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MicroRNA138 regulates keratin 17 protein expression to affect HaCaT cell proliferation and apoptosis by targeting hTERT in psoriasis vulgaris
Biomedicine & Pharmacotherapy 85 (2017) 169–176
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Original article
MicroRNA138 regulates keratin 17 protein expression to affect HaCaT cell proliferation and apoptosis by targeting hTERT in psoriasis vulgaris Shi-Jun Fenga,* , Rui-Qi Chub , Jing Maa , Zheng-Xiang Wanga , Guang-Jing Zhanga , Xiu-Fang Yanga , Zhi Songc , Yun-Yi Mac a b c
Department of Dermatology, Cangzhou Central Hospital, Cangzhou 061001, PR China Department of Dermatology, Affiliated Hospital of Hebei University, Baoding 071000, PR China Department of Dermatology, Jingzhou Central Hospital, Jingzhou 434020, PR China
A R T I C L E I N F O
Article history: Received 19 August 2016 Received in revised form 7 November 2016 Accepted 18 November 2016 Keywords: Psoriasis vulgaris MicroRNA-138 Keratin 17 hTERT HaCaT cells Proliferation Apoptosis
A B S T R A C T
The purpose of this study is to explore the how microRNA-138 (miR-138) affects the expression of keratin 17 (K17) and psoriasis development. Twenty-eight skin lesions from patients with psoriasis vulgaris and twenty-four normal skin tissues from healthy controls were collected. The HaCaT cells were assigned into blank, negative control (NC), miR-138 mimic, miR-138 inhibitor, hTERT siRNA and miR-138 inhibitor + hTERT siRNA groups. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the miR-138 expression. The hTERT and K17 protein expression were testified by Western Blotting. MTT assay, flow cytometry with PI single staining and Annexin V/PI double staining were performed to detect the cell proliferation activity, cell cycle and apoptosis, respectively. Compared with the healthy skin, the expression of miR-138 decreased in the psoriatic skin, but hTERT and K17 protein expressions increased. The miR-138 mimic and hTERT siRNA groups showed significantly decreased hTERT and K17 protein expressions, inhibited cell proliferation, increased number of cells at G1 phase and elevated apoptosis rate in comparison to the rest three groups. The hTERT and K17 protein expressions in the miR138 inhibitor group were up-regulated with promoted cell proliferation and reduced apoptosis rate as compared with the other four groups. In the miR-138 inhibitor + hTERT siRNA group, the hTERT and K17 protein expressions, cell proliferation and apoptosis were intermediate between the miR-138 inhibitor and hTERT siRNA groups. These findings indicated that the expression of miR-138 was lower in the psoriatic skin, which was negatively correlated to K17 expression. MiR-138 may regulate K17 protein expression to affect HaCaT cell proliferation and apoptosis by targeting hTERT gene. © 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Psoriasis is a T-cell mediated, chronic, inflammatory skin disease, of which psoriasis vulgaris is the most common type with major clinical symptoms of scaly debris and invasive erythema along with different degrees of itching [1]. The prevalence of psoriasis worldwide is about 2%–3% and it mostly occurs in adults over 39 years old [2]. Currently, the etiology and pathogenesis of psoriasis are not clear, but it has been reported that some factors, including genetic factors, environmental factors, immunological mechanisms, new blood vessel formation, lipid metabolism disorders and unhealthy mentality, bear significant impacts on
* Corresponding author at: Department of Dermatology, Cangzhou Central Hospital, No. 16, Western Xinhua Street, Cangzhou, 061001, Hebei Province, PR China. E-mail address: [email protected] (S.-J. Feng). http://dx.doi.org/10.1016/j.biopha.2016.11.085 0753-3322/© 2016 Elsevier Masson SAS. All rights reserved.
the occurrence of psoriasis [3]. It has been reviewed that general treatments of psoriasis are topical treatments, ultraviolet radiation b, psoralen/ultraviolet-A radiation (PUVA), methotrexate, cyclosporin-a, acitretin and biological agent therapies [4]. As the exploration into the development and progression of psoriasis, new therapies concerning cytokines, signaling molecules, and genes such as microRNAs (miRs) target gene have become potential therapeutic targets for psoriasis vulgaris [5,6]. MiRs are banded to the 30 untranslated regions (UTR) of mRNAs, which inhibits translation process and leads to accelerated turnover or degradation of the miRNA transcript [7]. Accordingly, abnormal expressions of miRs have been demonstrated to be associated with the occurrence of cancers [8,9], heart diseases [10], inflammatory diseases [11], and other medical conditions, which suggest that miRs can act as potential targets for medical diagnosis. The role of certain specific miRs in psoriasis has also been studied recently, such as hsa-miR-99a [5], miR-146a [12], and miR-125b
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[13]. As a member of miRs, miR-138 is involved in a variety of physical processes such as tumor metastasis and differentiation, DNA damage and cell senescence through regulating target genes [14,15]. Previous evidences have shown that the overexpression of miR-138 can induce a decrease in expression of human telomerase reverse transcriptase (hTERT) protein, which regulats telomerase activity in various cancers, such as malignant neuroblastoma, human anaplastic thyroid cancer and cervical cancer [11,16,17]. Interestingly, psoriasis is commonly characterized with the hyperproliferative state of keratinocytes and highly activated telomerase [18]. In addition, Keratin 17 (K17), compared to in normal epidermis, is reported to be abnormally expressed in the suprabasal keratinocytes of psoriatic lesions, [19]. K17 belongs to the group of human type I (acidic) epithelial keratins which help to keep the integrity of the epidermis by providing mechanical support to keratinocytes [20,21]. However, the functions of miR138 and hTERT gene are still not well understood in the specific skin diseases, such as skin lesions of psoriasis vulgaris. In this regard, we performed this study to explore the effects of miR-138 expression on psoriasis development by targeting the hTERT gene and we investigated how miR-138 affects the expression of K17, which may be a potential target for diagnosis and therapy of psoriasis vulgaris. 2. Material and methods 2.1. Study subjects We recruited 28 patients with psoriasis vulgaris (15 males and 13 females; average age: 31.4 9.7 years old) who were admitted into the Department of Dermatology of Cangzhou Central Hospital between June 2013 and January 2015. Patients who met the following criterion were eligible for this study: 1) patients pathologically diagnosed as psoriasis vulgaris; 2) patients with no consumption of drugs, such as immunosuppressant, glucocorticoid or vitamin A acid within three months before sample collection; 3) patients with no external use of drugs or light therapy for psoriasis vulgaris within at least 1 month before sample collection; 4) patients without other types of skin diseases, other autoimmune diseases, tumors or other serious diseases. Patients in pregnancy, lactation or menstrual period were excluded. Additionally, a total of 24 patients receiving surgery at the Department of Burn were recruited as the control group, consisting of 11 males and 13 females with the mean age of 32.2 10.1 years old. The inclusion criteria of the control group were: 1) no history of psoriasis and family history of disease; 2) no other immunological diseases; 3) no tumor or other severe diseases. Psoriatic skin and healthy skin were stored in liquid nitrogen. There was no significant difference of gender and age between the case and control groups (both P > 0.05). The experiment was approved by the Ethics Committee of Cangzhou Central Hospital and all subjects signed informed consent. 