Kruppel-like factor 4 upregulates the resistance to apoptosis induced by tumor necrosis factor α in synovial fibroblasts with rheumatoid arthritis

RASF culture

The human RASF cell line MH7A (Jennio, Guangxhou, China) was thawed and cultured in fresh Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Life Technologies) which was replaced every 3 days. Upon reaching 80% confluency, the adherent cells were digested with 0.25% trypsin, split 1:2 into a fresh flask, and then cultured in DMEM +10% FBS.

KLF4 small interfering RNA transfection

Three pairs of KLF4 small interfering RNA (siRNA): klf4-homo-1286 (GGUCAUCAGCGUCAGCAAATT), klf4-homo-638 (GGAGAAGACACUGCGUCAATT), klf4-homo-1831 (CCACCCACACUUGUGAUUATT), and a scrambled control siRNA (scRNA) (sense oligonucleotide: 5’-UUC UCC GAA CGU GUC ACG UTT-3’) were designed and synthesized commercially by Genepharma Inc (Shanghai, China). KLF4 siRNA or control siRNA was transfected into MH7A cells using a Lipofectamine RNAiMax kit (Invitrogen). Cells were collected 48 h post-transfection and then the efficacy of gene silencing was determined by quantitative polymerase chain reaction (qPCR).

Detection of cell proliferation by MTT assay

Cells were seeded into the wells of a multiwell plate and treated with 20 μL MTT solution (5 mg/mL). After 4 h of culture, the culture medium was discarded and 150 μL DMSO added to each well. The plate was shaken for 10 min in the dark to allow the crystals to fully dissolve, and the absorbance (OD value) measured at 490 nm. The metabolism of cells was recorded relative to that of the normal control group, defined as 100%. Cell growth in the treated group (%) = (OD of treated group/OD of untreated group) ^∗ 100%.

Detection of cell apoptosis by flow cytometry

Treated cells were digested by trypsin and then prepared as a suspension of individual cells. Apoptosis was measured using an annexin V-FITC cell apoptosis detection kit (Thermo scientific, Waltham, MA, USA), in which cells were stained with an annexin V antibody able to specifically bind phospholipid serine in the cell membrane. The 10× binding buffer was diluted to 1× using distilled water then placed on ice. Cells were washed twice in cold phosphate-buffered saline (PBS) (800–1000 r/min for 5 min), resuspended in 490 μL cold binding buffer (10⁵–10⁶ cells/mL), and then stained with 5 μL annexin V-FTIC and 5 μL propidium iodide. Cells were gently shaken and incubated for 10 min on ice in the dark. Cell apoptosis was measured by flow cytometry.

Change in cell apoptosis measured by Hoechst 33258 staining

The culture medium was removed from treated cells which were fixed in 0.5 mL fluorescent fixation solution for 15 min. The cells were washed 3 times in PBS (3 min each wash), stained with 0.5 mL Hoechst 33258 solution (Beyotime, Nantong, China) for 5 min, and then washed twice in PBS (3 min each wash). Fluorescence quenching and mounting solutions were added to a glass slide, onto which a cover slip was placed face down. Cell apoptosis was observed using a fluorescence microscope.

Western blot analysis

Treated cells were collected and lysed in radioimmune precipitation assay lysis buffer (Beyotime) for 30 min. Total protein was extracted and then quantified using a bicinchoninic acid assay (Beyotime). Equal quantities (20 μg) of protein sample were boiled, separated using 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto a polyvinylidene fluoride membrane (Millipore, MA, USA). The membrane was blocked with 5% skimmed milk for 2 h, incubated with primary antibody (anti-GAPDH monoclonal, 1:3000, Santa Cruz; rabbit anti-caspase-3 polyclonal, 1:1000, CST; rabbit anti-bax polyclonal, 1:1000, CST or rabbit anti-bcl-2 polyclonal. 1:1000, CST) overnight at 4°C, rinsed for 30 min, incubated with peroxidase-labeled secondary antibody for 1 h, stained with enhanced chemiluminescence (Millipore), and then visualized using a chemiluminescence imaging system. Grayscale values of the protein bands were calculated using Image J software.

