Normal human B lymphoblastoid cell lines (HCC1954 BL and NCI-BL2009) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). 293T cells were purchased from Hunan Fenghui Biotechnology Co., Ltd. (Hunan, China). Human DLBCL cell lines (SU-DHL-4 and Farage) were purchased from ATCC. Gibco™ RPMI-1640 medium, fetal bovine serum (FBS), penicillin and streptomycin were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). The RNA extraction reagent, TRIzol, and the transfection reagent, Lipofectamine 2000, were purchased from Thermo Fisher Scientific, Inc. (Invitrogen™; Waltham, MA, USA). The PrimeScript RT reagent kit and SYBR Fast qPCR Mix were purchased from Takara (Dalian, China). Rabbit anti-human MEK1 (cat. no. 12671), p-MEK1 (cat. no. 98195) and Bcl-2 (cat. no. 4223) antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Mouse anti-human ERK1/2 (cat. no. ab36991), p-ERK1/2 (cat. no. ab126423) and PCNA antibodies (cat. no. ab70472) were purchased from Abcam (Cambridge, MA, USA). The Annexin V/PI apoptosis detection kit and Cell Cycle detection kit were purchased from Biyuntian (Jiangsu, China). siRNA-MEK1 and siRNA-NC were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A cell proliferation detection kit (Click-iT^® EdU Alexa Fluor 488 Flow Cytometry Assay kit) was obtained from Molecular Probes (Eugene, OR, USA). The Dual-Glo^® Luciferase Assay System and pMIR luciferase reporter gene plasmid were provided by Promega (Madison, WI, USA).

A total of 72 patients diagnosed with DLBCL who received treatment at our hospital between September 2011 and April 2015 and had complete follow-up data were enrolled, including 38 males and 34 females (age range, 19–77 years; median age, 58 years; and average age, 50.7 years). Patients who did not undergo previous radiotherapy and chemotherapy were included, and patients who underwent antitumor treatment pre-operatively were excluded. The tumor tissue specimens were collected intra-operatively, immediately frozen in liquid nitrogen, and stored at −80°C. The tissue specimens were staged according to the Ann Arbor staging scheme as follows: I, invasion of a lymph node or an extranodal organ or part; II, diaphragmatic side, involving two or more lymph nodes or external limitations infringing on an extranodal organ or part; III, violation of both surfaces of the diaphragm, the junction, or additional limitations to infringe upon an extranodal organ or part or the spleen; and IV, diffuse invasion or disseminated extranodal organ or part of one or more. Among the participants, 42 were stages I–II and 30 were stages III–IV. The distribution of patients according to the Lymphoma International Prognostic Index (IPI) was as follows: 0–1, n=25; 2, n=22; 3, n=16; and 4–5, n=9. There were 28 patients with B symptoms, and 44 patients had no B symptoms.

The lymphatic tissue samples from 30 patients with reactive hyperplasia of lymph nodes were designated as the control group. All of the patients signed informed consent, which was approved by the Ethics Committee of Zhangzhou Affiliated Hospital of Fujian Medical University (protocol no. 2010032).

HCC1954 BL, NCI-BL2009, SU-DHL-4, and Farage cells were cultured in RPMI-1640 medium containing 10% FBS and 1% penicillin-streptomycin. The cells were passaged at a 1:4 ratio, and cells with suitable growth in the logarithmic phase were used in all experiments.