2.2. Quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA were extracted using miRNAeasy Mini Kit (Qiagen Company, Hilden, Germany). The concentration and purity of all RNA samples were detected by ultraviolet spectrophotometer at 260 nm and 280 nm. Optical density (OD) 260/OD280 ratio between 1.7–2.1 demonstrated a higher purity of RNA. Total RNA samples were reverse transcribed to synthesize complementary DNA (cDNA). qRT-PCR was performed on an ABI7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). The PCR reaction conditions: Pre-degeneration at 95 C for 10 min, denaturation at 95 C for 10 s, annealing at 60 C for 20 s, extension at 72 C for 34 s with a total of 40 cycles. MiR-138 upstream primer
sequence: 50 -GGTGTCGTGGAGTCGGCAA-30 , downstream primer sequence: 50 -AACTTCACAACACCAGCTTA-30 ; U6 upstream primer sequence: 50 -CTCGCTTCGGCAGCACA-30 , downstream primer sequence: 50 -AACGCTTCACGAATTTGCGT-30 . U6 gene was used as the internal reference. 2DDCt referred to the ratio of targeted gene expression between the experiment and control groups, with formula as DDCT = DCt experiment_group-DCt control_group (DCt = CtmiRNA CtU6). Ct referred to the cycle threshold that the realtime fluorescence intensity took to reach the expected threshold. 2.3. Western blotting The skins of psoriasis patients and healthy individuals were collected and treated with neutral protease at 4 C overnight. After complete grinding, cell protein lysate was added and samples were centrifuged at 2000 rpm for 2 min. Then, the supernatant was obtained, the concentration of which was examined using bicinchoninic acid (BCA) kit (Thermo Fisher Scientific, California, USA). The supernatant was boiled for 5–10 min and then stored at 20 C. The extracted protein underwent sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) for 1 h and then was transferred to polyvinylidene fluoride (PVDF) membrane for 20 min and sealed in milk solution for 2 h. With the addition of rabbit anti-human K17 and mouse anti-human, the membrane was maintained overnight at 4 C. Subsequently, HRP-labeled goat antirabbit IgG antibody and HRP-labeled rabbit anti-mouse IgG antibody were added and samples were incubated at 37 C for 1 h. The primary and second antibodies were purchased from Santa Cruz Biotechnology, CA, USA. Chemiluminescent visualization was performed in dark condition according to the instruction of electrochemical luminescence (ECL) color kit. Inner reference was at GAPDH. 2.4. HaCaT cell culture HaCaT cells (Wuhan Procell life Technology Co., Ltd., Hubei, China) were cultured in dulbecco's modified eagle medium (DMEM)/F12 (Gibco, Gaitherburg, MD, USA) containing 10% fetal calf (Hyclone, MA, USA) at 37 C in a humidified incubator with 5% CO2. The nutrient solution was refreshed every 2 days. When cells reached 80% confluence, they were passaged and digested with 0.25% pancreatin for 8 min, supplemented with 10% serumcontaining culture medium. When reaction was terminated, the cells were seeded into culture bottle. 2.5. Luciferase reporter gene assay DNA extraction was conducted according to the instruction of TIAN amp Genomic DNA Kit (TIANGEN Biotech (Beijing) Co., Ltd., China). Luciferase reporter vector was constructed. The luciferase reporter vectors of wild type 30 UTR (Wt-30 -UTR) and mutant type 30 UTR (Mut-30 -UTR) of hTERT gene were co-transfected with miR138 mimic or negative control, respectively, into the HaCaT cells. Luciferase activity of samples was detected using luciferase reporter assay system (E1910) (Promega Corporation, Madison, WI, USA). After 48 h transfection, the old medium was removed and sample was then washed by phosphate buffered saline (PBS) twice. With the addition of 100 ml passive lysis buffer (PLB) in each well, samples were slightly shaken at room temperature for 15 min, after which the cell lysates was collected. We set a program pre-reading at 2 s and program reading at 10 s, and set the injection volume of LARII Stop & Glo1 Reagent at 100 ml each time. Then the LARII Stop & Glo1 Reagent and luminotrons or plates with prepared cell lysates (20 ml of each sample) were put into the lumiaggregometer.
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2.6. HaCaT cell transfection and grouping After digested by 0.25% trypsin, the HaCaT cells was diluted to about 10 104 per ml in the culture medium and incubated at 37 C. Cells were divided into the following groups: 1) the blank group (without transfection), 2) the normal control (NC) group (transfected with miR-138 negative control sequence, purchased from Shanghai Gene Pharmaceutical Technology Co., Ltd, China), 3) the miR-138 mimic group (transfected with miR-138 mimic, purchased from Shanghai Gene Pharmaceutical Technology Co., Ltd, China), 4) the miR-138 inhibitor group (transfected miR-138 inhibitor, purchased from Shanghai Gene Pharmaceutical Technology Co., Ltd, China), 5) the hTERT siRNA group (transfected with hTERT siRNA, purchased from Shanghai Gene Pharmaceutical Technology Co., Ltd, China), and the miR-138 inhibitor + hTERT siRNA group (co-transfected with miR-138 inhibitor and hTERT siRNA, purchased from Shanghai Gene Pharmaceutical Technology Co., Ltd, China). Mixed with liposome 2000 (Thermo Fisher Scientific Inc., Shanghai, China) diluted by DMEM/F12 medium and segments in each group, cells were incubated for 20 min and then were transferred into 6-well plate. Six hours later, serumcontaining DMEM/F12 was adopted and cells were collected after 24, 36 and 48 h culture. MiR-138 expressions in cells after transfection were detected by qRT-PCR. 2.7. Western blotting After a 48 h transfection, cells were collected and 50 ml RIPA lysate (Thermo Fisher Scientific Inc., Shanghai, China) with 1% protease inhibitor was added to every 5 105 cells in ice condition for 30 min. After centrifuged at 12,000 r/min for 10 min at 4 C, the supernatant was obtained to detect the protein concentration by BCA protein assay. Expressions of hTERT and K17 proteins in the transfected cells were detected by Western Blotting. 2.8. MTT assay When cell density reached about 80%, cells were washed by PBS twice and digested with 0.25% trypsin and made into single cell suspension. Then, cells were inoculated in a 96-well plate with density of 3 103–6 103 cells/well with 200 ml in each well. After 48 h of culture, each well was added with 20 ml MTT solution (5 mg/ml, Sigma-Aldrich Chemical Company, St Louis MO, USA). Cells were cultured at 37 C for 4 h, after which the culture solution was aspirated. Then, 150 ml dimethyl sulfoxide (DMSO) (SigmaAldrich Chemical Company, St Louis MO, USA) were added to each well and slightly shaken for 10 min. Enzyme linked immunosorbent assay (ELISA) was used to determine the OD value of each well at wavelength of 490 nm at each time point. MTT curve was drawn with the OD value setting as the longitudinal coordinate and the interval setting (12, 24 and 48 h) as the horizontal coordinate.
