The MicroRNA Role Research On The Systemic Scleroderma Pathogenesis

Background and ObjectivesSystemic Scleroderma is an autoimmunity connective tissue disease, which is a complex disease involved in multiple factor, its etiology and pathogenesis is unclear. In clinically, it is characterized by excessive and widespread fibrosis in the skin and internal organs, vasculopathy and abnormal activation of the immune system. And fibrosis is the most remarkable clinic character in the process of scleroderma, it is also the major threaten of influencing seriously on the life quality of patients, even the patients’life. In previous studies, the pathogenesis of SSc was focused mainly on fibrosis, vessels change, the immune system abnormalty, twins, heredity, race, chimeras, and others, including genome-wide studies using gene-chip and protein-chip technology, but all can’t elucidate the SSc’s pathogenesis.MiRNAs are short 18- to 25-nt noncoding single strand RNAs that serve a regulatory function and participate in many vital processes, including early development, cell proliferation, cell differentiation, apoptosis, substance metabolism and others. These miRNAs have been demonstrated to suppress the expression of specific genes at the translation level by complementary binding to the 3′noncoding region of target mRNA, which play important role in the pathogenesis of many disease. MicroRNA arrays are a newly developed high-throughput screening technology that can be used to detect different expression levels of miRNAs in patients and the healthy subjects at the specified time and space. which hsa been applied to the study of some complex diseases, especially in the study of malignant tumor. It was demonstrated from a lot of study that microRNA plays the important role on the pathogenesis, metastasis, relapse, of malignant tumor, and the pathogenesis of psoriasis, SLE, malignant melanoma and so on, and some disease-specificity microRNA were found, for example: miR-203 is the psoriasis-specificity microRNA, miR-140 expression is related to osteosarcoma, and so on. As a sort of complex autoimmunity disease, SSc hsa been similar to tumor, psoriasis, SLE. So we presumed that microRNA may also play important role in the pathogensis and otherelse of SSc. With microRNA chip technology and bioinformatics methods, For the first time in the world, we screened different expression microRNA between SSc patient’s skin lesion and health people, then analyzed and predicted the screening results so as to get the microRNA, which is intimate or related or specific to SSc. The study result may provide the important reference for studying molecular mechanism of microRNA in the SSc, finding new diagnosis biomarker and new therapy target.Objects and MethodsThe objects were divided to two group, SSc group (SSc G.) and Control group (Ctrol. G.). the selected criterion of SSc G. was the patients who fulfilled the American College of Rheumatology classification criteria for SSc, had suffered from the SSc within 2 years, had not received recent hormonal systemic therapy or no more than 3 month, had systemic skin sclerosis and some signs of lung fibrosis but without obvious clinical manifestations. The Ctrol. G. was skin tissues of circumcision and the normal skin bordering a melanin pigmented naevus ectomy. Incise enough skin tissue from the two group patients, extract their total RNA, Isolate microRNA and fluorescent Labeled, Then hybridizing, cleaning and scanning. The scanning results and the different expression microRNA were analyzed with LuxScan 3.0 image analysis software and SAM 2.1 software. The different expression microRNA were demonstrated randomly with Real-time PCR to ensure the reliability of chip results.The process of bioinformatics analysis: firstly, establishing screening base of the emphsais target gene; Secondly, searching the target genes in the microRNA database (Target Scan), and get their function annotation; Thirdly, comparing the target genes with the genes in the screening base of the emphsais target gene, choose out the genes whose function are related to the ECM, fibrosis, vassular abnormality, immunologic abnormality, or which are in the screening base of the emphsais target gene, it is regarded as emphsais target genes; Fourthly, logging the website of NCBI, searching the emphsais target gene and the SSc, the searching formula is“SSc and emphsais target gene”, then reading the literature and analyzing which is relate to SSc or not; If there have many target genes regulated by the same microRNA are related to SSc or participant in the pathogenesis of SSc, then we would initially predict this microRNA correction with SSc pathogenesis.Results 1.We firstly selected out 24 different expression microRNA were from SSc patient’s skin lesion, in which the 9 microRNAs were upregulation and the 15 microRNAs downregulation. the three differential expression of microRNAs (microRNA—hsa-let-7g, hsa-miR-206 and hsa-miR-125b) were selected outrandomly, and demonstrated with real-time PCR to ensure the reliability of chip results. The verification results were consistent with the chip results. 2.The 6 differential expression of microRNAs were demonstrated that they may be related to SSc pathogenesis with bioinformatics methods and document analysis(http://www.ncbi.nlm.nih.gov/pubmed/). (www.gogene.org, now this website is maintenance, can’t open it, you can inquire out the target mRNA regulated by microRNA to http://www.targetscan.org ), in which, The 7 genes in the 584 target genes regulated by hsa-miR-206 were correlation with SSc, The 24 genes in the 819 target genes regulated by hsa-let-7g were correlation with SSc, The 12 genes in the 502 target genes regulated by hsa-miR-133a were correlation with SSc, The 8 genes in the 604 target genes regulated by hsa-miR-125b were correlation with SSc, The 7 genes in the 251 target genes regulated by hsa-miR-140-5p were correlation with SSc, The 14 genes in the 480 target genes regulated by hsa-miR-23b were correlation with SSc.Conclusions1.MicroRNA not only participant in the process of SSc pathogenesis, but play the important role in the SSc etiopathogenisis;2.The hsa-miR-206 six miRNAs are closely correlation with SSc’pathogenesis and they may have more important function in the SSc pathogenesis, development and turnover;3.The expression abnormalty of microRNA may be the important molecular event during the process of SSc genesis, development and disease evolution, especially in the early stage of SSc, which may take more important function;4.The significant different expression microRNAs may be potentially diagnosis marker, therapy target or important causative agent, which will provide the important reference for studying SSc molecular mechanism.