Preliminary Research On The Relevance Of Circulating MiRNAs And Steroid-induced Osteonecrosis Of The Femoral Head

Background:Osteonecrosis of femoral head is a common disease, which occurs mainly in young adults. The exact pathogenesis is still unknown. Lack of specific manifestations and effective diagnostic methods, makes it hard to diagnose at early stage. Many patients eventually undergo hip replacement surgery. However, the high costs of the surgery, the probable severe complications, and the limited prosthesis life, shows that joint replacement surgery might not be most suitable for the young patients. For most patients is concerned, to postpone the disease progress, avoid the femoral head collapse and prevent function loss through early-stage surgery or drug intervention, may be a better option. That depends on the clarification of the pathogenesis and the development of the diagnostics research.Many research showed that, the occurrence of ONFH was related to endothelial cell injury, abnormal stem cell differentiation, bone cell apoptosis and hypercoagulablestate. As endogenous noncoding RNAs found in eukaryotes, miRNAs regulate apoptosis, cells differentiation and metabolism. This suggests miRNAs may participate in the occurrence of ONFH. Studies showed that miRNAs was stable in blood circulation, and serum miRNAs expression changed specifically in many diseases, such as prostate cancer, pancreatic cancer, breast cancer, myocardial infarction and diabetes. Thus, miRNAs might also be specific in circulation of ONFH patients, and could be used as plasmic markers for the diagnosis of early stage of ONFH.In this study, we presume that the specific miRNA was also present in the plasma of ONFH patients, might regulate the pathogenesis of ONFH, and was likely to be diagnostic markers for the disease. We use deep sequencing technology and bioinformatics analysis to identify the ONFH-specific plasmic miRNAs, which will help to clarify the pathogenesis of disease, and also provide new ideas for the diagnostics research of ONFH. Objectives:1. To build plasma miRNA library for patients of steroid-induced ONFH and the controls.2. To achieve deep sequencing of plasma miRNAs by using Illumina high throughput sequencing technology.3. To make bio informatics analysis and identify the specific plasmic miRNAs of ONFH patients.4. To make database retrieval for the specific miRNAs and analyze their possible functions.Methods:1. Peripheral blood plasma was collected from3steroid-induced ONFH patients which basic disease was SLE,3SLE patients and3healthy volunteers, of whom, the gender and age was matched. MiRNeasy Mini Kit (Qiagen) was used to extract the RNA from plasma. Then a small RNA library was built.2. The second generation of high-throughput sequencing platform, Illumina HiSeq-2000was used to complete the one-way end direct sequencing of Small RNA for the9samples. Then a miRNA database was established for each sample.3. The9samples was divided into3groups, each group includes an ONFH patient, a SLE patient and a volunteer, of whom the gender and age was matched. The expression differences of plasmic miRNAs among the3samples and find the specific miRNAs of the ONFH patient in each group. Then miRNAs which was always specific in this3groups was selected from the results.4. Internet and database was retrieve for the selected candidate miRNA, and the possible function of them was analysed.Results:1. Adequate plasmic miRNAs was extracted from the9samples and the miRNA library was built successfully.2. Deep sequencing of plasmic miRNA from9samples was realised by using Illumina high-throughput sequencing technology.3. Bio informatics analysis was made and27ONFH-specific plasmic miRNAs was found, of which15miRNAs was up-regulated and12miRNAs was down-regulated.Conclusions:1. This is the first time to study the expression changes of plasmic miRNAs in ONFH patients. The miRNA library was built and deep sequencing of plasmic miRNA was achieved successfully by using Illumina high-throughput sequencing technology.2.27ONFH-specific plasmic miRNAs was identified. Though the result is preliminary, it lays a foundation for the diagnostic marker research of early-stage ONFH. Further researches are necessary to identify the sensitivity and specificity of the specific miRNAs used as diagnostic marker.3. This result suggests miRNAs may participate in the occurrence of ONFH. Further investigations are indispensable to illustrate the functioning pathway and mechanism of how these miRNAs contribute to ONFH. The result will be useful for understanding the pathogenesis of ONFH.