OBJECTIVE: To study siRNA inhibited the expression of mRNA and protein of NF-kappaBp65 in the Hep -2 cell line. METHOD: Hep-2 were transfected with p65SiRNA. Western blotting was used to examine the protein levels of NF-kappaBp65. RT-PCR method was adopted to determine the mRNA expression of NF-kappaBp65. MTT method was adopted to investigate the proliferation of the Hep-2 cells after the transfection of p65siRNA. RESULT: The western blotting result showed that the level of NF-kappaBp65 protein was gradually declined after transfection of p65siRNA. The RT-PCR result showed that transfection with p65siRNA caused special degradation of the p65mRNA in Hep-2 cells at 24, 48 and 72 h. After transfection with p65siRNA. CONCLUSION p65siRNA has significant inhibition effects on the proliferation of the Hep-2 cells and expression of purpose gene mRNA and protein. The inhibition effects are time depended.
Cell Line, Tumor ; Cell Proliferation ; Humans ; RNA, Messenger ; genetics ; RNA, Small Interfering ; Transcription Factor RelA ; genetics

OBJECTIVE: To study the impacts of RelA antisense oligonucleotides on proliferation in laryngeal carcinoma Hep-2 cell. METHOD: RelA antisense oligonucleotides was designed, which was transferred into laryngeal carcinoma Hep-2 cell. MTT was used to detect the growth-inhibiting ratio at different transferred timepoints. Hep-2 cell which was transferred 48 h was used to do colony assay, and expression of RelA was detected by Reverse Transcription PCR and Western blot. RESULT: MTT results showed that RelA antisense oligonucleotides could significantly suppress the proliferation of Hep-2 cell, and the suppression-ratio elevated with time. There were statistical difference compared with control groups. The number of cells colony was reduced in RelA antisense oligonucleotides group compared with control groups, which had statistic significance. RT-PCR and Western blot results demonstrated that RelA antisense oligonucleotides could significantly inhibit the expression of messenger RNA and protein in Hep-2 cell. CONCLUSION RelA antisense oligonucleotides can inhibit the expression of messenger RNA and protein, and induce the cell proliferation and increase the number of cells colony in Hep-2 cell.
Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Laryngeal Neoplasms ; genetics ; pathology ; Oligonucleotides, Antisense ; genetics ; pharmacology ; Transcription Factor RelA ; genetics

OBJECTIVETo observe the effect of TSP-2, the antibody of Toll-like receptor 2 extracellular domain, on the expression and DNA-binding activity of nuclear factor-κB (NF-κB) p65 protein in mice with ulcerative colitis (UC).

METHODSSixty BALB/c mice were randomized equally into normal control group, UC model group, TSP-2 treatment group, and rabbit IgG treatment group. In the latter 3 groups, the mice were fed with 5% DSS (C6H7Na3O14S3) solution for 7 days to induced UC, followed then by treatment with daily injections of TSP-2 or rabbit IgG as appropriate for 7 days. The disease activity index was recorded during the treatment. The colitis tissues were collected after the treatments for HE staining and detecting the expression and DNA-binding activity of NF-κB p65 in the colon mucosa by Western blotting and ELISA.

RESULTSThe DNA binding activity and expressions of NF-κB P65 protein increased significantly in UC model group (P<0.05). TSP-2 treatment group significantly decreased the disease activity index (P<0.05) and lowered the DNA-binding activity and expression of NF-κB P65 protein (P<0.05) in the UC mouse models, while rabbit IgG produced no such effects (P>0.05).

CONCLUSIONTSP-2 can suppress the DNA-binding activity and protein expressions of NF-κB P65 and regulate excessive immune response in the intestines to ameliorate ulcerative colitis in mice.

Animals ; Colitis, Ulcerative ; immunology ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Rabbits ; Thrombospondins ; pharmacology ; Transcription Factor RelA ; genetics ; metabolism

OBJECTIVE: To determine the effects of exogenous RASSF1A gene on the proliferation and expression of P65 and subunit of NF-kappaB, in lung adenocarcinoma cell line A549. METHODS: pcDNA3.0-RASSF1A and pcDNA3. 0 were introduced into A549 cell line by lipofectin transfection, and the A549 cells stably expressing RASSF1A gene were established by G418 selection. The expression of RASSF1A was detected by Western blotting. The cytobiologic characterizations of the positive clone were analyzed by methythiazoletertraolium (MTT) assay and cytometry. The expressing of P65 was analyzed by RT-PCR and Western blotting. RESULTS: A549 cells stably expressing RASSF1A protein were established by lipofection mediated transfection and selected for further study. Compared with the nontransfected and vector transfected cells, the positive clone cells grew more slowly. Flow cytometric data showed that more positive clone cells went into phase G0/G1 and fewer cells went into phase S. The expression of P65 in nuclear protein in positive clone cells was lower than that of the control group while there was no obvious difference between the expression of p65 mRNA and P65 protein in total protein among the 3 groups. CONCLUSION RASSF1A gene might suppress the proliferation of A549 cells through blocking the activity of P65 protein.
Adenocarcinoma ; genetics ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Lung Neoplasms ; genetics ; pathology ; RNA, Messenger ; biosynthesis ; genetics ; Transcription Factor RelA ; biosynthesis ; genetics ; Transfection ; Tumor Suppressor Proteins ; biosynthesis ; genetics

