| Systemic lupus erythematosus (SLE) is a chronic complex autoimmune disorder characterized by the presence of numberous autoantibodies. The manifestations of SLE can vary widely from person to person, with the potential involvement of virtually any bodily organ. The disease mainly involves the skin, joints, kidneys,, et al. Due to better diagnosis and treatment in the last decades, The5-year survival of patients with SLE has generally exceeded90%. However, the patients is still not optimistic under the condition of organic damage caused by SLE, especially renal failure or cardiovascular dysfunction after long-term follow-up. Although genetic factors and hormonal factors has been confirmed involved in the etiology of SLE, the two class factors can not account for all the cases of SLE. The results from recent studies suggested epigenetic regulation including DNA methylation, histone modifications and miRNAs have been implicated in the development of SLE. Our previous study showed that miRNA-126contributes to the regulation of DNA methylation in CD4+T cells of SLE. Therefore, we hypothesis that other more miRNAs involve in the pathogenesis of SLE..The first miRNA was observed in1993and as a part of epigenetics, miRNAs became highlighted since2005. They are small (19-25nt) single-stranded noncoding RNA molecules. Now lots of studies have confirmed miRNAs participated in human biological processes. Furthermore, some of them could induce autoimmune responses.In recent years, miRNA high-throughput deep sequencing appears to be widely used with the rapid development of miRNA identification techniques. Scientists combined sequecing with other molecular biological methods to clarify large-scale miRNAs and the function of their target genes. Compared to large limitation of Sanger sequencing, new ones set out many advantages such as high throughput and quality,saving time,operating easily, et al. So they are good tools for miRNA detection and difference analysis. Concretely, novel sequencing technology means454pyrosequencing,Solexa sequencing and SOLiD system and the three all take advantage of DNA and/or RNA researches.Therefore, we detect differences in microRNAs profiling of CD4+T Cells from healthy donors and patients with various clinical symptoms of SLE through Solexa sequencing, and then use Real-time qPCR to validate different miRNAs expression among SLE variable organ manifestations. Through this study, we will reveal the key miRNAs and lay foundations to further explore the mechanism about different disease manifestations of of SLE.Section â… Expression Profiling of miRNAs in CD4+T cells of SLE by High-throughput Deep SequencingObjective To investigate expression profiling of miRNAs and select different ones in CD4+T cells between healthy controls and SLE patients with different manifestations.Methods1. CD4+T cells was isolated by immunomagnetic beads from peripheral blood of4controls and12SLE patients.The first4patients had only skin lesion.Another4suffered from skin lesion and renal damage. And the last4ones got skin lesion,renal damage and arthritis;2. Total RNA of each sample was extracted by Trizol. Pooled4RNA samples from patients with similar organ, for example, Skin (S), Skin plus Kidney (S+K) or Skin plus Kidney and Joints (S+K+J).;3. Solexa sequencing was used to detect expression of various miRNAs and their differences among healthy donors and SLE patients were anlyze.Results After the transition from raw data,there are about1000,000clean reads of sRNA and the number of21-23nt-long miRNAs were all over60%of them in each group.3groups of patients mutually had38miRNAs expressed more and2expressed less than the healthy group (Iog2-ratio>1or<-1; P<0.01). Compared to controls,part of the miRNA expression profiles of all SLE groups showed miR-126and miR-181b were upregulated while miR-142-3p and miR-505were down regulated (P<0.01) MiR-451significantly raised in S and SK group (P<0.01).However,it was not up-regulated in SKJ group (P=0.0857). Furthermore, in the pairwise comparision among3groups of patients, miR-126and miR-181b increased according to the gradient of S>SKJ>SK(P<0.01or<0.05). In S group, miR-451raised more than those in SK group and SKJ group (P<0.01) while there was no difference between SK group and SKJ group (P=0.0528). The expression of miR-142-3p had the gradient of S<SK<SKJ (P<0.01).Finally,there was no significant difference in the expression of miR-505among different manifestations of SLE (P>0.05)Conclusion The aberrant expression of many miRNAs, such as miR-126, miR-181b,miR-451,miR-142-3p and miR-505,were found in CD4+T cells of patients with SLE.They may involve in pathogenesis of SLE. It is possible that miR-126and miR-142-3p lead to multiple manifestations of SLE through the pairwise comparision among3groups of patients. Section II Validation of Partial MicroRNAs in CD4+T CellsObjective To validate the expression of miR-181b,miR-451,miR-142-3p and miR-505in CD4+T cells from more healthy controls and SLE patients with different manifestations by RT-qPCR.Methods We used immunomagnetic beads to isolate CD4+T cells from peripheral blood of10controls30SLE patients with different manifestations including Skin (S), Skin plus Kidney (S+K) or Skin plus Kidney and Joints (S+K+J),10samples for each group. Total RNA of each sample was extracted by Trizol. The expression of miR-126,miR-181b,miR-451,miR-142-3p and miR-505were detected by Real-time qPCR.Results Compared to controls,miR-126and miR-181b were upregulated while miR-142-3p and miR-505were down regulated (P<0.01) in all SLE groups.The result was same to that of Solexa deep sequencing.Furthermore,in the pairwise comparision among3groups of patients, miR-126increased more in SKJ group than other2groups (P<0.05)while its expression had no difference between S group and SK group (P>0.05). miR-451increased according to the gradient of S>SKJ>SK (P<0.01or0.05). miR-142-3p was expressed in SKJ group less than S and SK group (P<0.01) and there was no difference between S group and SK group (P>0.05).miR-181b raised more in S group than other2groups (P<0.05) while its expression had no difference between SK group and SKJ group (P>0.05). there was no significant difference in the expression of miR-505among different manifestations of SLE (P>0.05)Conclusion The results of Real-time qPCR are basically consistent with those of high-throughput deep sequencing. MiR-181b,miR-451,miR-142-3p,miR-505may involve in pathogenesis of SLE.The results of Real-time qPCR show it is possible that the down regulation of miR-142-3p and the raised expression of miR-126and miR-451relate to multiple symptoms of SLE. |
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