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After a 48 h transfection, cells were digested and collected into the flow tube for centrifugation at 1000 r/min for 5 min with the supernatant aspirated. Then, cells were washed by cold PBS 3 times and the supernatant was aspirated. According to Annexin-V-FITC kit (Sigma-Aldrich Chemical Company, St Louis MO, USA) specifications, each tube was added with 5 ml Annexin-V-FITC and 150 ml binding buffer and cells were incubated at room temperature in the dark for 15 min after complete mixture. Then, 100 ml binding buffer and 5 ml PI were added and mixed (SigmaAldrich Chemical Company, St Louis MO, USA). Flow cytometry was performed to detect cell apoptosis. 2.10. Statistical analyses SPSS 19.0 software (SPSS, Inc., Chicago, IL, USA) was applied for statistical analysis. Continuous data were exhibited as mean standard deviation (x s) and t-test was used for comparison between two groups and one-way analysis of variance (ANOVA) for comparison among three groups. Pearson correlation analysis method was employed for correlation analysis. P < 0.05 was regarded as significant difference. 3. Results 3.1. Comparisons of miR-138 expression in the psoriatic skin and healthy control skin The qRT-PCR results showed that the expressions of miR-138 in the psoriatic skin and healthy control skin were 1.12 0.24 and 2.08 0.31, respectively, indicating that miR-138 was significantly down-expressed in patients with psoriasis vulgaris in comparison to the healthy individuals (P < 0.05) (Fig. 1). 3.2. Comparisons of hTERT and K17 protein expressions in the psoriatic skin and healthy control skin The results from Western Blotting (Fig. 2A) showed that hTERT and K17 protein expressions in the healthy control skin were 0.34 0.06 and 0.28 0.04, respectively. In the psoriatic skin, the expressions of these two proteins were 0.78 0.11 and 0.67 0.12, respectively. These results revealed that, compared with the healthy skin, the expressions of hTERT and K17 protein were much higher in the psoriatic skin (both P < 0.05) (Fig. 2B). 3.3. Correlation analysis of miR-138 with hTERT and K17 protein expressions The results of Pearson correlation analysis revealed that the expression of miR-138 was negatively related to the expression of hTERT (r = 0.623, P < 0.01, Fig. 3A), and also was negatively related
2.9. Flow cytometry HacaT cells at logarithmic phase were digested with pancreatin and centrifuged at 800 r/min for 5 min. Washed by PBS twice, cells were re-centrifuged at 800 r/min for 5 min and re-suspended by PBS, after which with the addition of 70% pre-cooling anhydrous ethanol, cells were maintained at 4 C in the dark overnight. On the next day, cells were collected after centrifugation and washed by PBS twice. Buffer solution was used to re-suspend cell deposition and 100 ml Rnase A was added for incubation at 37 C for 30 min. With the addition of 400 ml propidium iodide (PI) dye liquor, cells were incubated at room temperature in the dark for 30 min. The cell cycle was detected using flow cytometry (Becton, Dickinson and Company, NJ, USA).
Fig. 1. Comparisons of microRNA-138 expression in the psoriatic skin (n = 28) and healthy control skin (n = 24). Note: The miR-138 was significantly down-regulated in the psoriatic skin as compared to that in the healthy control skin; ** refers to P < 0.05 when compared with the healthy control skin.
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Fig. 2. Comparisons of hTERT And K17 protein expressions in the psoriatic skin (n = 28) and healthy control skin (n = 24). Note: A, Western Blotting graph about the hTERT and K17 protein expressions (line 1–3 refers to the psoriatic skin; line 4–6 refers to the healthy control skin); B, comparison of relative expressions of hTERT And K17 proteins in the psoriatic skin and healthy control skin; **refers to P < 0.05 when compared with the healthy control skin.
Fig. 3. Correlation analyses of microRNA-138 with hTERT and K17 protein expressions. A, Correlation analyses of microRNA-138 with hTERT protein expression in psoriatic skin; B, Correlation analyses of microRNA-138 with K17 protein expression in psoriatic skin; C, Correlation analyses of hTERT protein expression and K17 protein expression in psoriatic skin.
to the expression of K17 (r = 0.789, P < 0.01, Fig. 3B). But, the expression of K17 was positively related to the expression of hTERT (r = 0.683, P < 0.01, Fig. 3C). 3.4. MiR-138 targets hTERT By prediction and identification of target gene of miR-138 using bioinformatics technique-Target Scan (http://www.targetscan. org), we found that the sequence of 30 -UTR in hTERT matched with miR-138 (Fig. 4A). In order to prove the binding site of the miR-138 exerting predictive function on the change of the luciferase activity, reporter plasmid of the mutant sequence and the wild sequence on the binding site of 30 -UTR miR-138 deletion hTERT were designed respectively. The design of mutant site in the 30 -UTR of hTERT was showed in Fig. 4B. Using luciferase activity testing, mimic miR-138 and wild type (Wt-miR-138/hTERT) or
mutant (Mut-miR-138/hTERT) recombinant plasmids were transfected into the HaCaT cells. The results showed that miR-138 mimic had no significant effect on the luciferase activity of the Mut-miR138/hTERT plasmid group. However, the luciferase activity of the Wt-miR-138/hTERT group significantly decreased (P < 0.05) (Fig. 4C). 3.5. Comparisons of miR-138 expression among groups after transfection The qRT-PCR results indicated that miR-138 expression in the HaCaT cells in the miR-138 mimic group increased remarkably. However, the expressions of miR-138 in the miR-138 inhibitor and miR-138 inhibitor + hTERT siRNA groups decreased significantly. Compared with blank and NC groups, significant differences were observed among the rest three groups (all P < 0.05). There was no significant difference among the control, NC and hTERT siRNA groups (all P > 0.05). No significant difference in miR-138 expression was observed between the miR-138 inhibitor and miR-138 inhibitor + hTERT siRNA groups (P > 0.05) (Fig. 5). 3.6. Comparisons of hTERT and K17 protein expression among groups after transfection
Fig. 4. Effect of microRNA-138 on hTERT 30 UTR activity. A: The binding of microRNA-138 with hTERT 30 UTR region; B: The design of mutant site of 30 -UTR of hTERT gene; C: The relative luciferase activity of hTERT wild type or mutant 30 UTR in HaCaT cells following transfection with the miR-138 mimic; **refers to P < 0.05.
As shown in Fig. 6, hTERT and K17 protein expressions in the miR-138 inhibitor group were higher than those in the blank and NC groups (both P < 0.05). hTERT and K17 protein expressions in both miR-138 mimic and hTERT siRNA groups were significantly lower than those in the blank and NC groups (all P < 0.05). The expression levels of K17 and hTERT were not significantly different between the miR-138 mimic and siRNA hTERT groups (P > 0.05). While, hTERT and K17 protein expressions in miR-138 inhibitor + hTERT siRNA group significantly decreased when compared with miR-138 inhibitor group, but significantly increased as compared with the hTERT siRNA group (all P < 0.05). It revealed that hTERT
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Fig. 7. The proliferation curves of HaCaT cells after transfection at different time points by MTT assay. Note: The cell proliferation in the microRNA-138 mimic and hTERT siRNA groups were inhibited but promoted in the microRNA-138 inhibitor group, indicating that hTERT siRNA can reverse the increased cell proliferation caused by miR-138 inhibitor.
3.8. Comparisons of cell cycle and apoptosis among groups after transfection
Fig. 5. Comparisons of microRNA-138 expression in the HaCaT cells among groups after 48-h transfection. Note: Compared with the blank and NC groups, the microRNA-138 expression significantly increased in the microRNA-138 mimic and hTERT siRNA groups but decreased in the miR-138 inhibitor group; *refers to P < 0.05 when compared with the blank and NC groups, respectively.
siRNA can reverse the overexpression of hTERT and K17 caused by miR-138 inhibitor. There was no significant difference in hTERT and K17 expressions between the blank and NC groups (P > 0.05). 3.7. Comparisons of cell proliferation among groups after transfection MTT assay showed that the proliferation of the HaCaT cells in the miR-138 mimic and hTERT siRNA groups were strongly suppressed, and their corresponding OD values after 24 h were lower than those in the blank and NC group (all P < 0.05). There was no notable difference in HaCaT cell proliferation between the miR-138 mimic and hTERT siRNA groups (P > 0.05). Cell proliferation of the miR-138 inhibitor group was promoted, which was significantly different from that in the blank and NC groups (P < 0.05). Cell proliferation of the miR-138 inhibitor + hTERT siRNA group was significantly inhibited when compared with the miR138 inhibitor group, but significantly promoted when compared with the hTERT siRNA group (both P < 0.05), which demonstrated that hTERT siRNA can reverse the increased cell proliferation caused by miR-138 inhibitor. No significant difference in the cell viability between the blank and NC groups (P > 0.05) (Fig. 7).