The purpose of this paper was to study the transcriptional regulation of nuclear respiratory factor 1 (NRF1) on nuclear factor kappa B (NF-κB), a key molecule in lipopolysaccharide (LPS)-induced lung epithelial inflammation, and to clarify the mechanism of NRF1-mediated inflammatory response in lung epithelial cells. In vivo, male BALB/c mice were treated with NRF1 siRNA, followed with LPS (4 mg/kg) or 0.9% saline through respiratory tract, and sacrificed 48 h later. Expression levels of NRF1, NF-κB p65 and its target genes were detected by Western blot and real-time PCR. Nuclear translocation of NRF1 or p65 was measured by immunofluorescent technique. In vitro, L132 cells were transfected with NRF1 siRNA or treated with BAY 11-7082 (5 μmol/L) for 24 h, followed with treatment of 1 mg/L LPS for 6 h. Cells were lysed for detections of NRF1, NF-κB p65 and its target genes as well as the binding sites of NRF1 on RELA (encoding NF-κB p65) promoter by chromatin immunoprecipitation assay (ChIP). Results showed that LPS stimulated NRF1 and NF-κB p65. Pro-inflammatory factors including interleukin-1β (IL-1β) and IL-6 were significantly increased both in vivo and in vitro. Obvious nuclear translocations of NRF1 and p65 were observed in LPS-stimulated lung tissue. Silencing NRF1 resulted in a decrease of p65 and its target genes both in vivo and in vitro. In addition, BAY 11-7082, an inhibitor of NF-κB, significantly repressed the inflammatory responses induced by LPS without affecting NRF1 expression. Furthermore, it was proved that NRF1 had three binding sites on RELA promoter region. In summary, NRF1 is involved in LPS-mediated acute lung injury through the transcriptional regulation on NF-κB p65.
Acute Lung Injury/genetics* ; Animals ; Lipopolysaccharides/pharmacology* ; Male ; Mice ; NF-kappa B/metabolism* ; Nuclear Respiratory Factor 1/genetics* ; RNA, Small Interfering ; Transcription Factor RelA/metabolism*

OBJECTIVETo study the effect of chemokine receptor 7 (CCR-7) small interfering RNA (siRNA) on proliferation and invasion of squamous cell carcinoma of head and neck (SCCHN).

METHODSCCR-7 siRNA was co-transfected into SCCHN cell line PCI-4B using Lipofectamine 2000. CCR-7 protein level was detected by western blotting. SCCHN cell proliferation was detected by MTT, and the change of actin cytoskeleton observed by confocal laser scanning microscope. Transwell assays were used to determine chemotaxis and invasion of SCCHN cells. The activity and nuclear translocation of nuclear factor-kappa B (NF-kappa B) were detected by TransAM NF-kappa B p65 kit and fluorescence microscope respectively.

RESULTSAfter CCR-7 siRNA transfection, the protein level of CCR-7 was significantly decreased. The changes induced by CCL-19, including increased proliferation rate, polarized actin polymerization, increased chemotaxis rate and invasion rate, were all abolished by CCR-7 siRNA transfection. CCR-7 siRNA also diminished CCL-19-induced NF-kappaB activation and nuclear translocation.

CONCLUSIONSCCR-7 siRNA could inhibit expression of CCR-7 and diminish the increased proliferation and invasion of SCCHN induced by CCL-19 in vitro. CCR-7 siRNA may provide a potential treatment strategy for SCCHN.

Carcinoma, Squamous Cell ; genetics ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Head and Neck Neoplasms ; genetics ; pathology ; Humans ; Neoplasm Invasiveness ; RNA, Small Interfering ; Receptors, CCR7 ; genetics ; Transcription Factor RelA ; metabolism

OBJECTIVETo assess the effect of a high specific adenovirus vector-mediated shRNA targeting nuclear factor-κB (NF-κB) on cell proliferation of the endometrium of Macaca fascicularis.

