The Screening And Verification Of Micro Rna Expression Profiling In Systemic Lupus Erythematous

Background Systemic lupus erythematous(SLE) is an autoimmune disorder that primarily affects young women and is characterized by inflammation in several organs including the kidneys, skin, joints, blood, and nervous system, often with fluctuations in disease activity over time. There are many autoantibodies in serum of patients,such as antinuclear antibody. An epidemiological study of our country reveals the prevalence of SLE was 70/100 000. SLE is a protean disease with variable presentation, clinical course and prognosis. The array of clinical manifestations in SLE is intricate, suggesting that the pathogenesis of SLE may involve the interplay of multiple factors. The etiology and pathogenesis of systemic lupus erythematosus is not yet clear. The current researchers generally believe that the pathogenic factors of SLE include natural environmental factors(UV irradiation, dietary factors, contact with chemicals etc.), biological environment(bacteria, virus infection), social environmental factors(stress, trauma), genetic susceptibility and endocrine status and so on. These factors interact and influence each other, eventually lead to the disorder of immune function in patients with SLE. The therapeutic management of SLE is still a great debate. Despite the latest innovation agents or developing trials, there is not an integrated and common approach for treating SLE. Many of the symptoms still can not be completely or effectively controlled. Long term inflammation and drug-related side-effects may subsequently lead to permanent organ damage, a consequence which is intimately connected to decreased quality of life and mortality. So the research on systemic lupus erythematosus etiology and pathogenesis is particularly important. Studies have shown that T helper cells(Th) play an important role in the pathogenesis of SLE and other autoimmune diseases. Immunologically, SLE is the consequence of loss of self-tolerance and amplification of self-antigen-mediated hyperactivation of T and B lymphocytes.Micro RNAs(mi RNAs) are endogenous approximately 22 nt RNAs that can play important regulatory roles in animals and plants by targeting m RNAs for cleavage or translational repression. They bind to the 3’-untranslated region(UTR) of their target messenger RNAs(m RNAs) through complementary recognition, which then leads to m RNA degradation or repression of protein expression. Almost 30% protein coding genes of human is regulated by the mi RNAs, including apoptosis, cell differentiation and division, lipid metabolism, hematopoiesis and virus defense. The researchers found that, mi R-326 can inhibit Ets-1 transcription factor, and then promote the T helper 17 cell(Th17) differentiation. The expression of mi R-326 has been found correlated with SLE disease activity. Our previous studies have confirmed that, Th17 and related cytokines participate in the pathogenesis of SLE. We propose Th17 may serve as a therapeutic target for SLE. In addition, two genome-wide association studies of our research group participation have confirmed that Ets-1 is a susceptibility gene of SLE. Thus, we propose the role of Ets-1 in pathogenesis of SLE. Therefore, we hypothesized that mi RNA may be play an important role in the pathogenesis of SLE through regulation of some of the key target genes. Many studies have shown that several mi RNAs have been associated with autoimmune diseases, whether they are involved in the pathogenesis of SLE is not clear.Plasma mi RNA has the characteristics of content-rich, stable, easy to detect. It has been used as a new class of biomarkers for early screening of cancer and other chronic non-communicable diseases, diagnostic and prognostic assessment study. However, in SLE expression studies mostly in peripheral blood mononuclear cells(PBMC), the study about expression of mi RNA in plasma of SLE were few.Recent study shows that mi R-101 regulates the MAPK response by targeting MKP-1 m RNA 3’-UTR, and affects the secretion of the downstream inflammatory cytokines. The mitogen-activated protein kinase(MAPK) signaling pathway is the prototype that can regulate a variety of SLE-related cytokines like tumor necrosis factor-alpha(TNF-a), interleukin(IL-1), IL-6 and interferon(IFN). Whether IL-6 or TNF-a, their 3 ’UTR region contains binding sites of micro RNA-16. The studies have shown that micro RNA-16 overexpression causes the expression of IL-6, IL-8 and TGF-β is decreased. Therefore, it is our intent whether IL-6, IL-17 and TNF-a correlation of the expression level of mi R-16-5p and mi R-101-3p by ELISA assay.In summary, mi RNA expression levels of SLE patients and normal control plasma will be detected by mi RNA microarray. And then independent verification will be detected by Real-time PCR. Combined with demographic and clinical data, establish different clinical subtypes of SLE plasma mi RNA expression profiling. Explore the molecular mechanism of mi RNA in the pathogenesis of SLE. We analyze some of the abnormal expression of mi RNAs associated with three kinds of cytokines IL-6, IL-17 and TNF-alpha. Based on these findings, we postulate that aberrantly expressed plasma mi RNAs could be attractive as candidates for putative biomarkers of SLE and may help elucidate the possible pathogenesis of SLE.The study has two parts.Part 1 The screening and verification of micro RNA expression profiling in Systemic lupus erythematousObjectivesThe aim of this study was to evaluate the plasma expression levels of micro RNAs in patients with SLE and their correlations with clinical features and laboratory measurements. Investigate the pathogenesis of SLE and its relevance to the course of disease.Methods Our study adopts a case-control study. The mi RNA expression in plasma was detected by Mi RNA microarray. And then independent verification will be detected by Real-time PCR. Combined with demographic and clinical data, establish different clinical subtypes of SLE plasma mi RNA expression profiling. Explore the association between the clinical characteristics and abnormal expression mi RNA in SLE. SLE blood samples were collected from Department of Rheumatology and Immunology of the First Affiliated Hospital of Anhui Medical University and Anhui Provincial Hospital. All SLE patients were according to the 1997 revised American College of Rheumatology(ACR) classification criteria. Find the controls that match SLE patients according to Age and sex. The first phase, we collected 30 new cases of SLE and 30 healthy controls. The mi RNA expression in plasma was detected by Mi RNA microarray. The second phase, we collected 38 new cases of SLE and 40 healthy controls. The mi RNA expression in plasma was detected by Real-time PCR.Gene Pixpro 6.0 was used for Mi RNA microarray data analysis. Real-time quantitative reverse transcription–polymerase chain reaction(q RT-PCR) with sequence-specific primers was performed to detect the plasma levels of mi R-16-5p and mi R-101-3p in two compared groups. 