PI single staining assay revealed that HaCaT cells in the miR-138 mimic and hTERT siRNA groups were mainly stagnated at the G1 phase, while the proportion of G2 phase and S phase showed a decline, as compared to the blank and NC groups, indicating that the proliferation of HaCaT cells was remarkably inhibited (all P < 0.05). The number of HaCaT cells at G1 phase in miR-138 inhibitor group obviously declined, but proportion of cells at S phase and G2 phase significantly increased, as compared to the blank and NC groups, indicating that HaCaT cells proliferation was promoted (all P < 0.05). The number of cells at G1 phase in the miR138 inhibitor + hTERT siRNA group was significantly higher than that in the miR-138 inhibitor group, but lower than that in hTERT siRNA group (both P < 0.05); the proportion of S phase and G2 phase cells in miR-138 inhibitor + hTERT siRNA group was obviously lower than that in the miR-138 inhibitor group, while higher than that in the hTERT siRNA group (both P < 0.05). The results indicated that hTERT siRNA can reverse the accelerated cell proliferation caused by miR-138 inhibitor. There was no significant difference in cell cycle between the blank and NC groups (P > 0.05) (Fig. 8). Annexin V/PI double staining assay showed that after a 48 h transfection, cell apoptosis rate of the blank, NC, miR-138 mimic, miR-138 inhibitor, hTERT siRNA and miR-138 inhibitor + hTERT siRNA groups were (14.92 1.31)%, (15.64 1.48)%, (32.31 1.57)%, (9.54 0.89)%, (29.97 2.44)% and (18.25 1.78)%, respectively. The apoptosis rates of HaCaT cells in miR-138 mimic and hTERT siRNA group were significantly higher than those of the other four groups (all P < 0.05). And there was no difference in HaCaT cells apoptosis rate between the miR-138 mimic and hTERT siRNA groups (P > 0.05). However, the apoptosis rate of the miR-138 inhibitor group significantly decreased when compared with the other five groups (all P < 0.05). The apoptosis rate of the miR-138 inhibitor + hTERT siRNA group was intermediate between the miR138 inhibitor and hTERT siRNA groups, with significant differences (both P < 0.05), which indicated that hTERT siRNA can reverse the reduction of apoptosis caused by miR-138 inhibitor. There was no significant difference in cell apoptosis rate between the blank and NC groups (P > 0.05) (Fig. 9). 4. Discussion
Fig. 6. Comparisons of hTERT and K17 protein expressions in the HaCaT cells among groups after 48-h transfection. Note: Compared with the blank and NC groups, the hTERT and K17 protein expressions significantly decreased in the miR-138 mimic and hTERT siRNA groups but increased in the miR-138 inhibitor group; *refers to P < 0.05 when compared with the blank and NC groups, respectively.
The investigation aims to explore how miR-138 affects the development of psoriasis vulgaris and its effect on the expressions of K17. And we found that the lower expression of miR-138 bore a negative relationship with K17. MiR-138 might be involved in the aberrant proliferation of keratinocytes, thus upregulating the expression of K17 protein by targeting hTERT. Our miRNA expression profiles revealed that hTERT and K17 protein expressions were up-regulated in psoriatic lesion skin in
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Fig. 8. Comparisons of HaCaT cell cycles among groups after 48-h transfection detected by PI single staining. Note: HaCaT cells in the microRNA-138 mimic and hTERT siRNA groups were mainly stagnated at the G1 phase. The number of HaCaT cells at G1 phase in the microRNA-138 inhibitor group obviously declined, but proportion of cells at S phase and G2 phase significantly increased, indicating that hTERT siRNA can reverse the accelerated cell proliferation caused by microRNA-138 inhibitor; A, cell cycle in each transfection group; B, percentage of cell in the G1, S and G2 phases in each transfection group; *refers to P < 0.05 when compared with the blank and NC groups, respectively.
comparison to those in healthy skin, while miR-138 was downregulated. Further, miR-138 was negatively related to the expression levels of hTERT and K17. These results suggested that miR-138 may regulate the hTERT and K17, and overexpression of hTERT and
K17 may be associated with the development of psoriasis vulgaris. Mitomo et al. have also confirmed that miR-138 was negatively related to hTERT in human anaplastic thyroid carcinoma cell lines [22]. It’s supposed that abnormal proliferation of skin cells result
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Fig. 9. Comparisons of HaCaT cell apoptosis rates among groups after 48-h transfection detected by Annexin V/PI double staining. Note: Compared with the blank and NC groups, the microRNA-138 mimic and hTERT siRNA groups showed significantly increased apoptosis rates, while the microRNA-138 inhibitor group showed strongly decreased apoptosis rate, indicating that hTERT siRNA could reverse the reduction of cell apoptosis caused by microRNA-138 inhibitor.
from the sequence of pathological events in psoriasis vulgaris, while elevated expression of hTERT can cause an elevation in the activity of telomerase, thus enabling the activation and cell proliferation, which then migrate to skin and release inflammatory cytokines [23,24]. Further, the release of inflammatory factors, such as IFN-g, may up-regulate the K17 expression through signal transducer and activator of transcription 1 (STAT1) signaling pathway, producing autoimmune reactions against K17, thus resulting in vicious circles and lesions, such as inflammatory reaction and abnormal proliferation of keratinocytes, which leads to psoriasis vulgaris [4,25]. To verify the above-mentioned hypothesis, we conducted an in vitro study on the HaCaT cells. We found that the expression levels of hTERT and K17 were down-regulated in the miR-138 mimic group, while the expression levels of hTERT and K17 were upregulated in the miR-138 inhibitor group. Further, we employed miR-138 targeting technology and found that hTERT could act as a target of miR-138 and participated in the process of aberrant proliferation of keratinocytes. Meanwhile, there was a rise in the expression of K17. Abnormal regulation of miRNAs in cancer cells can affect the function of tumor suppressors and oncogenes and generally miRNAs inhibit the expression of its target genes by binding to the 30 -UTR [26,27]. The possible mechanism refers to the fact that miR-138 directly combines with the 30 UTR of target gene hTERT, which impedes the translation process of miRNA and down-regulates the expression of hTERT, thus regulating the abnormal proliferation of keratinocyte cells [16,23]. It’s reported that miR-138 can act as an inducer of decay to hTERT levels via binding a common site in the 30 -UTR of hTERT to regulate its expression and hTERT expression and function, which depends on the ratio of miR-138 [17]. And moreover, it’s proven that hTERT is generally up-regulated in most cancer cell types as well in immortalized cells; and HaCaT is a kind of immortal keratinocyte cell [28,29]. The reactivation of hTERT in human HaCaT skin keratinocytes cause sand maintains the rapid proliferation of cancer cell, thereby contributing to the progression of psoriasis vulgaris [16,30–32]. And it can be predicted that miR-138 can
inhibit the expression of K17 combined with the seed sequence of K17 30 UTR. Previous studies showed that antisense oligonucleotides targeting K17 can inhibit the cell proliferation of keratinocyte cells and the expression of K17 and notably improve the pathology in psoriasis vulgaris of severe combined immune deficiency (SCID) transplantation model [33,34]. It's confirmed that K17 can be a new target for therapy of psoriasis vulgaris [35]. In addition, the low expression of miR-138 in psoriatic lesion skin contributes to the up-regulation of hTERT. Besides, the expressions of hTERT and K17 in the miR-138 mimic and hTERT miRNA groups obviously decreased, and their growth was inhibited at G1 phase, with a higher apoptosis rate. Therefore, we speculated that hTERT is related to cell apoptosis by inhibiting the expression of K17 and the activity of telomerase, and thus slowing down the cell proliferation and contributing to apoptosis. 5. Conclusion In conclusion, our study demonstrated that miR-138 was downregulated in psoriatic lesion skins, and it was negatively related to the expression of K17. By targeting hTERT, miR-138 could participate in the process of aberrant proliferation of keratinocytes and increase the expression of K17. Thus, gene-targeted inhibitors are more sensitive with promising prospects. This research presented that genes with similar formula to miR-138 and hTERT inhibitor can down-regulate the expression of K17 and slow down the growth of epidermal cells, which could be a new therapeutic target for psoriasis. Competing interests The authors have declared that no competing interests exist. Acknowledgments We would like to give our sincere appreciation to the reviewers for their helpful comments on this article.
