The pSUPER. retro. neo (VEC-PRT-0003 linear) plasmid DNA and the retrovirus packaging cell line 293A were kindly provided by Dr Yang Yizeng (NIH Center for Molecular Studies in Digestive and Liver Diseases, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA). The human monocyte THP-1 cell line was kindly provided by the Department of Pharmacology, Xuanwu Hospital of Capital Medical University (Beijing, China), and the NIH3T3 cell line was provided by Institute of Neurology, Basic Medical Sciences of Peking University Health Science Center (Beijing, China). THP-1 and NIH3T3 cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and penicillin (100 U/ml) streptomycin (100 µg/ml). 293A cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS and penicillin (100 U/ml) streptomycin (100 µg/ml). DMEM and RPMI-1640 medium were purchased from Hyclone (GE Healthcare Life Sciences, Logan, UT, USA), and FBS was purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Two looped specific NF-κB P65 siRNA sequences and one scramble control sequence (list in Table I) were synthesized by Beijing Sunbiotech Co., Ltd. (Beijing, China) and ligated into pSUPER.retro.neo vector according to the protocol described in a previous study (4).

The NF-κB p65 siRNA vectors were transfected into 293A package cells using Lipofectamine2000™ (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions, and the virus suspension was collected following 24, 48 or 72 h. Virus titration was performed with NIH3T3 cells. The viral titer was 3.72×10⁶ colony forming units (CFU)/ml for p65 siRNA1, 3.56×10⁶ CFU/ml for p65 siRNA2 and 3.66×10⁶ CFU/ml for p65 si-Control.

Total RNAs were exacted from the cell lines at 80–90% confluence by using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.). A total of 1 µg RNA was reverse transcribed using a RevertAid first strand cDNA synthesis kit (Thermo Fisher Scientific, Inc.). The qPCR was performed using QuantiTect SYBR Green PCR kit (Qiagen, Inc., Valencia, CA, USA) according to the manufacturer's protocol. All the primers (listed in Table II) were obtained from Sanbio B.V. (Uden, The Netherlands). All the PCR reactions were initiated with incubation at 94°C for 2 min, followed by 29 cycles of 94°C for 30 sec, 58°C for 30 sec and 72°C for 2 min. Reactions were finished with a 72°C 10 min extension. Data were normalized using the 2^−ΔΔCq method (5).

Total protein were isolated from cultured cells by 300 µl ice cold lysis buffer containing 1% NP-40, 50 mmol/l Tris (pH 7.4), 150 mmol/l NaCl, 0.1% SDS, 0.5% deoxycholate, 200 µg/ml phenylmethanesulfonyl fluoride and 50 µg/ml aprotinin. Insoluble materials were removed by ultracentrifugation at 15,000 × g for 30 min for 4°C. The concentration of the extracted protein was measured spectrophotometrically with Coomassie G-250. Clarified protein lysates (50 µg) were electrophoretically resolved on denaturing SDS-PAGE (8–12%). The proteins were transferred onto polyvinylidene fluoride membranes using a wet transfer method following PAGE, and subsequently blocked with 3% bovine serum albumin (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 1 h at room temperature and washed with Tris-buffered saline and Tween-20 three times. Antibodies specific for NF-κB p65 (mouse anti-human IgG; 1:500; cat. no. sc-8008) and GAPDH (mouse anti-human IgG; 1:10,000; cat. no. sc-47724) were used to probe membranes for 2 h at room temperature, followed by peroxidase-conjugated secondary antibodies (goat anti-mouse IgG; 1:5,000; cat. no. sc-3697) for 1 h at room temperature and enhanced chemiluminescence detection (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). All primary and secondary antibodies were obtained from Santa Cruz Biotechnology, Inc. For quantification of band intensity, appropriate films were scanned and band densities were determined using Quantity One software (version 4.6.6; Bio-Rad Laboratories, Inc., Hercules, CA, USA), normalized against GAPDH, and presented as a ratio of control.

Secretory levels of IL-1β and TNF-α in culture supernatants were determined by the Quantikine ELISA kit (R&D Systems, Inc. Minneapolis, MN, USA, cat. nos. DLB50 and DTA00C). The color generated was determined by measuring the OD at 450 nm using a spectrophotometric microplate reader. A standard curve was run on each assay plate using serial dilutions of recombinant IL-1β and TNF-α. The experiment was repeated three times and results were presented as the mean value.

