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Objective To investigate the mRNA and protein expression levels of telomeric-repeat binding factor-1 (TRF1) and TRF2 in the peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus (SLE),and the relations between these gene expression levels and clinical data of SLE patients were explored.Methods According to disease activity,these SLE patients were divided into the active group (40 cases) and the stable group (67 cases).These patients were also grouped as renal damage group (46 cases) and renal damage-free group (61 cases) based on their renal conditions.Healthy individuals (41 cases) were also included as control.Real-time quantitative polymerase chain reaction (RT-qPCR) was employed to study the mRNA expression of TRF1 and TRF2.The protein levels of TRF1 and TRF2 were measured by Western Blot (WB).Independent-Samples t test or one-way analysis of variance (ANOVA) in conjunction with the Least-Significant Difference method (LSD method) wasperformed if the data were in normal distributions;otherwise,the Kruskal-Wallis test was applied.Spearman's correlation analysis was also used for statistical analysis.Results The mRNA and protein expression levels of TRF1 and TRF2 in the PBMCs of the active group (TRF1:0.003 1±0.003 3;TRF2:0.010 5±0.064 8) and renal damage group (TRF1:0.002 3 ±0.002 6;TRF2:0.004 3 ±0.003 3) were significantly increased compared to the stable group (TRF1:0.001 2±0.001 1;TRF2:0.004 2±0.008 6),the renal damage-free group (TRF1:0.001 3±0.001 8;TRF2:0.003 4±0.007 2) and healthy (TRF1:0.001 2±0.003 0;TRF2:0.003 4±0.002 7) individuals respectively (P＜0.05).In SLE patients,the expression levels of TRF1 mRNA were correlated with erythrocyte sedimentation rate (r=0.365,P＜0.05);the expression levels of TRF2 mRNA were correlated with SLEDAI score (r=0.270,P＜0.05),erythrocyte sedimentation rate (r=0.304,P＜0.05),creatinine (r=0.258,P＜0.05) and 24-hour urinary protein (r=0.344,P＜0.05).Conclusion Altered expression of TRF1 and TRF2 might be involved in the pathogenesis of Systemic lupus erythematosus.The positive correlation between TRF2 and SLEDAI score,24-hour urinary protein suggest that TRF2 might be usedas a biomarker for disease activity or renal damage in
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Objective: Cellular proliferate inhibition, senescence or apoptosis are induced by telomere shortening through the activation of P53 pathway, but so far, little is known of the mechanism. This study aimed to clarify the molecular regulation of P53 through telomere pathway by the investigation of molecular interaction between P53 and the main telomere associated protein telomeric repeat binding protein 1(TRBP1) in vitro. Methods: Glutathione S-transferase (GST) alone and 4 different human P53-GST fusion proteins were expressed in E. coli. and purified through glutathione Sepharose TM 4B by affinity chromatography, P53s were wild type P53 (1-393), N terminal truncated form P53 2C (95-393), C terminal truncated form P53 N5 (2-293) and single amino acid mutant P53 R175H (175 arginine to histidine). Glutathione Sepharose TM 4B, purified GST alone and P53 fusions were mixed with human breast cancer cell line MCF-7 cellular protein extracts through in vitro binding assay-pull down, the molecular interaction between P53 and TRBP1 were detected by Western blot. Results: SDS-PAGE and Coomassie brilliant blue staining showed that the molecular weights of all the purified proteins were as expected and purities were over 90%. Western blot of TRBP1 showed that both wild type P53 and P53 R175H could bind to TRBP1 of MCF-7 cells, and their binding capacities are similar, whereas GST alone and Glutathione Sepharose TM 4B beads couldn’t. Compared with both of them, the interaction between P53 2C and TRBP1 enhanced dramatically, but between P53 N5 and TRBP1 reduced significantly.Conclusion: P53 can interact with TRBP1 directly and in vitro, C terminus of P53 (293-393) is the structural domain of their interaction. This C terminus domain dependent interaction between P53 and TRBP1 may be related to the cellular activities induced by telomere dynamic changing.