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Original article
MicroRNA138 regulates keratin 17 protein expression to affect HaCaT cell proliferation and apoptosis by targeting hTERT in psoriasis vulgaris Shi-Jun Fenga,* , Rui-Qi Chub , Jing Maa , Zheng-Xiang Wanga , Guang-Jing Zhanga , Xiu-Fang Yanga , Zhi Songc , Yun-Yi Mac a b c
Department of Dermatology, Cangzhou Central Hospital, Cangzhou 061001, PR China Department of Dermatology, Affiliated Hospital of Hebei University, Baoding 071000, PR China Department of Dermatology, Jingzhou Central Hospital, Jingzhou 434020, PR China
A R T I C L E I N F O
Article history: Received 19 August 2016 Received in revised form 7 November 2016 Accepted 18 November 2016 Keywords: Psoriasis vulgaris MicroRNA-138 Keratin 17 hTERT HaCaT cells Proliferation Apoptosis
A B S T R A C T
The purpose of this study is to explore the how microRNA-138 (miR-138) affects the expression of keratin 17 (K17) and psoriasis development. Twenty-eight skin lesions from patients with psoriasis vulgaris and twenty-four normal skin tissues from healthy controls were collected. The HaCaT cells were assigned into blank, negative control (NC), miR-138 mimic, miR-138 inhibitor, hTERT siRNA and miR-138 inhibitor + hTERT siRNA groups. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the miR-138 expression. The hTERT and K17 protein expression were testified by Western Blotting. MTT assay, flow cytometry with PI single staining and Annexin V/PI double staining were performed to detect the cell proliferation activity, cell cycle and apoptosis, respectively. Compared with the healthy skin, the expression of miR-138 decreased in the psoriatic skin, but hTERT and K17 protein expressions increased. The miR-138 mimic and hTERT siRNA groups showed significantly decreased hTERT and K17 protein expressions, inhibited cell proliferation, increased number of cells at G1 phase and elevated apoptosis rate in comparison to the rest three groups. The hTERT and K17 protein expressions in the miR138 inhibitor group were up-regulated with promoted cell proliferation and reduced apoptosis rate as compared with the other four groups. In the miR-138 inhibitor + hTERT siRNA group, the hTERT and K17 protein expressions, cell proliferation and apoptosis were intermediate between the miR-138 inhibitor and hTERT siRNA groups. These findings indicated that the expression of miR-138 was lower in the psoriatic skin, which was negatively correlated to K17 expression. MiR-138 may regulate K17 protein expression to affect HaCaT cell proliferation and apoptosis by targeting hTERT gene. © 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Psoriasis is a T-cell mediated, chronic, inflammatory skin disease, of which psoriasis vulgaris is the most common type with major clinical symptoms of scaly debris and invasive erythema along with different degrees of itching [1]. The prevalence of psoriasis worldwide is about 2%–3% and it mostly occurs in adults over 39 years old [2]. Currently, the etiology and pathogenesis of psoriasis are not clear, but it has been reported that some factors, including genetic factors, environmental factors, immunological mechanisms, new blood vessel formation, lipid metabolism disorders and unhealthy mentality, bear significant impacts on
* Corresponding author at: Department of Dermatology, Cangzhou Central Hospital, No. 16, Western Xinhua Street, Cangzhou, 061001, Hebei Province, PR China. E-mail address: [email protected] (S.-J. Feng). http://dx.doi.org/10.1016/j.biopha.2016.11.085 0753-3322/© 2016 Elsevier Masson SAS. All rights reserved.
the occurrence of psoriasis [3]. It has been reviewed that general treatments of psoriasis are topical treatments, ultraviolet radiation b, psoralen/ultraviolet-A radiation (PUVA), methotrexate, cyclosporin-a, acitretin and biological agent therapies [4]. As the exploration into the development and progression of psoriasis, new therapies concerning cytokines, signaling molecules, and genes such as microRNAs (miRs) target gene have become potential therapeutic targets for psoriasis vulgaris [5,6]. MiRs are banded to the 30 untranslated regions (UTR) of mRNAs, which inhibits translation process and leads to accelerated turnover or degradation of the miRNA transcript [7]. Accordingly, abnormal expressions of miRs have been demonstrated to be associated with the occurrence of cancers [8,9], heart diseases [10], inflammatory diseases [11], and other medical conditions, which suggest that miRs can act as potential targets for medical diagnosis. The role of certain specific miRs in psoriasis has also been studied recently, such as hsa-miR-99a [5], miR-146a [12], and miR-125b
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[13]. As a member of miRs, miR-138 is involved in a variety of physical processes such as tumor metastasis and differentiation, DNA damage and cell senescence through regulating target genes [14,15]. Previous evidences have shown that the overexpression of miR-138 can induce a decrease in expression of human telomerase reverse transcriptase (hTERT) protein, which regulats telomerase activity in various cancers, such as malignant neuroblastoma, human anaplastic thyroid cancer and cervical cancer [11,16,17]. Interestingly, psoriasis is commonly characterized with the hyperproliferative state of keratinocytes and highly activated telomerase [18]. In addition, Keratin 17 (K17), compared to in normal epidermis, is reported to be abnormally expressed in the suprabasal keratinocytes of psoriatic lesions, [19]. K17 belongs to the group of human type I (acidic) epithelial keratins which help to keep the integrity of the epidermis by providing mechanical support to keratinocytes [20,21]. However, the functions of miR138 and hTERT gene are still not well understood in the specific skin diseases, such as skin lesions of psoriasis vulgaris. In this regard, we performed this study to explore the effects of miR-138 expression on psoriasis development by targeting the hTERT gene and we investigated how miR-138 affects the expression of K17, which may be a potential target for diagnosis and therapy of psoriasis vulgaris. 2. Material and methods 2.1. Study subjects We recruited 28 patients with psoriasis vulgaris (15 males and 13 females; average age: 31.4 9.7 years old) who were admitted into the Department of Dermatology of Cangzhou Central Hospital between June 2013 and January 2015. Patients who met the following criterion were eligible for this study: 1) patients pathologically diagnosed as psoriasis vulgaris; 2) patients with no consumption of drugs, such as immunosuppressant, glucocorticoid or vitamin A acid within three months before sample collection; 3) patients with no external use of drugs or light therapy for psoriasis vulgaris within at least 1 month before sample collection; 4) patients without other types of skin diseases, other autoimmune diseases, tumors or other serious diseases. Patients in pregnancy, lactation or menstrual period were excluded. Additionally, a total of 24 patients receiving surgery at the Department of Burn were recruited as the control group, consisting of 11 males and 13 females with the mean age of 32.2 10.1 years old. The inclusion criteria of the control group were: 1) no history of psoriasis and family history of disease; 2) no other immunological diseases; 3) no tumor or other severe diseases. Psoriatic skin and healthy skin were stored in liquid nitrogen. There was no significant difference of gender and age between the case and control groups (both P > 0.05). The experiment was approved by the Ethics Committee of Cangzhou Central Hospital and all subjects signed informed consent. 2.2. Quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA were extracted using miRNAeasy Mini Kit (Qiagen Company, Hilden, Germany). The concentration and purity of all RNA samples were detected by ultraviolet spectrophotometer at 260 nm and 280 nm. Optical density (OD) 260/OD280 ratio between 1.7–2.1 demonstrated a higher purity of RNA. Total RNA samples were reverse transcribed to synthesize complementary DNA (cDNA). qRT-PCR was performed on an ABI7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). The PCR reaction conditions: Pre-degeneration at 95 C for 10 min, denaturation at 95 C for 10 s, annealing at 60 C for 20 s, extension at 72 C for 34 s with a total of 40 cycles. MiR-138 upstream primer
