Screening and Functional Study of the Genes inducing Malignant Degeneration of Neurofibromatosis Type 1

Neurofibromatosis type 1(NF1) is one of the most common inherited autosomal dominant disorders, with an estimated incidence of 1 per 3,500 births. NF1 is caused by mutations in the NF1 gene which consists of 57 exons and encodes a GTPase activating protein(G AP), neurofibromin. NF1 is clinically characterized by cafe-au-lait(CAL) spots, neurofibromas, freckling of the axillary or inguinal region, Lisch nodules, optic nerve glioma, and bone dysplasias. NF1 is notable for the existence of a gradual malignant degeneration of normal and/or benign tumor cells. Three pathological phenotypes of fibroblast cells, normal, benign and malignant, are exist within an individual despite they bare an identical germ line mutation. The influence of tumor-related genes such as TP53, NK4, and ARF, and the impact of stochastic events have been suggested as causes of this variability. However, there has been no clear evidence to clarify the cell-fate decision factor in NF1. For whole gene expression comparison among normal fibroblasts, benign and malignant tumor cells, we used ACP(annealing controlled primer) based PCR method, GeneFishing DEG(differentially expressed gene) screening, which substantially improves the specificity and sensitivity of PCR and eliminates the false positives and poor reproducibility of previous DEG discovery methods such as cDNA microarray. In this study, we compared mRNA expression levels among cultured cells from the pathologically normal, benign, and malignant tissues of a Korean patient with NF1. Using 120 sets of ACP primers, we found total 64 DEGs that include 37 DEGs showing up-regulation in malignant cells and 27 DEGs showing down-regulation in malignant cells. To confirm the existence of genetic alterations in NF1 tumor cells, we carried out CGH(comparative genomic hybridization) array in NF1 cells, but no genetic alterations have been found in the NF1 cells. We have cloned and sequenced 20 DEGs that showed clear differences in expression pattern among cell types. We performed a validation study on the 8 DEGs by RT-PCR using gene-specific primers and then by Western blot analysis. Finally we selected 3 genes, TAGLN, SERPINE1, and IFITM1, as the key gene(s) involved in malignant degeneration of NF1 in this patient. Two genes, TAGLN and SERPINE1, showed up-regulation of expression in the tumor cells, while IFITM1 showed down-regulation of expression in the tumor cells. In order to clarify the cellular functions of theses 3 genes in the malignant NF1 cells, we have used RNA interference(RNAi) for inhibition of gene expression, and vector-mediated gene expression for induction of gene expression. Proliferation rate of the malignant NF1 cells was reduced by RNAi for both TAGLN and SERPINE1 genes and by over-expression of IFITM1. Finally, mitochondria fragmentation and cell death was observed in the malignant NF1 cells, by over-expression of IFITM1. A further functional analysis of them may lead to a better understanding the mechanisms of tumoregenesis and malignant degeneration of NF1.

Author dc:creator

• 박, 용예

Contributors dc:contributor

• 김, 현주
• 대학원 의학과
• 106506

• NF1
• neurofibromin
• DEG
• RNAi
• TAGLN

Language dc:language

ko

Identifier

http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000002121
000000002121

OAI identifier oai:identifier

oai:repository.ajou.ac.kr:201003/1651