The whole genome DNA of 293T cells was used as a template, and PCR amplification contained the MEK1 3′-UTR region of the miR-101 binding site. SacI and HindIII enzyme cutting sites and protective bases were added to the 5′-terminal of the upstream and downstream primers, respectively. The primer sequences for MEK1 were as follows: Sense, 5′-CGAGCTCAAGCAACAAAGAGCGAGTCCCGA0-3′ and antisense, 5′-CCAAGCTTGCAAAGCATGCTTCACATGCACT-3′ (SacI and HindIII enzyme sites are underlined; the former is a protective base). The amplified product was the 10–465 bp range of the 3′-UTR region of MEK1. The total PCR amplification reaction volume consisted of the following: 2X KOD buffer 10 µl; d NTP (2 mM) 4 µl; primer F (10 µM) 0.5 µl; primer R (10 µM) 0.5 µl; KOD FX 0.4 µl; template DNA 2 µl; and ddH₂O 3.6 µl. The cycling conditions were as follows: 95°C for 10 min, followed by 30 cycles at 94°C for 30 sec; 58°C for 30 sec; and 72°C for 10 min, with 4°C infinity. After PCR amplification, SacI and HindIII double enzymes were used to cut the PCR products and pMIR Noblets. The conditions for the enzyme digestion were 37°C for 4 h. The purified product was recovered by 1.5% agarose gel electrophoresis, and the purified PCR products were incubated at 16°C for overnight connection. The products were transformed into DH5α competent cells, inoculated on plates containing penicillin, and incubated at 37°C overnight. Then, a single positive clone was selected and incubated in Escherichia coli culture medium at 37°C overnight to extract the plasmids. The sequence was identified, and designated as pMIR-MEK1-WT. Using the wild-type plasmid as a template, miR-101 and MEK1 3′-UTR binding region mutations for meaningless sequence design of MEK1 point mutation primers were as follows: forward, 5′-CTCTTGTGTTAATAAATATTACTGTCT-3′ and reverse, 5′-AAGTGTCATATATTAAAAATGACAAGA-3′ (mutant sequences are underlined). The detailed construction steps were the same as the wild-type, and the constructed mutant plasmid was designated as pMIR-MEK1-MUT.

The 293T (1×10⁵) cells were inoculated in 24-well plates. After being cultured in DMEM containing 10% FBS for 24 h, the cells were divided into 4 transfection groups: pMIR-MEK1-WT+miR-101 mimic group; pMIR-MEK1-WT+miR-NC group; pMIR-MEK1-MUT+miR-101 mimic group; and pMIR-MEK1-MUT+miR-NC group. Each group had 3 duplicate pores. The specific transfection additions were as follows: 0.8 µg of gene recombinant plasmid; 0.02 µg of pRL-SV40 plasmid; and 20 pmol miR-101 mimic or miR-NC in 50 µl of Opti-MEM. The mixture was then stored for 5 min at room temperature. Lipofectamine™ 2000 (2 µl) was added to 50 µl of Opti-MEM, and was placed at room temperature for 5 min after gentle mixing. The two mixtures were combined after mixing for 20 min at room temperature. The 4 groups of cells were added to 24-hole plates. DMEM fresh medium was replaced by culture medium at 37°C for 6 h, and the cultures were continued for 48 h. A Dual-Glo Luciferase Assay System kit was used to detect luciferase activity, as follows: 100 µl of Passive Lysis Buffer cell lysate was added to each hole of a 24-well plate; the plates were slowly shaken at room temperature for 15 min; 20 µl of cell lysate was added to 100 µl of luciferase assay reagent II (LAR II), mixed, and the fluorescence of the firefly luciferase activity was detected. Fluorescein (100 µl) was added to the detection kit. The Renilla luciferase activity of firefly luciferase activity to Renilla luciferase activity/relative activity value was analyzed.

The day before transfection, SU-DHL-4 and Farage cells were cultured in 10-cm culture dishes, ensuring that the transfection cell density was 50–60% on the day of fusion, and the transfected cells were divided into the following 4 groups: miR-NC group; miR-101 mimic group; siRNA-NC group; and siRNA-MEK1 group. The miR-NC, miR-101 mimic, siRNA-NC and siRNA-MEK1 group cells were diluted with Opti-MEM and incubated at room temperature for 5 min. Each transfected nucleotide was then mixed with Lipofectamine 2000 and gently inverted by mixing, and incubated for 30 min at room temperature. The cell culture medium was removed and washed twice in PBS to remove serum, and then replaced with Opti-MEM serum-free cell culture medium. The transfection complexes were added to the culture medium and mixed. After culturing for 6 h, they were replaced with RPMI-1640 medium containing 10% FBS and 1% penicillin. After being cultured for 72 h, they were trypsinized. Cells were harvested for gene, protein expression, and flow cytometric assays.

