The cultured Hut78 human CTCL cell line (23) was purchased from American Type Culture Collection (Manassas, VA, USA). The non-malignant T-cell line (normal T lymphocytes as control cells), were established in the medical laboratory of The First Hospital of Zibo City (Zibo, China) from patients with mycosis fungoides and Sézary syndrome according to Woetmann's methods (24). Fresh blood samples were obtained from a 46-year-old male patient with mycosis fungoides and Sézary syndrome at the First Hospital of Zibo City (Zibo, China) in April 2015. The fresh blood was diluted with an equal volume of PBS and was gently dropped onto the lymphocyte separation medium (Anhui Haoyang Chemical Group Co., Ltd., Fuyang, China) which was in a 15-ml centrifuge tube. The non-malignant T-cell line was obtained from the interlayer between the blood and lymphocyte separation medium following 15-ml tube centrifugation for 15 min at 500 × g and 25°C. Briefly, in 5% CO₂ at 37°C, this non-malignant T-cell line was cultured in RPMI 1640 medium, containing 10% (v/v) heat-inactivated fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), L-glutamine and antibiotics (100 IU/ml of penicillin, 100 µg/ml of streptomycin). The present study was performed with the approval of the First Hospital of Zibo City. Signed written consent was obtained from the patient prior to recruitment to the study.

The following reagents and instruments were used in the present study: miRcute miRNA isolation kit, miRcute miRNA cDNA first-strand synthesis kit, miRcute miRNA quantitative fluorescence detection kit, SuperReal PreMix (SYBR Green), and TIANScript II cDNA first-strand synthesis kit; all were purchased from Tiangen Biotech Co., Ltd. (Beijing, China). The RT-qPCR instrument (BIOER FQD-96A), GATA-3 (cat. no. ab106625), TOX (cat. no. ab155768) and β-actin (cat. no. ab8227) primary antibodies and secondary antibody (cat. no. ab7090; Abcam, Cambridge, MA, USA), TRIzol reagent (Yisheng Biology, Shanghai, China), BCA protein assay reagent kit (Zhongke Ruitai, Beijing, China), and serum RNA extraction miRNeasy serum/plasma kit (Jianlun Biology, Guangzhou, China) were also used. All plasmids/agomiR were designed and synthesized by Shanghai Biological Technology Co., Ltd. (Shanghai, China). Pre-miR-135a, miR-135a mimic, miR-135a inhibitor, and their negative controls were purchased from GeneChem Co. (Shanghai, China; Table I). Xfect transfection reagents were purchased from Takara Biotechnology Co., Ltd. (Dalian, China).

The Hut78 cells and normal T lymphocytes were collected, and total RNAs were extracted with TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) using the phenol-chloroform extraction method. The RNA integrity was examined by gel electrophoresis, and the RNA purity was assessed according to the 260/280 ratio by spectrophotometry. Total RNA (2 µg) underwent RT to synthesize cDNA using Oligo-dT (10 µM) and Super Pure dNTPs (10 mM), according to the protocol of the TIANScript II RT kit (cat. no. KR107; Tiangen Biotech Co., Ltd.). The sequences of the GATA-3, GAPDH, miR-135a, and U6 primers used for RT-qPCR analysis are shown in Table II. GAPDH and U6 were used as internal controls for GATA-3 and miR-135a, respectively. The reaction mixture was prepared as follows, according to SuperReal PreMix Plus (SYBR Green) (cat. no. FP205; Tiangen Biotech Co., Ltd.): 10 µl SYBR Ex Taq II, 0.4 µl ROX Reference Dye, 0.8 µl each primer (final concentration, 250 nmol/l), 7 µl ddH₂O and 1.0 µl cDNA. The PCR procedure for GATA-3 was as follows: Pre-denaturation for 10 min at 95°C, followed by 40 cycles of denaturation for 30 sec at 95°C, annealing for 20 sec at 55°C, and extension for 30 sec at 72°C. The reaction conditions for miR-135a were pre-denaturation at 95°C for 5 min, 40 cycles of denaturation at 95°C for 20 sec, and annealing at 60°C for 30 sec. The relative levels of GATA-3 and miR-135a were calculated using the 2^−ΔΔCq method (25).

Total protein was extracted by protein lysis, and its concentration was measured using the BCA protein assay reagent kit. Subsequently, the proteins (20 µg) were separated by 10% SDS-polyacrylamide electrophoresis and transferred onto a PVDF membrane. The membrane was blocked by 5% skim milk for 2 h. Following blocking, the primary antibodies (1:1,000) targeting GATA-3, TOX, and β-actin were added and incubated overnight at 4°C. Subsequently, the secondary antibody (1:10,000) was added and incubated at room temperature for 1 h. The membrane was developed in ECL luminescent liquid, and the developed film was scanned using a GT 2500 scanner (Epson America, Inc., Long Beach, CA, USA) and analyzed using ImageJ 1.50i (National Institutes of Health, Bethesda, MD, USA). The relative expression level of GATA-3 with respect to β-actin was calculated based on the grey value that was obtained from Image J software.