METHODSThe adenoviral vector NF-κB-p65-shRNA and the empty vector were separately trasnfected in cultured endometrial cells of Macaca fascicularis. The changes in the expression of the target gene protein and apoptotic proteins, cell proliferation, and cell cycle distribution were observed after the transfection.

RESULTSCompared with the control cells, infection of the endometrial cells with the NF-κB-p65-shRNA adenovirus significantly increased the expression levels of apoptotic proteins, promoted apoptosis of the endometrial cells, and reduced the cells in division?stage.

CONCLUSIONSNF-κB-p65 shRNA adenovirus can effectively promote apoptosis of endometrial cells and inhibit the proliferation of endometrial cells of Macaca fascicularis.

Adenoviridae ; Animals ; Apoptosis ; Cell Proliferation ; Cells, Cultured ; Endometrium ; cytology ; Female ; Genetic Vectors ; Macaca fascicularis ; RNA, Small Interfering ; genetics ; Transcription Factor RelA ; genetics ; Transfection

Combined radiation-wound injury (CRWI) is characterized by blood vessel damage and pro-inflammatory cytokine deficiency. Studies have identified that the direct application of leptin plays a significant role in angiogenesis and inflammation. We established a sustained and stable leptin expression system to study the mechanism. A lentivirus method was employed to explore the angiogenic potential and peripheral inflammation of irradiated human umbilical vein endothelial cells (HUVECs). Leptin was transfected into human placenta-derived mesenchymal stem cells (HPMSCs) with lentiviral vectors. HUVECs were irradiated by X-ray at a single dose of 20 Gy. Transwell migration assay was performed to assess the migration of irradiated HUVECs. Based on the Transwell systems, co-culture systems of HPMSCs and irradiated HUVECs were established. Cell proliferation was measured by cell counting kit-8 (CCK-8) assay. The secretion of pro-inflammatory cytokines (human granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-1α, IL-6, and IL-8) was detected by enzyme-linked immunosorbent assay (ELISA). The expression of pro-angiogenic factors (vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF)) mRNA was detected by real-time quantitative polymerase chain reaction (RT-qPCR) assay. Relevant molecules of the nuclear factor-κB (NF-κB) and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways were detected by western blot assay. Results showed that leptin-modified HPMSCs (HPMSCs/ leptin) exhibited better cell proliferation, migration, and angiogenic potential (expressed more VEGF and bFGF). In both the single HPMSCs/leptin and the co-culture systems of HPMSCs/leptin and irradiated HUVECs, the increased secretion of pro-inflammatory cytokines (human GM-CSF, IL-1α, and IL-6) was associated with the interaction of the NF-κB and JAK/STAT signaling pathways. We conclude that HPMSCs/leptin could promote angiogenic potential and peripheral inflammation of HUVECs after X-ray radiation.
Cell Proliferation ; Cells, Cultured ; Cytokines/biosynthesis* ; Female ; Human Umbilical Vein Endothelial Cells/radiation effects* ; Humans ; Inflammation/etiology* ; Leptin/pharmacology* ; Mesenchymal Stem Cells/physiology* ; Neovascularization, Physiologic/physiology* ; Placenta/cytology* ; Pregnancy ; STAT3 Transcription Factor/genetics* ; Transcription Factor RelA/genetics* ; X-Rays

Animals ; Disease Models, Animal ; Liver ; pathology ; Liver Failure, Acute ; metabolism ; pathology ; Male ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Transcription Factor RelA ; metabolism

Ependymoma is the third most common pediatric primary brain tumor. Ependymomas are categorized according to their locations and genetic abnormalities, and these two parameters are important prognostic factors for patient outcome. For supratentorial (ST) ependymomas, RELA fusion-positive ependymomas show a more aggressive behavior than YAP1 fusion-positive ependymomas. Extracranial metastases of intra-axial neuroepithelial tumors are extremely rare. In this paper, we report a case of aggressive anaplastic ependymoma arising in the right frontoparietal lobe, which had genetically 1q25 gain, CDKN2A homozygous deletion, and L1CAM overexpression. The patient was a 10-year-old boy who underwent four times of tumor removal and seven times of gamma knife surgery. Metastatic loci were scalp and temporalis muscle overlying primary operation site, lung, liver, buttock, bone, and mediastinal lymph nodes. He had the malignancy for 10 years and died. This tumor is a representative case of RELA fusion-positive ST ependymoma, showing aggressive behavior.
Brain Neoplasms ; Buttocks ; Child ; Ependymoma* ; Genetics ; Humans ; Liver ; Lung ; Lymph Nodes ; Male ; Neoplasm Metastasis* ; Neoplasms, Neuroepithelial ; Neural Cell Adhesion Molecule L1 ; Scalp ; Supratentorial Neoplasms ; Transcription Factor RelA