To elucidate the molecular mechanisms underlying photodamage and skin carcinogenesis by UVB, several groups have investigated the microRNA expression profiles of UVB-irradiated cells using mi-croRNA microarray [6][7][8] . For instance, Guo et al. [6] investigated the differential expression profiles of miR-NAs in NIH 3T3 cells in response to UVB irradiation. Furthermore, changes in miRNA expression and stress granule formation were found to be most pronounced within the first hours after UVB irradiation, suggesting that miRNA-mediated gene regulation operates earlier than most transcriptional responses [7] . The function of the miRNA response may have some relationship with DNA damage response and cell proliferation [7][8][9] . In our preliminary study, we assessed the effect of UVB irradiation on miRNA expression changes in HaCaT keratinocytes and found that 3 miRNAs (miR-188-5p, miR-223 and miR-22) were down-regulated, while  3 miRNAs (miR-125a-5p, miR-146a and miR-141) were up-regulated in UVB-irradiated cells compared with untreated cells. These results indicated that miR-NAs were potentially involved in the pathogenesis of UVB-induced photodamage.
MiR-141 is a member of the miR-200 family and is an important regulator of the epithelial to mesenchymal transition [10][11][12] . In addition to the role of miR-141 in the phenotypic conversion of normal cells, dysregulation of miR-141 expression occurs in multiple types of cancer cells and is linked to tumor progression [13][14][15] . However, the role of miR-141 expression in UVB-treated cells is not fully understood. The tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has been shown to be a target of miR-141 in nasopharyngeal carcinoma cells [15] . Furthermore, both UVA and UVB have been shown to downregulate PTEN in primary human keratinocytes, human HaCaT keratinocytes, human dermal fibroblasts and mouse skin [16,17] . Decreased PTEN expression after UV irradiation may promote the survival of epidermal keratinocytes [17] . Previous work suggests that PTEN downregulation can occur via two mechanisms: 1) PTEN is cleaved by active caspase in apoptotic cells; 2) PTEN transcription is suppressed in surviving cells independent of caspase activation [17] . In this study, we find that UVB suppresses the expression of PTEN by upregulating miR-141 in HaCaT cells.

Cell culture and UVB irradiation
The immortalized keratinocyte HaCaT cell line was obtained from Dr. Gu, Department of Dermatology, Changhai Hospital, Shanghai, China. Cells were maintained in Roswell Park Memorial Institute (RPMI)-1640 medium with 10% fetal bovine serum and 100 units/mL penicillin/streptomycin at 37°C in 5% CO 2 . At 70%-80% confluence, cells were cultured in serum-free RMPI-1640 medium 24 h before UVB irradiation. Subconfluent cultures were sham or UVirradiated in phosphate-buffered saline (PBS) through the plastic dish covered with a solar simulator (Sigma, USA). The 1 kW xenon arc lamp was adjusted to 8×10 -5 W/cm 2 and metered with a research radiometer fitted with a UVB probe at 285±5 nm. Sham cultures were handled identically except that they were shielded with aluminum foil during the irradiation.

miRNA isolation and real-time RT-PCR
Total RNA was extracted from each sample using a TRI Reagent Kit (Ambion, TX, USA) according to the manufacturer's instructions. Expression of miR-141 after UVB irradiation at different dosages (30, 60 and 90 mJ/cm 2 ) was analyzed using the TaqMan MicroR-NA Assays, TaqMan MicroRNA RT Kit and Taqman 29 Universal PCR Master Mix without UNG Amperase (Applied Biosystems, CA, USA) on an Applied Biosystems 7500 Fast Real Time PCR System. The reactions were incubated in a 96-well plate at 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The Applied Biosystems 7500 Fast software was used to analyze the Ct values of different miRNAs normalized to an endogenous control (U6).
The normalized values (dCT) from Group 1 were then compared to Group 2, yielding miRNA differential expression profiles. The relative amount of miRNAs was determined with the 2 -ΔΔCt method.
Annexin V/fluorescein isothiocyanate staining Apoptosis levels were detected 24 h after UVB irradiation in all groups. Treated cells were isolated, washed, and stained using annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI). Apoptosis was measured by performing flow-cytometric analysis using an ELITE ESP flow cytometer (Beckman-Coulter, USA).