sequence: 50 -GGTGTCGTGGAGTCGGCAA-30 , downstream primer sequence: 50 -AACTTCACAACACCAGCTTA-30 ; U6 upstream primer sequence: 50 -CTCGCTTCGGCAGCACA-30 , downstream primer sequence: 50 -AACGCTTCACGAATTTGCGT-30 . U6 gene was used as the internal reference. 2DDCt referred to the ratio of targeted gene expression between the experiment and control groups, with formula as DDCT = DCt experiment_group-DCt control_group (DCt = CtmiRNA CtU6). Ct referred to the cycle threshold that the realtime fluorescence intensity took to reach the expected threshold. 2.3. Western blotting The skins of psoriasis patients and healthy individuals were collected and treated with neutral protease at 4 C overnight. After complete grinding, cell protein lysate was added and samples were centrifuged at 2000 rpm for 2 min. Then, the supernatant was obtained, the concentration of which was examined using bicinchoninic acid (BCA) kit (Thermo Fisher Scientific, California, USA). The supernatant was boiled for 5–10 min and then stored at 20 C. The extracted protein underwent sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) for 1 h and then was transferred to polyvinylidene fluoride (PVDF) membrane for 20 min and sealed in milk solution for 2 h. With the addition of rabbit anti-human K17 and mouse anti-human, the membrane was maintained overnight at 4 C. Subsequently, HRP-labeled goat antirabbit IgG antibody and HRP-labeled rabbit anti-mouse IgG antibody were added and samples were incubated at 37 C for 1 h. The primary and second antibodies were purchased from Santa Cruz Biotechnology, CA, USA. Chemiluminescent visualization was performed in dark condition according to the instruction of electrochemical luminescence (ECL) color kit. Inner reference was at GAPDH. 2.4. HaCaT cell culture HaCaT cells (Wuhan Procell life Technology Co., Ltd., Hubei, China) were cultured in dulbecco's modified eagle medium (DMEM)/F12 (Gibco, Gaitherburg, MD, USA) containing 10% fetal calf (Hyclone, MA, USA) at 37 C in a humidified incubator with 5% CO2. The nutrient solution was refreshed every 2 days. When cells reached 80% confluence, they were passaged and digested with 0.25% pancreatin for 8 min, supplemented with 10% serumcontaining culture medium. When reaction was terminated, the cells were seeded into culture bottle. 2.5. Luciferase reporter gene assay DNA extraction was conducted according to the instruction of TIAN amp Genomic DNA Kit (TIANGEN Biotech (Beijing) Co., Ltd., China). Luciferase reporter vector was constructed. The luciferase reporter vectors of wild type 30 UTR (Wt-30 -UTR) and mutant type 30 UTR (Mut-30 -UTR) of hTERT gene were co-transfected with miR138 mimic or negative control, respectively, into the HaCaT cells. Luciferase activity of samples was detected using luciferase reporter assay system (E1910) (Promega Corporation, Madison, WI, USA). After 48 h transfection, the old medium was removed and sample was then washed by phosphate buffered saline (PBS) twice. With the addition of 100 ml passive lysis buffer (PLB) in each well, samples were slightly shaken at room temperature for 15 min, after which the cell lysates was collected. We set a program pre-reading at 2 s and program reading at 10 s, and set the injection volume of LARII Stop & Glo1 Reagent at 100 ml each time. Then the LARII Stop & Glo1 Reagent and luminotrons or plates with prepared cell lysates (20 ml of each sample) were put into the lumiaggregometer.
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2.6. HaCaT cell transfection and grouping After digested by 0.25% trypsin, the HaCaT cells was diluted to about 10 104 per ml in the culture medium and incubated at 37 C. Cells were divided into the following groups: 1) the blank group (without transfection), 2) the normal control (NC) group (transfected with miR-138 negative control sequence, purchased from Shanghai Gene Pharmaceutical Technology Co., Ltd, China), 3) the miR-138 mimic group (transfected with miR-138 mimic, purchased from Shanghai Gene Pharmaceutical Technology Co., Ltd, China), 4) the miR-138 inhibitor group (transfected miR-138 inhibitor, purchased from Shanghai Gene Pharmaceutical Technology Co., Ltd, China), 5) the hTERT siRNA group (transfected with hTERT siRNA, purchased from Shanghai Gene Pharmaceutical Technology Co., Ltd, China), and the miR-138 inhibitor + hTERT siRNA group (co-transfected with miR-138 inhibitor and hTERT siRNA, purchased from Shanghai Gene Pharmaceutical Technology Co., Ltd, China). Mixed with liposome 2000 (Thermo Fisher Scientific Inc., Shanghai, China) diluted by DMEM/F12 medium and segments in each group, cells were incubated for 20 min and then were transferred into 6-well plate. Six hours later, serumcontaining DMEM/F12 was adopted and cells were collected after 24, 36 and 48 h culture. MiR-138 expressions in cells after transfection were detected by qRT-PCR. 2.7. Western blotting After a 48 h transfection, cells were collected and 50 ml RIPA lysate (Thermo Fisher Scientific Inc., Shanghai, China) with 1% protease inhibitor was added to every 5 105 cells in ice condition for 30 min. After centrifuged at 12,000 r/min for 10 min at 4 C, the supernatant was obtained to detect the protein concentration by BCA protein assay. Expressions of hTERT and K17 proteins in the transfected cells were detected by Western Blotting. 2.8. MTT assay When cell density reached about 80%, cells were washed by PBS twice and digested with 0.25% trypsin and made into single cell suspension. Then, cells were inoculated in a 96-well plate with density of 3 103–6 103 cells/well with 200 ml in each well. After 48 h of culture, each well was added with 20 ml MTT solution (5 mg/ml, Sigma-Aldrich Chemical Company, St Louis MO, USA). Cells were cultured at 37 C for 4 h, after which the culture solution was aspirated. Then, 150 ml dimethyl sulfoxide (DMSO) (SigmaAldrich Chemical Company, St Louis MO, USA) were added to each well and slightly shaken for 10 min. Enzyme linked immunosorbent assay (ELISA) was used to determine the OD value of each well at wavelength of 490 nm at each time point. MTT curve was drawn with the OD value setting as the longitudinal coordinate and the interval setting (12, 24 and 48 h) as the horizontal coordinate.
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After a 48 h transfection, cells were digested and collected into the flow tube for centrifugation at 1000 r/min for 5 min with the supernatant aspirated. Then, cells were washed by cold PBS 3 times and the supernatant was aspirated. According to Annexin-V-FITC kit (Sigma-Aldrich Chemical Company, St Louis MO, USA) specifications, each tube was added with 5 ml Annexin-V-FITC and 150 ml binding buffer and cells were incubated at room temperature in the dark for 15 min after complete mixture. Then, 100 ml binding buffer and 5 ml PI were added and mixed (SigmaAldrich Chemical Company, St Louis MO, USA). Flow cytometry was performed to detect cell apoptosis. 2.10. Statistical analyses SPSS 19.0 software (SPSS, Inc., Chicago, IL, USA) was applied for statistical analysis. Continuous data were exhibited as mean standard deviation (x s) and t-test was used for comparison between two groups and one-way analysis of variance (ANOVA) for comparison among three groups. Pearson correlation analysis method was employed for correlation analysis. P < 0.05 was regarded as significant difference. 3. Results 3.1. Comparisons of miR-138 expression in the psoriatic skin and healthy control skin The qRT-PCR results showed that the expressions of miR-138 in the psoriatic skin and healthy control skin were 1.12 0.24 and 2.08 0.31, respectively, indicating that miR-138 was significantly down-expressed in patients with psoriasis vulgaris in comparison to the healthy individuals (P < 0.05) (Fig. 1). 3.2. Comparisons of hTERT and K17 protein expressions in the psoriatic skin and healthy control skin The results from Western Blotting (Fig. 2A) showed that hTERT and K17 protein expressions in the healthy control skin were 0.34 0.06 and 0.28 0.04, respectively. In the psoriatic skin, the expressions of these two proteins were 0.78 0.11 and 0.67 0.12, respectively. These results revealed that, compared with the healthy skin, the expressions of hTERT and K17 protein were much higher in the psoriatic skin (both P < 0.05) (Fig. 2B). 3.3. Correlation analysis of miR-138 with hTERT and K17 protein expressions The results of Pearson correlation analysis revealed that the expression of miR-138 was negatively related to the expression of hTERT (r = 0.623, P < 0.01, Fig. 3A), and also was negatively related
2.9. Flow cytometry HacaT cells at logarithmic phase were digested with pancreatin and centrifuged at 800 r/min for 5 min. Washed by PBS twice, cells were re-centrifuged at 800 r/min for 5 min and re-suspended by PBS, after which with the addition of 70% pre-cooling anhydrous ethanol, cells were maintained at 4 C in the dark overnight. On the next day, cells were collected after centrifugation and washed by PBS twice. Buffer solution was used to re-suspend cell deposition and 100 ml Rnase A was added for incubation at 37 C for 30 min. With the addition of 400 ml propidium iodide (PI) dye liquor, cells were incubated at room temperature in the dark for 30 min. The cell cycle was detected using flow cytometry (Becton, Dickinson and Company, NJ, USA).