2-△Ct was used to compute the expression value of mi R-16-5p and mi R-101-3p. Epi Data 3.1 software was used for the database establishment, double entry and error detection. The Statistical Package for Social Sciences, version 10.01 was used for data analysis. P values(2-tailed) less than 0.05 were considered statistically significant.Results(1)The results of micro RNA microarray screening found that 97 genes were up-regulated and 182 genes were down-regulated. P values(2-tailed) less than 0.05 were considered statistically significant. Ultimately, we have found that there are 32 genes up-regulated and 126 genes down-regulated.(2)Expand the sample size. Real-time PCR with sequence-specific primers was performed to detect the plasma levels of mi R-16-5p and mi R-101-3p in two compared groups. The expression of mi R-16-5p and mi R-101-3p were increased in the plasma from SLE patients(P= 0.001, 0.005). These results are consistent with the experimental results of the micro RNA microarray screening.(3) The mi R-16-5p and mi R-101-3p expression were higher in SLE patients with rash compared to patients without rash(P=0.017, 0.041).Iinterestingly, the mi R-16-5p and mi R-101-3p expression were lower in SLE patients with thrombocytopenia compared to patients without thrombocytopenia(P=0.032, 0.029).(4) There was no significant difference for two mi Rs expression between active and inactive SLE patients(P>0.05). In addition, there was no significant difference between SLE disease activity score and the expression levels of two mi Rs.(5) Furthermore,mi R-16-5p expression was positively correlated with the expression of mi R-101-3p in SLE patients(r=0.675,P<0.001).Conclusions We have found that there are 32 genes up-regulated and 126 genes down-regulated from micro RNA microarray screening. Subsequent verification experiment showed that the expression of mi R-16-5p and mi R-101-3p were increased in the plasma from SLE patients. These results are consistent with the experimental results of the micro RNA microarray screening. In addition, we also found the expression levels of two mi Rs associated with SLE patients with rash and thrombocytopenia. Suggesting that they are hoped to find their use as novel biomarkers in the diagnosis,therapy and prognosis of diseases.Part 2 Study on the relationship between the expression level of mi R-16-5p and mi R-101-3p and IL-6, IL-17 and TNF-α level in SLE plasmaObjectives In this part, by using the method of ELISA, we analysed the relationship between the expression level of mi R-16-5p and mi R-101-3p and IL-6, IL-17 and TNF-α level in SLE plasma.Methods SLE patients in part 1 and part 2 were the same. In this study, we have chosen 37 SLE patients. Before study, all subjects provided written informed consent. 5 ml venous blood from the SLE patients was collected in EDTA-containing tubes. All the blood samples were processed immediately for the isolation of plasma within 3 h after collection. We collected demographics and clinical data by professional person. Disease activity was evaluated using the SLE Disease Activity Index(SLEDAI) score. The plasma levels of IL-6, IL-17 and TNF-α were detected by ELISA. To analyze it’s associations with activity and disease manifestations, additionally, to explore the correlations within mi R-16-5p and mi R-101-3p expression levels.Results(1) IL-6, IL-17 and TNF-α plasma levels in SLE patients are(18.15±7.82)pg/m L,(20.42±10.11)pg/m L and(33.66±15.18)pg/m L.(2) Association of plasma IL-6, IL-17 and TNF-α levels and clinical data of SLE patients: plasma IL-6 level were significantly associated with urinary tube(t=3.388, P=0.009); plasma IL-17 level were significantly associated with myositis(t=-5.354, P<0.001) and thrombocytopenia(t=-2.267, P=0.030); plasma TNF-α level were significantly associated with discoid erythema(t=5.499, P<0.001).(3) No significant associations were found between plasma IL-6, IL-17 and TNF-α levels and SLEDAI.(4) Plasma TNF-α levels were significantly associated with mi R-16-5p expression levels in SLE patients without butterfly erythema(r=0.928, P <0.001), SLE patients with arthritis(r=-0.486, P=0.014) and positive anti-ribosomal P protein(r=-0.488, P=0.047). No significant associations were found between plasma IL-6, IL-17 and TNF-α levels and mi R-16-5p expression levels.(5) Plasma IL-17 levels were significantly associated with mi R-101-3p expression levels in SLE patients(r=0.329, P=0.047). Plasma IL-17 levels were significantly associated with mi R-101-3p expression levels in SLE patients with discoid erythema, low complement, positive ANA, urinary tube negative group, proteinuria negative group, pyuria negative group, negative anti-ribosomal P protein(all P<0.05). Plasma TNF-α level were significantly associated with mi R-101-3p expression levels in SLE patients with negative anti-ribosomal P protein(r=0.448,P=0.048).Conclusions In conclusion, plasma IL-17 levels were significantly associated with the expression levels of mi R-101-3p.Although no significant associations were found between these cytokines plasma levels(IL-6 and TNF-α) and two mi Rs expression levels, there were many associations between these cytokines plasma levels(IL-6 and TNF-α) and two mi Rs expression levels about clinical features and laboratory indicators.