Western blotting assay
PTEN, phospho-AKT and Bcl-xL protein levels were detected 24 h after UVB irradiation in all groups. Cellular lysate was prepared using radioimmunoprecipitation assay (RIPA) buffer with protease inhibitors and quantified using the bicinchoninic acid (BCA) protein assay (Pierce, IL, USA). Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, IN, USA) using a mini trans-blot (Hercules, CA, USA). Membranes were then blocked with PBST (PBS with 0.05% Tween 20) containing 5% non-fat dry milk for 1 h and incubated at 4°C overnight with anti-PTEN or anti-phospho-AKT polyclonal antibody (Santa Cruz Biotechnology, CA, USA), anti-Bcl-xL polyclonal antibody (Cell Signaling, CA, USA) in fresh blocking buffer. Membranes were then washed with PBST and incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, USA) for 1 h. The blots were developed using an enhanced chemiluminescence (ECL) kit (Pierce, IL, USA). Protein levels were normalized against β-actin (Sigma-Aldrich, MO, USA).

Anti-miRNA oligonucleotide transfection
HaCaT cells were transfected with 30 nM anti-miR TM miR-141 inhibitor or 30 nM of anti-miR TM miRNA inhibitor Negative control #1 (Control) (Ambion, TX, USA) using Lipofectamine 2000 (Invitrogen, USA) following the manufacturer's protocol. The cells were treated and analyzed 24 h after transfection.

Statistical analysis
Data were shown as mean±SE, the SPSS software package (Version 10.0; SPSS Inc., Chicago, IL) was used for the statistical analyses. All data were analyzed statistically using the Student's t-test, with P values less than 0.05 considered statistically significant.

Effects of UVB irradiation on miR-141 expression
The mechanism by which UVB radiation inhibits PTEN expression in HaCaT cells is unknown. Previously, PTEN was shown to be a target of miR-141 [15] . Therefore, we investigated miR-141 levels in UVBtreated cells. Our real-time RT-PCR results indicated that miR-141 expression was significantly upregulated at all UVB dosages examined (30, 60 and 90 mJ/cm 2 ; Fig. 1) compared to control-treated cells (0 mJ/cm 2 ).

CaT cells
As shown in Fig. 2, there was significant dosedependent decrease in the level of PTEN expression in UVB-treated HaCaT cells. Quantitative analysis

UVB can induce apoptosis in HaCaT cells
UVB had a dose-dependent cytotoxic effect on HaCaT cells, as revealed by our annexin V/PI staining assay results (Fig. 3). Treatment with 30-90 mJ/ cm 2 UVB induced significantly higher apoptosis rates (78.18% of cells were apoptotic at the 90 mJ/cm 2 UVB dose) compared with controls 24 h after irradiation.

Anti-miR-141 restored PTEN expression
To further confirm that miR-141 can affect PTEN protein levels in HaCaT cells, we transfected HaCaT cells with anti-miR-141 oligonucleotides (Fig. 4), then treated them with 30 mJ/cm 2 UVB. PTEN levels were determined by Western blotting 24 h after treatment. The results suggest that transfection of UVB-irradiated HaCaT cells with anti-miR-141 efficiently abolished the inhibitory effect of miR-141 (Fig. 5). Moreover, the annexin V/PI staining assay revealed that anti-miR-141 decreased apoptosis in UVB-irradiated Ha-CaT cells (Fig. 6), suggesting that miR-141 is a likely regulator of PTEN expression. In other words, anti-miR-141 can partially restore the expression of PTEN and reverse the pro-apoptosis effect of UVB ( Fig. 5 and Fig. 6).

Decreased PTEN expression induces AKT activation and Bcl-xL upregulation
Furthermore, in order to make clear the effect of PTEN in the apoptosis induced by UVB, we detected the phospho-AKT and Bcl-xL expression of each group, As shown in Fig. 2 and Fig. 5, the lever of phospho-AKT and Bcl-xL proteins were increased when the PTEN expression was down-regulated. These results indicated that PTEN-AKT-Bcl-xL could play an important part in the apoptosis of HaCaT cells and may be regulated by UVB and miR-141.

