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. 2015 Oct 15;8(10):19701-8.
eCollection 2015.

A Correlation Study on the Effects of DNMT1 on Methylation Levels in CD4(+) T Cells of SLE Patients

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Free PMC article

A Correlation Study on the Effects of DNMT1 on Methylation Levels in CD4(+) T Cells of SLE Patients

Jun Liang et al. Int J Clin Exp Med. .
Free PMC article

Abstract

Objective: To study the effect of DNMT1 on CD4(+) T cells in the peripheral blood of systemic lupus erythematosus (SLE) patients.

Methods: To investigate the differential expression of DNMT1 in CD4(+) T cells of SLE patients and healthy individuals, a DNMT1 lentiviral plasmid (pLenti6.3/V5-DNMT1) and a control plasmid (pLenti6.3/V5-GW/LacZ) were constructed and transfected into CD4(+) T cells from the peripheral blood of SLE patients. The transcriptional and translational expression of DNMT1, global genomic DNA methylation, and the production of IgG antibody in the CD4(+) T cells in the peripheral blood of SLE patients were assessed using qPCR analysis, western blotting, flow cytometry, and ELISA, respectively.

Results: The expression level of DNMT1 in SLE patients was significantly lower than that in normal humans. The expression of DNMT1 was found to be positively correlated with the methylation level of genomic DNA and negatively correlated with the IgG titration level. DNA sequencing results confirmed that the DNMT1 lentiviral plasmid was successfully constructed. After the CD4(+) T cells from the peripheral blood of SLE patients were transfected with the pLenti6.3/V5-DNMT1 plasmid, the transcription level of the DNMT1 gene was upregulated and abundance of DNMT1 protein significantly increased. Global genomic DNA methylation was enhanced, while the production of IgG antibody was reduced.

Conclusion: DNMT1 can inhibit the autoimmune response in SLE patients by reversing the abnormally low DNA methylation level in the CD4(+) T cells.

Keywords: CD4+ T cells; Lentivirus; SLE; methylation; pLenti6.3/V5-DNMT1.

Figures

Figure 1
Figure 1
A: PCR results showed that in CD4+ T lymphocytes from SLE patients, DNMT1 is significantly lower than that in normal population (P < 0.05); B: Western Blot results showed that in CD4+ T lymphocytes from SLE patients, the protein expression of DNMT1 was significantly lower than that in normal population; C: The DNA total methylation level of CD4+ T lymphocyte in SLE patients were significantly lower than that in healthy people; D: In SLE patients, the expression of DNMT1 and IgG antibody titers were negatively correlated (P < 0.05).
Figure 2
Figure 2
Synthesis process of plenti6.3/v5-DNMT1 viral vector.
Figure 3
Figure 3
A: PCR results showed that, after the transfection of plenti6.3/v5-DNMT1 viral vector into CD4+ T lymphocytes, the mRNA level of DNMT1 were markedly elevated; B: Western Blot results showed that, after the transfection of plenti6.3/v5-DNMT1 viral vector into CD4+ T lymphocytes, the protein level of DNMT1 were markedly elevated; C: Flow cytometry results showed that, after the transfection of plenti6.3/v5-DNMT1 viral vector into CD4+ T lymphocytes, the methylation level of total DNA raised; D: ELISA results show that, compared with the control group, the production of IgG antibody was reduced after the transfection of plenti6.3/v5-DNMT1 viral vector into CD4+ T lymphocytes (P = 0.021); E: 5-Aza-dc cell demethylation can significantly reverse the level of IgG antibody titers in CD4+ T lymphocytes.

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