Bioinformatics prediction was used to identify the upstream miRNA for GATA-3. The following gene prediction software programs were used for bioinformatics prediction: miRanda (http://www.microma.org/rnicroma/home.do), TargetScan (www.targetscan.org), PiTa (http://genie.weizmann.ac.il/pubs/mir07/mir07_data.html), RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/), and PicTar (http://pictar.mdc-berlin.de/). miR-135a was identified as the potential upstream gene of GATA-3.

The wild and mutant types of the miR-135a binding sequence in the 3′-untranslated region (3′-UTR) of the GATA-3 gene were constructed by in vitro chemical synthesis. The cleavage sites of Spe1 and HindIII were respectively added on both ends. The two DNA fragments were cloned into pMIR-REPORT luciferase plasmids. Using the liposome method, the plasmids with the wild-type 3′-UTR and mutant-type 3′-UTR sequences were transfected into 293T cells, which were purchased from the Cell Bank of the Institute of Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Subsequently, the agomiR-135a (100 nM) was transfected into cells and incubated for 24 h. The fluorescence values were measured using the GloMax 20/20 luminometer. The Renilla fluorescent activity was used as the internal control, and all procedures were performed in strict accordance with the dual luciferase assay kit instructions.

The plasmids pre-miR-135a, miR-135a mimic, miR-135a inhibitor, and their negative controls were transfected into cells using Xfect transfection reagents, according to the manufacturer's protocol.

Processed by SPSS18.0 (SPSS, Inc., Chicago, IL, USA), all data are presented as the mean ± standard deviation and a normality test performed. Multiple groups of measurement data were analyzed using one-way analysis of variance (26). P<0.05 was considered to indicate a statistically significant difference.

To detect the mRNA and protein expression levels of GATA-3 in the Hut78 CTCL cells and normal T lymphocytes, RT-qPCR and western blot analyses were performed, respectively. As shown in Fig. 1, the mRNA (Fig. 1A) and protein (Fig. 1B) levels of GATA-3 in the CTCL Hut78 cells were significantly increased compared with those in the normal T lymphocytes (P<0.01). Therefore, the expression level was GATA-3 is increased in the CTCL Hut78 cells.

To identify the upstream regulatory miRNA of GATA-3, bioinformatics prediction was performed. miR-135a was found to be the potential upstream gene of GATA-3. The wild and mutation types of the binding sequence are shown in Fig. 2A. To determine whether miRNA-135 directly targets GATA-3, a dual luciferase assay was performed. The fluorescence values were significantly decreased following co-transfection with agomiR-135a and the pMIR-REPORT plasmid (P<0.01; Fig. 2B). No statistically significant differences in fluorescence values were observed compared with the mutation group (P>0.05); however, miR-135a was shown to bind directly with GATA-3 at the 3′-UTR to regulate its expression.

To detect changes in the expression of miR-135a in the CTCL Hut78 cells, RT-qPCR analysis was performed. The expression level of miR-135a in the CTCL Hut78 cells was significantly decreased compared with that in the normal T lymphocytes (P<0.01; Fig. 3). Therefore, miR-135a may have a regulatory role in the pathological process of CTCL through GATA-3.

The proliferation curves of Hut78 CTCL cells were drawn according to the instructions of the Cell Counting Kit-8. The proliferation of the Hut78 CTCL cells transfected with the pre-miR-135a plasmid was increasingly inhibited on the third day (Fig. 4). On the fourth and fifth days, the proliferation of cells was further increased.

According to the bioinformatics prediction, one of the target genes of miR-135a was identified as GATA-3. The potential binding sequence for miR-135a was identified in the 3′-UTR of GATA-3 (Fig. 2A). As predicted, the results of the western blot analysis showed that the overexpression of miR-135a mimics decreased the expression of GATA-3, whereas miR-135a increased its expression upon transfection (Fig. 5A). Simultaneously, the expression level of TOX, which is a downstream gene of GATA-3, was also decreased in the miR-135a-overexpressing cells (Fig. 5A). Additionally the results of the RT-qPCR assay showed that the mRNA level of GATA-3 in the miR-135a mimics group was marginally decreased compared with its level in the other groups (Fig. 5B), and the protein expression of GATA-3 was substantially downregulated in the Hut78 cells. The primers for RT-qPCR analysis were located at the middle of GATA-3 mRNA, and the PCR product belonged to one of several exons in the GATA-3 pre-mRNA. The existence of GATA-3 pre-mRNA may have resulted in the marginal decrease in the mRNA expression of GATA-3 in the RT-qPCR assay. Furthermore, GATA-3 mRNA was destroyed by miR-135a following pre-mRNA maturation. Therefore, miR-135a led to the destruction of GATA-3 mRNA and inhibited the translation of GATA-3 mRNA.