MOLECULAR MEDICINE REPORTS 21: 1941-1949, 2020 TET2 is involved in DNA hydroxymethylation, cell proliferation and inflammatory response in keratinocytes XINXIN LIU1*, XIN WANG2*, NIAN LIU1*, KE ZHU1, SONG ZHANG1, XIAORU DUAN1, YUQIONG HUANG1, ZILIN JIN1, HIMANSHU JAYPAUL1, YAN WU1 and HONGXIANG CHEN1 1Department of Dermatology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hebei 430022; 2Department of Dermatology, Affiliated Hospital of Nantong University, Nantong, Jiangsu 226001, P.R. China Received July 4, 2019; Accepted January 29, 2020 DOI: 10.3892/mmr.2020.10989 Abstract. DNA methylation and hydroxymethylation are the Introduction most common epigenetic modifications associated with the cell cycle and the inflammatory response. The present study aimed Epigenetic processes, including DNA methylation, histone to investigate the role of 5-hydroxymethyl-cytosine (5-hmC) modification and non‑coding RNA regulation, are inherit- and ten‑eleven translocation‑2 (TET2) in keratinocytes. able changes that can affect gene expression without altering Following TET2 knockdown, dot blot analysis was performed the DNA sequence (1). In mammals, methylation at the to assess the levels of 5‑hmC in keratinocytes, using HaCaT carbon 5 position of cytosine [5‑methyl‑cytosine (5mC)] is cells. Subsequently, the viability and cell cycle of HaCaT cells the most common DNA modification, and it is involved in were assessed by MTT, Cell Counting Kit‑8 assay and flow various biological processes, including cell differentiation, cytometric assays. Cyclin‑dependent kinase inhibitor 2A and development and proliferation (2). 5mC is associated with proinflammatory cytokine protein and mRNA expression the regulation of a number of pathophysiological processes levels were also detected. The present results suggested that and states, including gene expression regulation, genomic TET2 may play an important role in regulating cellular prolif- imprinting, X‑chromosome inactivation, repression of eration by mediating DNA hydroxymethylation in HaCaT transposable elements, embryonic development and cell differ- cells. In addition, TET2 knockdown decreased the production entiation (2‑5). of proinflammatory cytokines, including lipocalin 2, S100 The ten‑eleven translocation (TET) family, which consists calcium binding protein A7, matrix metallopeptidase 9, C‑X‑C of Fe(II)‑ and 2‑oxoglutarate‑dependent dioxygenases, motif chemokine ligand 1, interferon regulatory factor 7 and has attracted increasing attention due to its key role in the interleukin‑7 receptor. The present study suggested that TET2 reversal of DNA methylation (3). It has been reported that regulated cell viability, apoptosis and the expression of inflam- TET proteins (TET1, TET2 and TET3) sequentially oxidize matory mediators in keratinocytes. Collectively, the results 5mC to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine indicated that TET2 knockdown may relieve inflammatory (5fC) and 5‑carboxylcytosine (5caC) in DNA (2,6,7). Previous responses in the skin. studies have reported that 5fC and 5caC may be converted into cytosine by a thymine‑DNA glycosylase/base excision repair‑dependent pathway (8‑10). In addition to mediating the formation of 5-hmC, TET proteins are involved in gene transcription repression via histone de‑acetylation, which represents a DNA methylation-independent epigenetic mecha- nism (11). Increasing evidence suggested that 5‑hmC, which is regu- Correspondence to: Professor Yan Wu or Professor Hongxiang Chen, lated by TET2, plays an important role in various diseases, Department of Dermatology, Union Hospital, Tongji Medical College, such as immune system disorders, hematopoietic diseases Huazhong University of Science and Technology, 1277 Jiefang Avenue, and cancers (5,6,8). Li et al (12), reported a predominant Wuhan, Hebei 430022, P.R. China role for TET2 in the differentiation of adult neural stem cells E‑mail: [email protected] E‑mail: [email protected] by increasing the levels of 5‑hmC. Sun et al (13), indicated that inhibiting the TET2 protein decreases the expression of *Contributed equally genes associated with cell death by mediating the formation of 5‑hmC, which aggravated ischemic damage in a rat model Key words: ten-eleven translocation-2, keratinocytes, hydroxy- of spinal cord injury. In addition, decreased 5‑hmC levels are methylation, proliferation, inflammation associated with the progression and poor survival of numerous malignancies, including melanoma, renal cell carcinoma, and gastric and ovarian cancer (14). 