DISCUSSION
Damage to skin cells from repeated UVB exposure can lead to cancer [18][19][20] . Cellular mechanisms exist to repair the DNA damage, or induce apoptosis to remove severely damaged cells [21,22] . The molecular  A B C events involved in the induction of skin cancer are under active investigation, and until recently most studies have focused on protein-coding genes. However, several studies have now shown that UVB can regulate the expression of many miRNAs related to cell proliferation, differentiation, apoptosis and DNA damage response [6][7][8] , indicating that miRNAs play an important role in UVB-mediated responses. The PTEN gene, located at 10q23.3, encodes a central domain with homology to the catalytic region of protein tyrosine phosphatases [23] . This gene is an important regulator of protein phosphatases and 3'-phosphoinositol phosphatases [24] . PTEN dephosphorylates phosphatidylinositol-3,4,5-triphosphate (PIP3), the second messenger produced by phosphoinositide 3-kinase (PI3K), to negatively regulate the activity of the serine/threonine protein kinase, Akt [25][26][27] . PTEN is inactivated in some malignant tumors, resulting in Akt hyper-activation, thereby promoting cell proliferation, inhibition of apoptosis, and enhancing cell invasion and radioresistance [28][29][30] . In addition, UVB irradiation has been shown to increase PTEN and Akt phosphorylation in human dermal fibroblasts, which in turn leads to UVB-induced secretion of MMP-1 and -3, enzymes crucial for UV irradiation-induced photoaging in human skin [31] . These results indicate that PTEN is a target of UVB radiation and that it is involved in the pathology of photodamage. In this study, we also found that phospho-AKT and Bcl-xL expression was increased when the PTEN expression was downregulated, suggesting that the PTEN-AKT signal pathway could play a role in photodamage induced by UVB radiation in HaCaT cells.
In our previous study, we assessed the effect of UVB irradiation on miRNA expression in HaCaT cells and found that miR-141 was up-regulated in UVB-irradiated cells compared to controls. Using TargetScan and GO-analysis to predict miR-141 targets, we found that miR-141 may be involved in photocarcinogenesis, hypomethylation and apoptosis, suggesting that miR-141 is involved in the pathogenesis of photodamage. In the present study, we confirmed that miR-141 expression increased in a dose-dependent manner in UVB-treated cells. Furthermore, we found that upregulation of miR-141 expression after UVB radiation is inversely correlated with PTEN expression in HaCaT cells, which is accompanied by increased apoptosis.
Recently, UVB has been shown to down-regulate PTEN in apoptotic cells through degradation by active caspases, and by suppressing PTEN transcription in surviving cells [17] . The seed sequence of miR-141 was shown to match the 3'UTR of PTEN, and PTEN-3'UTR luciferase reporter assays confirmed PTEN as a direct target of miR-141 [15] . To further investigate the mechanism underlying PTEN downregulation, we employed anti-miRs that efficiently and specifically silence endogenous miRNAs. Anti-miR-141 is a specific inhibitor of miR-141. Our results indicated that anti-miR-141 increased PTEN expression and decreased apoptosis in UVB-treated cells. Since inhibition of miR-141 has also been shown to reduce the apoptosis rate in nasopharyngeal carcinoma cells [15] , we suggest that decreased apoptosis rate in UVBtreated cells can be attributed to the downregulation of miR-141. However, the decreased apoptosis rate may also contribute to increased PTEN expression following miR-141 inhibition, as PTEN is cleaved by active caspase in apoptotic cells [17] .
Because gene expression is regulated via a complex cellular network, it is likely that many factors other than miR-141 are involved in the regulation of PTEN expression. However, we conclude that UVB suppresses the expression of PTEN by upregulating miR-141 in HaCaT cells. Furthermore, the results of this study indicate that miR-141 could serve as a potential gene therapy target for UVB-induced photodamage.