The Molecular Mechanism Of MiRNA-22Involved In The Cell Proliferation And Apoptosis Of Medulloblastoma

Medulloblastoma (MB) is the most common brain malignancy in children (12%-25%of all childhood central nervous system (CNS) tumours), which is WHO IV lesions. MBoriginates from infratentorially in the cerebellum or fourth ventricle. Children arevulnerable to MB at3or7years old, however, there is a rare incidence of MB in adult(0.4%-1%) and the median age is20-40years. The most classical symptoms are thoseassociated with raised intracranial pressure, headaches and vomiting. The destruction of thecerebellum may lead to truncal ataxia and nystagmus. The disease will deteriorate whenspread to the spinal subarachnoid space. The survival rates have been improved by themulti-modality treatments, however, severe side effects always accompaniedneuro-endocrine late effects, cognitive defects，neuropsychological disorders and growthretardation. Hence，exploring the pathological mechanism involved in the MB will providenovel effective targets for this devastating disease.MicroRNAs (miRNAs) are short non-coding RNAs of~20–24nucleotides in length，which have critical regulatory functions in a variety of biological processes, such as cellproliferation, apoptosis, differentiation, development and tumorigenesis, by regulating theexpression of multiple target genes. MiRNA expression profile was analysized by Ferretti etal using qRT-PCR in34MB samples and14normal cerebellums, the miR-22located in the17p13.3was down-regulated in the profile, however, the17p loss is a frequent event in theMB, which implicates that miR-22plays an important role in the pathogenesis of MB. Inorder to throw a light on this hypothesis, we amplified our sample size, conducted thefunctional analysis, screened the novel targets and validated it, the role of miR-22involvedin the pathogenesis of MB has primarily been treated.METHODS:In part I,we detected the expression of miR-22by qRT-PCT in MB tissues、primarycultured cells, and MB cell lines. Fluoresence in situ hybridization (FISH) was carried out to explore the loss of heterozygosity (LOH) occurred in chromosome17p13.3.In part II, we over-or down-regulated the expression of miR-22using the lentivirus inMB cell lines, the cell proliferation was analysized by CCK-8assay, the apoptosis wasdetected by flow cytometric analysis. At the same time the biological function ofoverexpressed miR-22was studied in the tumor-bearing nude mice. We conducted themicroarray analysis using the MB cells with or without the overexpressed miR-22. Thenovel targets of miR-22was also validated by the luciferase reporter assay system.In part III, we explored the expression of PAPST1by qRT-PCT in MB tissues、primarycultured cells, and MB cell lines. The proliferation of MB cells was analysized by CCK-8assay after knocking down of PAPST1by lentivirus.RESULTS:1. The expression of miR-22in MB（1） The downexpression of miR-22was observed in70%(19/27) MB samples,3MB cell lines(less than20%of the level in the normal cerebellum).（2） The LOH of17p13.3was detected in19%of MB tissues by FISH analysis.2. The role of miR-22in the proliferation and apoptosis of MB cells（1）The overexpression of miR-22inhibited the proliferation of MB cells, howeverthe downregulation of miR-22increased the cell proliferation.（2）MiR-22overexpression induced the cell apoptosis in MB.（3）MiR-22overexpression induced the growth arrest and cell apoptosis in the nudemice.（4）The result of microarray analysis indicates that overexpressed miR-22down-regulated the expression of PAPST1, the luciferase assay confirmed that PAPST1is a noveltarget of miR-22.3. The expression of PAPST1in MB and its effects on MB cell proliferation（1）The overexpressed PAPST1was detected in67%(18/27) MB samples, and2MBcell lines (over2folds of the level in normal cerebellum).（2）The result of PAPST1immunohistochemistry showed cytoplasmic browngranules, no immunoreactivity was detected in a normal cerebellum analysed.（3）The knock-down expression of PAPST1reduced the cell proliferation in MB. CONCLUSIONS:MiR-22is frequently down-regulated in MBs, the exepression level of miR-22isnegatively correlated with the cell proliferation in vitro, the forced expression of miR-22can induce the cell apoptosis and tumor growth arrest in vitro and in vivo.PAPST1is frequently overexpressed in medulloblastomas, which is a novel target ofmiR-22, the downregulation of PAPST1decreases the cell proliferation in MB cell lines.