1942 LIU et al: TET2 ROLE IN KERATINOCYTES The skin provides the first line of defense against skin injury Dot blot analysis. Genomic DNA from transfected HaCaT and invading pathogens, and keratinocytes are the main active cells was extracted using a DNeasy Blood and Tissue kit (cat. cell type in the epidermis (15,16). Recent studies indicated that no. 69504; Qiagen, Inc.), according to the manufacturer's keratinocytes actively regulate cutaneous immune reactions, protocol. DNA concentration (50 ng/µl) was quantified using which may play a role in inflammatory diseases affecting the a micro ultraviolet‑visible spectrophotometer. DNA denatur- skin, including psoriasis and atopic dermatitis (15,17). The ation was performed at 95˚C for 10 min. Denatured DNA present study suggested that TET2 knockdown in HaCaT kera- samples were immediately placed in an ice water bath for tinocytes promoted inflammation. Therefore, the effects of 5 min. Subsequently, samples (2 µl) were spotted on a nylon TET2 on epigenetic modifications and cellular functions were membrane and blocked with 5% skimmed milk for 1 h at room investigated in the present study. The results of the present temperature. The membranes were incubated with anti‑5‑hmC study indicated that the TET2 protein‑mediated formation primary antibody (cat. no. 39769; 1:10,000; Active Motif, Inc.) of 5-hmC increased the proportion of apoptotic cells and the overnight at 4˚C. After washing, the membrane was incubated expression of inflammatory factors in HaCaT cells. with a horseradish peroxidase‑conjugated secondary anti- body (anti‑rabbit IgG; 1:2,000; cat. no. 7074; Cell Signaling Materials and methods Technology, Inc.) for 1 h at room temperature. Detection was performed using a chemiluminescence kit (cat. no. G2014; Cell culture. The human keratinocyte HaCaT cell line (cat. Wuhan Servicebio Technology Co., Ltd.). no. GDC106; China Center for Type Culture Collection) was identified by STR profiling performed by Cobioer Biosciences Cell Counting Kit‑8 (CCK‑8) assay. HaCaT cells (5x105/ml) Co., Ltd. HaCaT cells were maintained in minimal essential were cultured in 96‑well plates and transfected with plasmid medium (HyClone; GE Healthcare Life Sciences) supple- or siRNA for 48 h at 37˚C in a humidified atmosphere with mented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) 5% CO2. Cell viability was assessed using the CCK‑8 kit and 1% penicillin‑streptomycin (HyClone; GE Healthcare (cat. no. WC0037; Wuhan Servicebio Technology Co., Ltd.), Life Sciences) at 37˚C with 5% CO2 in a humidified incu- according to the manufacturer's protocol. CCK‑8 solution was bator. The medium was replaced every 3 days. Keratinocytes added to each well and the plates were incubated for 1‑4 h from generations 3 to 4 were used for subsequent experi- at 37˚C. The absorbance was measured at a wavelength of ments. At 60‑80% confluency, cells were transfected. For 450 nm using a microplate reader. The assay was performed small interfering (si)RNA transfection, HaCaT cells were in triplicate. randomly divided into four groups: i) Negative control (NC) group (transfected with control siRNA); ii) si‑TET2‑01 group MTT assay. HaCaT cells (5x105/ml) were cultured in 96‑well (transfected with a siRNA targeting TET2); iii) si‑TET2‑02 plates and transfected with plasmid DNA or siRNA for 48 h at group (transfected with a second siRNA targeting TET2), 37˚C in a humidified atmosphere with 5% CO2. Cell viability and iv) si‑TET2‑03 group (transfected with a third siRNA was measured using an MTT assay (cat. no. G4101; Wuhan targeting TET2). For plasmid DNA transfection, HaCaT cells Servicebio Technology Co., Ltd.), according to the manufac- were randomly divided into two groups: i) NC group (trans- turer's protocol. MTT solution was added to each well and fected with the empty GV230 vector), and ii) TET2 group incubated for 4 h at 37˚C in a humidified atmosphere with (transfected with GV230 containing the coding sequence of 5% CO2. Subsequently, the supernatant was removed and TET2). DMSO was used to dissolve the purple formazan. The absor- bance of each well was measured at a wavelength of 570 nm siRNA transfection. siRNAs were designed and obtained from using a microplate reader. Cell viability was calculated as the Guangzhou RiboBio Co., Ltd. The sequences of the siRNAs were ratio of absorbance at a wavelength of 570 nm between each as follows: si‑TET2‑01, 5'‑CCAGAATAGTCGTGTGAGT‑3'; group and the control group. si‑TET2‑02, 5'‑GCTCTGAACGGTATTTAAA‑3'; si‑TET2‑03, 5'‑CGAGACTCATAATGTCCAA‑3'; and NC Flow cytometric analysis. Following transfection, HaCaT siRNA, 5'‑UUCUCCGAACGUGUCACGU‑3'. TET2 and cells (5x105/ml) were cultured for 48 h at 37˚C in a humidified control siRNAs (40 pmol/ml) were transfected into HaCaT atmosphere with 5% CO2. Subsequently, cells were trypsinized cells (5x105/ml) using Lipofectamine® 3000 (Invitrogen; (0.25% trypsin at 37˚C for 3‑5 min), harvested and washed Thermo