The Role Of Mirna In The Spinal Cord Development And Tumor Classification

MicroRNAs (miRNAs) are small (21～23 nucleotide) regulatory RNAs which areprocessed from longer hairpin transcripts by the enzyme Dicer and can be found innematodes, plants, insects and mammals. Recently, following the accumulation ofknowledge in their roles as post-transcriptional modulators of gene expression andregulators of developmental timing, the study of miRNA is the onset of intensive researchand becoming a hot point of biological theory and biomedical application study. Onbiological theory study, it is mainly about the issue how miRNAs take part in themodulation of posttranscriptional gene expression and the role on cell differentiation,proliferation, apoptosis, and development; in application area, miRNA drugs or themiRNAs as the drug target are becoming a hot point in the development of biomedicine.The miRNA expression profile in specific tissues probably can be used as a kind of newmarker in the diagnosis, classification, stage and prognosis of tumor.PartⅠ. The role of miRNAs in fetal spinal cord developmentObjects: To profile and verify the miRNAs specially expressed in fetal spinal cord. Thebasic function of miRNAs in cell differentiation, proliferation and development wasanalyzed to provide basement in the further research of normal development andtaratogenia of fetal nervous system.Methods: Firstly, an oligonucleotide DNA microarray (OMA) for genome-wide miRNAprofiling method was developed and was used in 18 organic samples from 12 gestationalweeks old (G12w) and 24 gestational weeks old (G24w) aborted human fetuses to findmiRNAs which control nervous system development. Global miRNA expression patternsof different organs were verified using Cluster analysis and Northern blot; Timed-pregnantrats were gavage-fed all-trans-retinoic acid to produce spina bifida model and a controlgroup received olive oil. Fetuses were obtained on 13.5 d, 15.5 d, 17.5 d and 19.5 d, andexamined of the malformations macroscopically and microscopically. MiRNAs expressionprofile was analyzed by Northern blot. In situ apoptosis detection and miRNA in situ hybridization methods on sections of paraffin embed tissues were employed to explore themechanism of miRNA in nerve cell differentiation, proliferation and development.Result:(1) The whole genomic miRNA profiles of human fetal organs were gotten. The OMArevealed that 72%～83% of 158 miRNAs were expressed in human fetal organs. A series ofmiRNAs were found specifically and higher expressed in the specific human fetal organand specific time. After cluster analysis, we firstly found that miR-9/9~*, miR-124a,miR-125b and miR-128 in nervous system and confirmed by Northern blot consistently,which may play a key role in nervous system development.(2) The apoptosis cell number increased in sacral cord induced by retinoic acid(RA),which indicated that the treatment of RA probably can induce cell abnormally death in thelate stage of spinal cord development; Bcl-2 vs miR-9/9~* and P53 vs mR-124a. 125b hadthe same time and spatial model of expression; furthermore, RA can weaken the polarity ofmiR-9/9~* and cause absence of miR-124a/125b expression in motor neuron and dorsal rootganglion.Conclusion:(1) It was firstly found that specific miRNAs expression profile in human fetal nervoussystem, which indicated that it play an important role in the development of human fetalcerebral and spinal cord.(2) The mainly dysregulation of miRNA specifically expressed in nervous systeminduced by RA is disorder of time and spatial expression model, which indicate thefunction of the miRNAs in the normal development of spinal cord and influence of celldeath. In addition, the results also provide the clue that miR-9/9~* and miR-124a/125b onthe modulation Bcl-2 and P53 pathway can play a role in the development of sensory andmotor neuron.PartⅡ. The role of miRNAs in the classification of gilocytoma and retinoblastoma.Objects: To screen and construct the miRNA expression profiles of gliocytoma andretinoblastoma tumor tissues and to study the possibility of miRNA as a new biomarker intumor grading, classification and prognosis.Methods: Collect fresh samples of gliocytoma and retinoblastoma according WHO clinically graded. Formalin-fixed, paraffin-embedded (FFPE) tissues microarray(TMA)samples were used to do miRNA profile analysis, and clinical protein biomarkers ofKi-67, VEGF and GFAP were analyzed for gliocytoma samples at the same time, to findthe possible relationship between them.Result:(1) MiRNA expression profile in gliocytoma tissues indicated that the expression of miR-9and miR-21 were increased and miR-124a and miR-125b were decreased. From thediagnosis of pathology, the correlation of miRNA expression pattern with the pathologygrades was found; in additional, the expression of miRNAs and Ki-67, VEGF and GFAPbiomarkers are negative relationship.(2) The result of miRNA profile in retinoblastoma tissues indicated that the total level ofmiRNAs expression was upregulated compared with normal retina tissues. There were 11miRNAs overexpressed in retinoblastoma tumor with the change>2-folds, in whichfour-fold improved miRNAs in retinoblastoma were miR-373~*, miR-503 and miR-320.The results were then verified using Northern Blot analysis and ISH method.Conclusion:(1) According to the result of characteristic miRNA expression pattern, the conclusion wasgotten that miRNA can be used as a new kind of biomarker in staging and gradeclassification of cancer. At the same time, in addition to the clinical course, pathologicdiagnosis and cluster analysis, the miRNA expression pattern can be used to judge theprognosis and sensitivity to treatment of the patients. The miR-9, miR-21, miR-124a andmiR-125b are probably the key biomarkers and conduce to diagnosis, treatment andprognosis of gliocytoma.(2) In the study of miRNA expression in retinoblastoma, it is the first time to identify themiRNA expression level in human retinoblastoma tissues was higher than normal retinatissues, which indicates that the role of miRNAs in retinoblastoma tumorigenesis isdifferent from that in other solid tumors. From which, all of miR-373~*, miR-503 andmiR-320 are the most prominent miRNAs, which probably be as the key modulators in thetumorgenetics of retinoblastoma.