Fig. 1. Comparisons of microRNA-138 expression in the psoriatic skin (n = 28) and healthy control skin (n = 24). Note: The miR-138 was significantly down-regulated in the psoriatic skin as compared to that in the healthy control skin; ** refers to P < 0.05 when compared with the healthy control skin.
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Fig. 2. Comparisons of hTERT And K17 protein expressions in the psoriatic skin (n = 28) and healthy control skin (n = 24). Note: A, Western Blotting graph about the hTERT and K17 protein expressions (line 1–3 refers to the psoriatic skin; line 4–6 refers to the healthy control skin); B, comparison of relative expressions of hTERT And K17 proteins in the psoriatic skin and healthy control skin; **refers to P < 0.05 when compared with the healthy control skin.
Fig. 3. Correlation analyses of microRNA-138 with hTERT and K17 protein expressions. A, Correlation analyses of microRNA-138 with hTERT protein expression in psoriatic skin; B, Correlation analyses of microRNA-138 with K17 protein expression in psoriatic skin; C, Correlation analyses of hTERT protein expression and K17 protein expression in psoriatic skin.
to the expression of K17 (r = 0.789, P < 0.01, Fig. 3B). But, the expression of K17 was positively related to the expression of hTERT (r = 0.683, P < 0.01, Fig. 3C). 3.4. MiR-138 targets hTERT By prediction and identification of target gene of miR-138 using bioinformatics technique-Target Scan (http://www.targetscan. org), we found that the sequence of 30 -UTR in hTERT matched with miR-138 (Fig. 4A). In order to prove the binding site of the miR-138 exerting predictive function on the change of the luciferase activity, reporter plasmid of the mutant sequence and the wild sequence on the binding site of 30 -UTR miR-138 deletion hTERT were designed respectively. The design of mutant site in the 30 -UTR of hTERT was showed in Fig. 4B. Using luciferase activity testing, mimic miR-138 and wild type (Wt-miR-138/hTERT) or
mutant (Mut-miR-138/hTERT) recombinant plasmids were transfected into the HaCaT cells. The results showed that miR-138 mimic had no significant effect on the luciferase activity of the Mut-miR138/hTERT plasmid group. However, the luciferase activity of the Wt-miR-138/hTERT group significantly decreased (P < 0.05) (Fig. 4C). 3.5. Comparisons of miR-138 expression among groups after transfection The qRT-PCR results indicated that miR-138 expression in the HaCaT cells in the miR-138 mimic group increased remarkably. However, the expressions of miR-138 in the miR-138 inhibitor and miR-138 inhibitor + hTERT siRNA groups decreased significantly. Compared with blank and NC groups, significant differences were observed among the rest three groups (all P < 0.05). There was no significant difference among the control, NC and hTERT siRNA groups (all P > 0.05). No significant difference in miR-138 expression was observed between the miR-138 inhibitor and miR-138 inhibitor + hTERT siRNA groups (P > 0.05) (Fig. 5). 3.6. Comparisons of hTERT and K17 protein expression among groups after transfection
Fig. 4. Effect of microRNA-138 on hTERT 30 UTR activity. A: The binding of microRNA-138 with hTERT 30 UTR region; B: The design of mutant site of 30 -UTR of hTERT gene; C: The relative luciferase activity of hTERT wild type or mutant 30 UTR in HaCaT cells following transfection with the miR-138 mimic; **refers to P < 0.05.
As shown in Fig. 6, hTERT and K17 protein expressions in the miR-138 inhibitor group were higher than those in the blank and NC groups (both P < 0.05). hTERT and K17 protein expressions in both miR-138 mimic and hTERT siRNA groups were significantly lower than those in the blank and NC groups (all P < 0.05). The expression levels of K17 and hTERT were not significantly different between the miR-138 mimic and siRNA hTERT groups (P > 0.05). While, hTERT and K17 protein expressions in miR-138 inhibitor + hTERT siRNA group significantly decreased when compared with miR-138 inhibitor group, but significantly increased as compared with the hTERT siRNA group (all P < 0.05). It revealed that hTERT
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Fig. 7. The proliferation curves of HaCaT cells after transfection at different time points by MTT assay. Note: The cell proliferation in the microRNA-138 mimic and hTERT siRNA groups were inhibited but promoted in the microRNA-138 inhibitor group, indicating that hTERT siRNA can reverse the increased cell proliferation caused by miR-138 inhibitor.
3.8. Comparisons of cell cycle and apoptosis among groups after transfection
Fig. 5. Comparisons of microRNA-138 expression in the HaCaT cells among groups after 48-h transfection. Note: Compared with the blank and NC groups, the microRNA-138 expression significantly increased in the microRNA-138 mimic and hTERT siRNA groups but decreased in the miR-138 inhibitor group; *refers to P < 0.05 when compared with the blank and NC groups, respectively.
siRNA can reverse the overexpression of hTERT and K17 caused by miR-138 inhibitor. There was no significant difference in hTERT and K17 expressions between the blank and NC groups (P > 0.05). 3.7. Comparisons of cell proliferation among groups after transfection MTT assay showed that the proliferation of the HaCaT cells in the miR-138 mimic and hTERT siRNA groups were strongly suppressed, and their corresponding OD values after 24 h were lower than those in the blank and NC group (all P < 0.05). There was no notable difference in HaCaT cell proliferation between the miR-138 mimic and hTERT siRNA groups (P > 0.05). Cell proliferation of the miR-138 inhibitor group was promoted, which was significantly different from that in the blank and NC groups (P < 0.05). Cell proliferation of the miR-138 inhibitor + hTERT siRNA group was significantly inhibited when compared with the miR138 inhibitor group, but significantly promoted when compared with the hTERT siRNA group (both P < 0.05), which demonstrated that hTERT siRNA can reverse the increased cell proliferation caused by miR-138 inhibitor. No significant difference in the cell viability between the blank and NC groups (P > 0.05) (Fig. 7).