Data are presented as the mean ± standard deviation. All data analyses were performed using SPSS software version 13.0 for Windows (SPSS, Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) and independent-samples t-tests were conducted. Tukey's test was used for post-hoc testing following the ANOVA. All experiments were performed at least three times with similar results. P<0.05 was considered to indicate a statistically significant difference.

In a previous study, the authors successfully constructed the p65 siRNA retroviruses using gene recombination technology. In the present study, the effect of p65 siRNA retroviruses on the expression of NF-κB p65 mRNA in THP-1 cells was investigated. Fig. 1A revealed the levels of p65 mRNA at 0, 4, 8, 12 and 24 h following infection with p65 siRNA retroviruses, as measured by RT-qPCR. Statistical analysis demonstrated that the p65 mRNA expression levels were significant reduced at 4, 8, 12 and 24 h following infection with p65 siRNA1 and p65 siRNA2 retroviruses, while no significant difference in THP-1 cells infected with siControl retroviruses (Fig. 1B).

To further investigate the inhibitory effects of p65 siRNA retroviruses on the NF-κB signaling pathway, the expression of NF-κB p65 protein was detected using western blot analysis. The level of p65 protein in THP-1 cells infected with p65 siRNA retroviruses at 0, 4, 8, 12, 24, 48 and 72 h was presented in Fig. 2A. Statistical analysis revealed that the protein expression levels of p65 in THP-1 cells was significantly decreased from 12 h to 72 h following infection with p65 siRNA1 retroviruses, while decreased from 8 h to 72 h following infection with p65 siRNA2 retroviruses. There was no significant difference of p65 protein expression at different time in THP-1 cells following infection with siControl retroviruses (Fig. 2B).

The RT-qPCR and western blot analysis revealed that the siRNA2 was the most effective in decreasing p65 expression, so p65 siRNA2 was used for further research.

In order to investigate the effect of p65 siRNA retroviruses on the pro-inflammatory cytokines release stimulated by LPS, RT-qPCR was used to detect the expression of IL-1β in THP-1 cells. The level of IL-1β mRNA in THP-1/p65(−) and THP-1/siControl cells stimulated by LPS (0.1 µg/ml) at 0, 4, 8, 12 and 24 h is presented in Fig. 3. Statistical analysis revealed that the mRNA expression levels of p65 was significantly decreased at 12 h and 24 h in THP-1/p65(−) cells following stimulation with LPS, while increased at 12 h and 24 h in THP-1/siControl cells (Fig. 3B and D).

To further evaluate whether p65 siRNA retroviruses could suppress IL-1β production, ELISA was used to measure the secretion of IL-1β in THP-1/p65(−) and THP-1/siControl cells stimulated by LPS. As presented in Fig. 3E, treatment with LPS resulted in a significant increase in the secretion of IL-1β at 24 h and 48 h in THP-1/siControl cells. However, the secretion of IL-1β sustained a lower level and there was no significant difference at 0 h, 12 h, 24 h and 48 h in THP-1/p65(−) cells following stimulation with LPS (Fig. 3E).

To explore the effect of p65 siRNA retroviruses on the production of TNF-α, THP-1 differentiated macrophage-like (THP-1/M) cells were used. When the THP-1 cells were pretreated with PMA (100 nM/ml) for 48 h, the THP-1 cells were almost completely induced to differentiate into macrophage-like cells (Fig. 4).

The level of TNF-α mRNA in THP-1/M/p65(−) and THP-1/M/siControl cells stimulated by LPS (0.1 µg/ml) at 0, 4, 12 and 24 h was presented in Fig. 4A and C, as measured by RT-qPCR. Statistical analysis revealed that the mRNA expression levels of TNF-α was significantly decreased in THP-1/M/p65(−) cells at 4, 12 and 24 h stimulated by LPS, while increased in THP-1/M/siControl cells at 4, 12 and 24 h stimulated by LPS (Fig. 4B and D).

In addition, to confirm whether the inhibition of TNF-α mRNA correspond to a decrease in TNF-α protein, the secretion of TNF-α in THP-1/M/p65(−) and THP-1/M/siControl cells stimulated by LPS (0.1 µg/ml) was measured by ELISA. As demonstrated in Fig. 4E, treatment with LPS resulted in a significant increase in the secretion of TNF-α at 12, 24 and 48 h in THP-1/M/siControl cells. However, the secretion of TNF-α sustained a lower level and there was no significant difference at 0, 12, 24 and 48 h in THP-1/M/p65(−) cells stimulated by LPS (Fig. 4E).