One milliliter of TRIzol was added to every 20 mg of tissue or every 3×10⁶ cells. After full lysis, the tissues or cells were added to chloroform for extraction. After stratification, the RNA supernatant was transferred to a new EP tube. After washing with isopropanol and 70% ethanol, DEPC water was dissolved to obtain RNA. The RNA was reversed transcribed into cDNA using PrimeScript RT reagent kit with cDNA as the template and qPCR detecting gene expression. The reverse transcription reaction system contained 0.5 µl (50 µM), 0.5 µl of random 6 mers (100 µM), 0.5 µl of PrimeScript RT Enzyme Mix, 1.0 µg of RNA, 2 µl of 5X PrimeScript Buffer, and RNase-free H₂O₂ was added to a total volume of 10 µl. The reverse transcription conditions were 37°C for 15 min and 85°C for 5 sec. The PCR reaction system contained SYBR Fast qPCR Mix (10.0 µl), 0.8 µl of forward primer (10 µM), 0.8 µl of reverse primer (10 µM), 2.0 µl of cDNA, and 6.4 µl of RNase-free dH₂O. The PCR reaction consisted of 95°C pre-denaturation for 10 min, followed by 40 cycles at 95°C for 5 sec, 60°C for 20 sec, and extension at 72°C for 15 sec. Real-time PCR was performed on Bio-Rad CFX96/CFX Connect™ (Bio-Rad Laboratories, Inc., Hercules, CA, USA) to test the relative expression.

Total protein was extracted by RIPA from cells or tissues. A total of 40 µg of protein was separated by 8–10% sodium lauryl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 5% concentration gel for 3 h and transferred to nitrocellulose membranes. Next, the membranes were blocked with 5% skim milk at room temperature for 60 min and incubated with the primary antibody at 4°C overnight (MEK1, MMP-2, anti ERK1/2, ERK1/2, Bcl-2 and β-actin; the dilution ratios were 1:2,000, 1:1,000, 1:2,000, 1:1,000, 1:2,000, 1:10,000 and PBST, respectively). After thrice-washing, the membranes were incubated with horseradish peroxidase (HRP)-labeled secondary antibody (1:20,000) for 60 min, then thrice-washed. Finally, protein expression was detected by enhanced chemiluminescence (ECL). Data were analyzed with ImageJ software k1.45 (National Institutes of Health, Bethesda, MD, USA).

Each transfection group of cells was resuspended in complete medium. After a 120-min incubation with 10 µM EdU, the cells were continuously cultured for 48 h, and then the cells were collected after trypsinization. The cells were washed during 250 × g centrifugation 1 time with PBS containing 1% BSA, and then 100 µl of Click-iT fixative was added for 15 min and washed during 250 × g centrifugation 1 time with PBS containing 1% BSA. The cells were added to 100 µl of 1X Click-iT saponin-based permeabilization for 15 min at room temperature and 500 µl of PBS, CuSO₄, Alexa Fluor 488, and buffer was added before incubation in the dark at room temperature for 30 min. The cells were added to 3 ml of 1X Click-iT saponin-based permeabilization and washed during 250 × g centrifugation 1 time. The cells were then added to 500 µl of 1X Click-iT saponin-based permeabilization and wash reagent and tested on a Beckman Kurt FC 500 MCL/MPL flow cytometry device (Beckman Coulter, Inc., Brea, CA, USA) to evaluate cell proliferation.

The transfected cells were digested by trypsin and collected after 250 × g centrifugation, then washed 2 times in PBS. Cells were incubated in 5 µl of Annexin V-FITC and 5 µl of PI in the dark for 10 min. Next, the cells were resuspended in 400 µl of binding buffer and tested on a Beckmann FC 500 MCL/MPL flow cytometry device to evaluate cell apoptosis.

All data analyses were performed using SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA). The measurement data are depicted as the mean ± standard deviation and were compared by t-tests. For two-group comparisons, a Chi-square test was used, and when making comparisons among multiple groups, one way ANOVA test was used to determine the relationship between the level of miR-101 expression and the clinical characteristics of the patients. The Mann-Whitney U test was used to compare the level of miR-101 and MEK1 mRNA expression in the lymphoid tissues of the two groups. The survival curve was drawn with the Kaplan-Meier method, and the survival rate was compared using a log-rank test. A P<0.05 was considered statistically significant.

MicroRNA.org online prediction showed the targeted binding site between miR-101 and 3′-UTR of MEK1 mRNA (Fig. 1A). A dual luciferase assay revealed that miR-101 mimic transfection significantly decreased the relative luciferase activity of 293T cells transfected with wild-type pMIR-MEK1-WT, while the relative luciferase activity of 293T cells transfected with mutant pMIR-MEK1-MUT was not affected (Fig. 1B), indicating the regulatory relationship between miR-101 and MEK1.