PI single staining assay revealed that HaCaT cells in the miR-138 mimic and hTERT siRNA groups were mainly stagnated at the G1 phase, while the proportion of G2 phase and S phase showed a decline, as compared to the blank and NC groups, indicating that the proliferation of HaCaT cells was remarkably inhibited (all P < 0.05). The number of HaCaT cells at G1 phase in miR-138 inhibitor group obviously declined, but proportion of cells at S phase and G2 phase significantly increased, as compared to the blank and NC groups, indicating that HaCaT cells proliferation was promoted (all P < 0.05). The number of cells at G1 phase in the miR138 inhibitor + hTERT siRNA group was significantly higher than that in the miR-138 inhibitor group, but lower than that in hTERT siRNA group (both P < 0.05); the proportion of S phase and G2 phase cells in miR-138 inhibitor + hTERT siRNA group was obviously lower than that in the miR-138 inhibitor group, while higher than that in the hTERT siRNA group (both P < 0.05). The results indicated that hTERT siRNA can reverse the accelerated cell proliferation caused by miR-138 inhibitor. There was no significant difference in cell cycle between the blank and NC groups (P > 0.05) (Fig. 8). Annexin V/PI double staining assay showed that after a 48 h transfection, cell apoptosis rate of the blank, NC, miR-138 mimic, miR-138 inhibitor, hTERT siRNA and miR-138 inhibitor + hTERT siRNA groups were (14.92 1.31)%, (15.64 1.48)%, (32.31 1.57)%, (9.54 0.89)%, (29.97 2.44)% and (18.25 1.78)%, respectively. The apoptosis rates of HaCaT cells in miR-138 mimic and hTERT siRNA group were significantly higher than those of the other four groups (all P < 0.05). And there was no difference in HaCaT cells apoptosis rate between the miR-138 mimic and hTERT siRNA groups (P > 0.05). However, the apoptosis rate of the miR-138 inhibitor group significantly decreased when compared with the other five groups (all P < 0.05). The apoptosis rate of the miR-138 inhibitor + hTERT siRNA group was intermediate between the miR138 inhibitor and hTERT siRNA groups, with significant differences (both P < 0.05), which indicated that hTERT siRNA can reverse the reduction of apoptosis caused by miR-138 inhibitor. There was no significant difference in cell apoptosis rate between the blank and NC groups (P > 0.05) (Fig. 9). 4. Discussion
Fig. 6. Comparisons of hTERT and K17 protein expressions in the HaCaT cells among groups after 48-h transfection. Note: Compared with the blank and NC groups, the hTERT and K17 protein expressions significantly decreased in the miR-138 mimic and hTERT siRNA groups but increased in the miR-138 inhibitor group; *refers to P < 0.05 when compared with the blank and NC groups, respectively.
The investigation aims to explore how miR-138 affects the development of psoriasis vulgaris and its effect on the expressions of K17. And we found that the lower expression of miR-138 bore a negative relationship with K17. MiR-138 might be involved in the aberrant proliferation of keratinocytes, thus upregulating the expression of K17 protein by targeting hTERT. Our miRNA expression profiles revealed that hTERT and K17 protein expressions were up-regulated in psoriatic lesion skin in
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Fig. 8. Comparisons of HaCaT cell cycles among groups after 48-h transfection detected by PI single staining. Note: HaCaT cells in the microRNA-138 mimic and hTERT siRNA groups were mainly stagnated at the G1 phase. The number of HaCaT cells at G1 phase in the microRNA-138 inhibitor group obviously declined, but proportion of cells at S phase and G2 phase significantly increased, indicating that hTERT siRNA can reverse the accelerated cell proliferation caused by microRNA-138 inhibitor; A, cell cycle in each transfection group; B, percentage of cell in the G1, S and G2 phases in each transfection group; *refers to P < 0.05 when compared with the blank and NC groups, respectively.
comparison to those in healthy skin, while miR-138 was downregulated. Further, miR-138 was negatively related to the expression levels of hTERT and K17. These results suggested that miR-138 may regulate the hTERT and K17, and overexpression of hTERT and
K17 may be associated with the development of psoriasis vulgaris. Mitomo et al. have also confirmed that miR-138 was negatively related to hTERT in human anaplastic thyroid carcinoma cell lines [22]. It’s supposed that abnormal proliferation of skin cells result
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Fig. 9. Comparisons of HaCaT cell apoptosis rates among groups after 48-h transfection detected by Annexin V/PI double staining. Note: Compared with the blank and NC groups, the microRNA-138 mimic and hTERT siRNA groups showed significantly increased apoptosis rates, while the microRNA-138 inhibitor group showed strongly decreased apoptosis rate, indicating that hTERT siRNA could reverse the reduction of cell apoptosis caused by microRNA-138 inhibitor.
from the sequence of pathological events in psoriasis vulgaris, while elevated expression of hTERT can cause an elevation in the activity of telomerase, thus enabling the activation and cell proliferation, which then migrate to skin and release inflammatory cytokines [23,24]. Further, the release of inflammatory factors, such as IFN-g, may up-regulate the K17 expression through signal transducer and activator of transcription 1 (STAT1) signaling pathway, producing autoimmune reactions against K17, thus resulting in vicious circles and lesions, such as inflammatory reaction and abnormal proliferation of keratinocytes, which leads to psoriasis vulgaris [4,25]. To verify the above-mentioned hypothesis, we conducted an in vitro study on the HaCaT cells. We found that the expression levels of hTERT and K17 were down-regulated in the miR-138 mimic group, while the expression levels of hTERT and K17 were upregulated in the miR-138 inhibitor group. Further, we employed miR-138 targeting technology and found that hTERT could act as a target of miR-138 and participated in the process of aberrant proliferation of keratinocytes. Meanwhile, there was a rise in the expression of K17. Abnormal regulation of miRNAs in cancer cells can affect the function of tumor suppressors and oncogenes and generally miRNAs inhibit the expression of its target genes by binding to the 30 -UTR [26,27]. The possible mechanism refers to the fact that miR-138 directly combines with the 30 UTR of target gene hTERT, which impedes the translation process of miRNA and down-regulates the expression of hTERT, thus regulating the abnormal proliferation of keratinocyte cells [16,23]. It’s reported that miR-138 can act as an inducer of decay to hTERT levels via binding a common site in the 30 -UTR of hTERT to regulate its expression and hTERT expression and function, which depends on the ratio of miR-138 [17]. And moreover, it’s proven that hTERT is generally up-regulated in most cancer cell types as well in immortalized cells; and HaCaT is a kind of immortal keratinocyte cell [28,29]. The reactivation of hTERT in human HaCaT skin keratinocytes cause sand maintains the rapid proliferation of cancer cell, thereby contributing to the progression of psoriasis vulgaris [16,30–32]. And it can be predicted that miR-138 can
inhibit the expression of K17 combined with the seed sequence of K17 30 UTR. Previous studies showed that antisense oligonucleotides targeting K17 can inhibit the cell proliferation of keratinocyte cells and the expression of K17 and notably improve the pathology in psoriasis vulgaris of severe combined immune deficiency (SCID) transplantation model [33,34]. It's confirmed that K17 can be a new target for therapy of psoriasis vulgaris [35]. In addition, the low expression of miR-138 in psoriatic lesion skin contributes to the up-regulation of hTERT. Besides, the expressions of hTERT and K17 in the miR-138 mimic and hTERT miRNA groups obviously decreased, and their growth was inhibited at G1 phase, with a higher apoptosis rate. Therefore, we speculated that hTERT is related to cell apoptosis by inhibiting the expression of K17 and the activity of telomerase, and thus slowing down the cell proliferation and contributing to apoptosis. 5. Conclusion In conclusion, our study demonstrated that miR-138 was downregulated in psoriatic lesion skins, and it was negatively related to the expression of K17. By targeting hTERT, miR-138 could participate in the process of aberrant proliferation of keratinocytes and increase the expression of K17. Thus, gene-targeted inhibitors are more sensitive with promising prospects. This research presented that genes with similar formula to miR-138 and hTERT inhibitor can down-regulate the expression of K17 and slow down the growth of epidermal cells, which could be a new therapeutic target for psoriasis. Competing interests The authors have declared that no competing interests exist. Acknowledgments We would like to give our sincere appreciation to the reviewers for their helpful comments on this article.
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