Analysis Of Circulating Mirna In Exosome Based On High-Throughput Sequencing And Its Applications For Pregant Related Diseases

MicroRNAs (miRNAs) are a class of single-stranded, non-coding RNA molecules that are 18-22 nucleotides in length, are encoded by endogenous genes. MiRNAs participate in a series of critical physiological processes, and are also associated with the incidence of multiple diseases, including the development of tumors. MiRNAs were found to be present in human plasma and serum in a remarkably stable and cell independent form, making their potential as novel non-invasive biomarkers for physiological and pathophysiological conditions, including cancer, of growing interest. Circulating nucleic acid is a family of extracellular nucleic acid existing in body fluid, and circulating miRNAs are thought to be stable in plasma/serum, the content and proportion of circulating miRNAs are related to pre-eclampsia, gestational diabetes mellitus and cancers. The source of circulating miRNAs and reason for stability are unkown. Exosomes, a type of small (40-100 nm) vesicles are released by cells and exist in body fluid. Exosomes carry lots of specific protein and also some certain miRNAs. Exosomes could protect circulating miRNAs by package miRNAs in itself.Pregnancy is a highly regulated complex physiological process related with hormone as well as other molecules. Recently, numerous evidences showed that specific miRNAs associated with regulation of pre-implantation, embryonic stem cells differentiation, and pregnancy relative disease. Placenta special miRNAs could be detected in matemal blood. Pre-eclampsia and gestational diabetes mellitu are common disease in pregnancy but the pathogenesis is not clear. Endometrial cancer is malignancy in female reproductive system, discrepancy of hormone and mRNA are used to elaborate pathogenesis. Although high-throughput sequencing technology has widely been used in miRNA researches, this technology still has disadvantages, such as unsuitable for minute nucleic acid sample. Hence establishment of high-throughput sequencing technology of minute nucleic acid sample is important for non-invasive diagnosis of circulating nucleic acid. Meanwhile magnanimity miRNA data are produced by high-throughput sequencing, making full use of the information is beneficial to tracing pathogenesis of disease and helpful to improve specificity and sensitivity of diagnose.Here, we established an extracting method for miRNAs in exosome firstly, and studied distribution characteristics of DNA in exosome and stability of miRNA in exosome. Then we sequenced miRNAs in placenta and plasma of pre-eclampsia and gestational diabetes mellitu to analyze differential miRNAs. These results may be the basis for a further study to find new pregnancy-related biomarkers.1. Characterization of circulating nucleic acids in exosomeCells release various types of membrane vesicles into the extracellular space. These extracellular vesicles contain various functional proteins, microRNAs (miRNAs) and mRNAs. The vesicle structure could protect miRNAs from ribonuclease. We characterize circulating exosome in plasma, and provide foundational work for related researches.We chosed a precipitate kit independent of ultracentrifugation to purify exosome, and the resluts of electron microscope indicate diameter of exosome is about 50-70nm. Then we compared concentration of RNA extracted from exosomes and plasma under different conditions and found that RNA in exosome are more stable than in plasma. Comparison of individual miRNA level in exosomes and plasma by RT-PCR also verified this consequence. The miRNA stability also was impacted by freeze/thaw cycles, but exosome RNA tended to be less affected by freeze/thaw cycles2. Preparation of sequencing library for circulating miRNAsSince the concentration of circulating miRNAs are very low in plasma, we optimized sequencing library construction based on single random strand.3’ pre-adenylated adaptors with two random nucleotides in 5’ end.PCR were carried out with a universal primer and miRNA specific primer?, and results indicated that random strand has no preference over different kinds of microRNAs. Our method also were highly consistent with existing commercial kits. Next we sequenced miRNA in plasma and exosomes, the reads and types of miRNAs in plasma were obviously fewer than in exosomes. MiRNAs in exosomes are idea samples for constructing library because they are intact.3. Comparative analysis of circulating miRNAs sequenced from pre-eclampsia and gestational diabetes mellituPregnancy is a highly regulated complex physiological process related with many diseases, such as pre-eclampsia and gestational diabetes mellitu. We studied the profile of circulating miRNAs in pre-eclampsia and gestational diabetes mellitu with high-throughput sequencing. For pre-eclampsia, miRNA profiles of placenta and serum (maternal serum and umbilical cord serum) were compared.11 miRNAs in placenta were found to be expressed associated with PE; 22 miRNAs in maternal serum and 8 miRNAs in umbilical cord serum were associated with PE. The differential miRNAs may participate in pathway in pre-eclampsia, and circulating miRNAs are potential biomarkers for diagnose. Meanwhile, we compared the profile of exosome miRNAs between gestational diabetes mellitu and normal pregnancy. We found 28 discrepant miRNAs,12 were up-regulated and 16 were down-regulated. Further we made target prediction, functional analysis, and pathway analysis for 5 abundant miRNAs, and found that this 5 miRNAs might regulate transcription of 5 genes so as to impact 3 pathways. Our results not only explained mechanism of gestational diabetes mellitu, but also provided reliable biomarkers for the disease.4. Analysis of miRNA cluster/family and isomiRs based on high-throughput sequencingMiRNA genes are arranged as clusters on the chromosome. When members of a gene cluster exhibit sequence similarity and homologous regulation, they form miRNA gene families, which further increase the diversity of miRNA gene cluster distribution. Previous studies have generally focused on the expression and regulation of single miRNAs, while studies on the expression and function of clustered miRNAs are infrequent and examining the expression profile of miRNA clusters at the genome-wide level are particularly limited. We firstly investigated the differential expression of miRNAs in ISK and HEC-1B cells. Based on the cluster comparison definition used in the present study, it was observed that there were 5 miRNA clusters in the ISK cell line in which expression levels were significantly upregulated compared with those of the HEC-IB cell line, and 3 miRNA clusters in which expression levels were significantly down-regulated in ISK cells as compared with HEC-1B cells. We also chosed 9 abundant miRNAs to study distribution profile of isomiR, mir-17、mir-18a、mir-19b in mir-17 cluster were similar in distribution pattern, others were varied.Elevated placental-specific circulating mircoRNAs in plasma of patients with preeclampsia could be sensitive biomarkers for monitoring certain placental disease. And these highly expressed placenta-specific miRNAs (mainly from C19MC) in plasma, usually had consistent isomiR spectrum compared with those in placenta where the source placenta-specific circulating miRNAs originated. To combine the two evidences together, we could get specific and proportional biomarkers to supervisory control preeclampsia, even pathological pregnancies or cancers.