Based on quantitative RT-PCR (qRT-PCR), it was shown that the expression of miR-101 in tumor tissues from DLBCL patients was significantly decreased (Mann-Whitney U value=367, P<0.05) when compared with proliferative lymph node tissues in the control group (Fig. 2A), while the expression of MEK1 mRNA was elevated in the tumor tissues from DLBCL patients compared with the control group (Mann-Whitney U=269, P<0.05; Fig. 2A). Western blot analysis showed that the expression levels of MEK1 and p-MEK1 protein were significantly increased in the DLBCL tissues when compared with levels in the control tissues; representative illustrations are shown in Fig. 2B. Based on the median miR-101 expression, DLBCL patients were divided into a miR-101 high-expression group and a miR-101 low-expression group. The relationship between clinical features and the level of miR-101 expression was analyzed. According to χ² test analysis, the number of patients with high expression of miR-101 was higher in patients with clinical stage I–II and the number of patients with low expression of miR-101 was significantly higher in patients with clinical stage III–IV (P=0.017). The proportion of patients with an IPI score >3 points was higher in the low miR-101 expression group and the proportion of patients with an IPI score <3 was significantly higher in the high miR-101 expression group (P=0.026), yet patient age, gender, no B symptoms, and miR-101 expression had no apparent relationship (P>0.05; Table I). Survival analysis showed that the survival and prognosis of patients with high expression of miR-101 were significantly better than the patients with low miR-101 expression (log-rank test χ²=5.684, P=0.017). Survival and prognosis of patients with low expression MEK1 mRNA were better than patients with high expression of MEK1 mRNA (log-rank test χ²=5.360, P=0.021; Fig. 2C).

qRT-PCR detection showed that compared with normal lymphoblastoid cells (HCC1954 BL and NCI-BL2009), the expression of miR-101 in SU-DHL-4 and Farage cells was significantly decreased and MEK1 mRNA expression was significantly increased (Fig. 3A). Western blot detection showed that compared with normal lymphoblastoid cells (HCC1954 BL and NCI-BL2009), the expression of MEK1 and p-MEK1 protein in SU-DHL-4 and Farage cells was significantly increased (Fig. 3B). The expression of miR-101 in SU-DHL-4 and Farage cells was significantly increased in the miR-101 mimic transfection group compared with the miR-NC group while MEK1 mRNA expression was significantly decreased (Fig. 3C). Compared with the siRNA-NC group, the expression of MEK1 mRNA in the SU-DHL-4 and Farage cells of the siRNA-MEK1 transfected group was significantly decreased (Fig. 3C). Western blot analysis showed that transfection of miR-101 mimic or siRNA-MEK1 significantly reduced MEK1, p-MEK1 and p-ERK1/2 protein expression in the SU-DHL-4 and Farage cells (Fig. 3D).

Flow cytometric analysis showed that the proliferative rate of SU-DHL-4 and Farage cells was significantly higher than the HCC1954 BL and NCI-BL2009 cells (Fig. 4A). The EdU staining flow test showed that the EdU-positive rate of SU-DHL-4 and Farage cells in the miR-101 mimic transfected group was significantly lower than the miR-NC group. Compared with the siRNA-NC group, the EdU-positive rates of SU-DHL-4 and Farage cells in the siRNA-MEK1 transfected group were significantly lower (Fig. 4B and C).

Flow cytometric analysis showed that the apoptosis rate of SU-DHL-4 and Farage cells was significantly lower than that noted in the HCC1954 BL and NCI-BL2009 cells (Fig. 5A). The flow cytometry test showed that the apoptosis rate of SU-DHL-4 and Farage cells in the miR-101 mimic transfected group was significantly higher than that noted in the miR-NC group. Compared with the siRNA-NC group, the apoptosis rate of the SU-DHL-4 and Farage cells in the miR-101 minic and siRNA-MEK1 group was significantly higher (Fig. 5B and C). Using western blot analysis we found that miR-101 targets Bcl-2, PARP and caspase-3 expression to regulate the apoptosis of lymphoma cells. When miR-101 expression was increased, expression of the anti-apoptotic factor Bcl-2 protein was significantly decreased, and PARP and caspase-3 expression was increased (Fig. 5D).