Real-time PCR

Treated cells were collected and total RNA extracted using Trizol. First-strand cDNA was reverse transcribed using a RevertAid first-strand cDNA synthesis kit (Thermo scientific) in a 20 μL reaction system containing 1 μg RNA, 40 U/μL MMuLV2 reverse transcriptase, 100 nm Oligo (dT), 1 mm dNTP mix, 0.1 m DTT, 40 U/μL RNAse inhibitor, and DEPC deionized water. Reverse transcription was performed at 42°C for 60 min and then 70°C for 10 min. The synthesized cDNA was amplified using a quantitative PCR kit ((Applied Biosystems, Foster City, CA) in a 20 μL reaction system containing 10 μL SYBR green PCR master mix, 0.4 μm cDNA, 0.2 μm each of forward and reverse primers, and deionized water. Amplification was performed at 95°C for 5 min, then 95°C for 15 s and 60°C for 1 min for 40 cycles and then finally 72°C for 7 min. The CT value of each sample was converted into gene expression using the 2^-△△CT method for template and control samples (Table 1).

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Table 1.

Primer sequences of genes used in this study.

Gene	Primer sequence
klf4	5’-GCAATATAAGCATAAAAGATCACCT-3’
	5’-AACCAAGACACCAACCACC-3’
bax	5’-CCCGAGAGGTCTTTTTCCGAG-3’
	5’-CCAGCCCATGATGGTTCTGAT-3’
bcl-2	5’-GGTGGGGTCATGTGTGTGG-3’
	5’-CGGTTCAGGTACTCAGTCATCC-3’
gapdh	5’-GGACCTGACCTGCCGTCTAG-3’
	5’-GTAGCCCAGGATGCCCTTGA-3’

Statistical analysis

Data were statistically analyzed using GraphPad Prism 5 and expressed as mean ± standard deviation. Comparisons between two groups were analyzed using a t test, while one-way analysis of variance (ANOVA) was used to compare multiple groups.

p < 0.05 were considered statistically significant.

Effects of TNFα on RASF proliferation and apoptosis

The MTT results indicated that RASF proliferation increased following TNFα stimulation, being most significant after 4 h and 12 h (p < 0.05) (Figure 1(a)). Fluorescence staining and flow cytometry indicated that RASF apoptosis declined after TNFα stimulation, being most significantly inhibited after 24 h (p < 0.05) (Figure 1(b) and (c)).OPEN IN VIEWER

Effect of TNFα on apoptosis-related gene and protein expression in RASFs

Western blot analysis indicated that TNFα stimulation significantly downregulated caspase-3 protein expression in RASFs after 12 h (p < 0.05) but induced no change in the bcl2/bax ratio (Figure 2(a)). Quantitative PCR demonstrated that the bcl-2/bax ratio increased significantly, peaking 24 h after TNFα stimulation (p < 0.05) (Figure 2(b)).OPEN IN VIEWER

KLF4 siRNA sequence and concentration selection

The PCR results demonstrated that KLF4 mRNA was markedly downregulated in RASFs after transfection with klf4-homo-1286 (p < 0.05). The effect was most significant at a concentration of 20 nm. There was also a decreasing trend in KLF4 mRNA expression following transfection with klf4-homo-638 and klf4-homo-1831, although this change was not statistically significant. Therefore, the klf4-homo-1286 sequence was used for subsequent gene silencing experiments (Figure 3).OPEN IN VIEWER

Effect of KLF4 silencing on RASF proliferation and apoptosis

The MTT assay demonstrated that TNFα-induced cell proliferation decreased significantly following transfection with KLF4 siRNA (p < 0.05) but not scRNA (Figure 4(a)). Fluorescence staining revealed that transfection with KLF4 siRNA, but not scRNA, promoted TNFα-induced cell apoptosis (Figure 4(b)). Likewise, flow cytometric analysis demonstrated that TNFα-induced cell apoptosis increased significantly after KLF4 siRNA transfection (p < 0.05) but did not change significantly following scRNA transfection (Figure 4(c)).OPEN IN VIEWER

Effect KLF4 silencing on apoptosis-related gene and protein expression in RASFs

Western blot analysis demonstrated that KLF4 siRNA transfection significantly increased TNFα-induced capase-3 protein expression and decreased bcl-2/bax mRNA expression (p < 0.05), whereas scRNA transfection had no significant effect on TNFα-induced capase-3 or bcl-2/bax expression (Figure 5(a)). Similarly, qPCR indicated that KLF4 siRNA transfection markedly decreased TNFα-induced bcl-2/bax mRNA expression (p < 0.05), while scRNA transfection had no significant impact on TNFα-induced bcl-2/bax expression (Figure 5(b)).OPEN IN VIEWER