Screening And Function Of MicroRNA In Gastrointestinal Stromal Tumors

Gastrointestinal stromal tumors(GISTs)represent the most common mesenchymal tumors of the gastrointestinal(GI)tract,which accounts for approximately 1 to 4%of gastrointestinal tumors.Activating mutations in either the KIT or PDGFRA gene are the principal oncogenic triggers with the former accounting for more than 80%of cases.These mutually exclusive mutations cause ligand independent auto-phosphorylation of the type III tyrosine kinase receptors,activating crucial growth and survival signalling cascades.MicroRNAs(miRNAs)are a family of 21-25-nucleotide small RNAs that negatively regulate gene expression at the post-transcriptional level.The functions of miRNAs are not limited to the regulation of developmentally timed events.Instead,they have diverse expression patterns and probably regulate many aspects of development and physiology.Recently,miRNA have been found to be involved in the regulation of multiple aspects of tumor development and progression.To date,however,only a few groups have studied microRNA(miRNA)expression in GISTs.Therefore,the research of global pattern of miRNA expression in GISTs may contribute to detect the molecular mechanisms in the development of GISTs and it could be considered as a tool for future therapeutic strategies for GISTs.This study is divided into two parts.Part ?:KIT and PDGFRA mutations in gastrointestinal stromal tumorsObjective:To investigate the mutation of KIT and PDGFRA gene in gastroiniestinal stromal tumors and their possible relevance with clinical pathologic characteristies and prognosis.Methods:85 cases of formalin-fixed paraffin-embedded(FFPE)samples of GISTs were selected and Genomic DNAs were isolated routinely.PCR amplification and direct sequencing were performed to determine the mutant sites of KIT or PDGFRA gene in the CD 117-positive GISTs and CD 117-negative GISTs.The relationship between the clinicalpathologic characteristics and gene mutation were also investigated.Results:Among 85 cases of GISTs,the mutations of KIT gene were indentified in 61.2%patients as follows:78.8%in exon 11,7.3%in exon 9,3.8%in exon 13 and none in exon 17.50 cases of C-KIT gene mutation were occurred in 60 cases of CD 117-positive GISTs,except 2 cases occurred in CD 117-negative GISTs.PDGFRA mutations were indentified in 4.7%of all the 85 cases and 16%in CD 117-negative GISTs with none of the positive cases expressed detectable CD117 protein,which almost involved in exon 18 with mutations D842V.Morphorlogically,75%of PDGFRA mutations present in epithelioid or mixed phenotype GISTs with low degree of risk and better prognosis.C-KIT oncogenic mutations are genegrally detected in the maiority of spindle phenotype GISTs.In contrast to PDGFRA mutations,the mutations of C-KIT gene in GISTs showed a significant correlation with histological type,tumor recurrence and metastasis.The exon 11 mutations of C-KIT gene in GISTs demonstrated a significant relationship with primary tumor site and histological type.The GISTs with exon 11 deletion mutation showed a high degree of malignancy and poor prognosis in our study.Conclusions:C-KIT oncogenic mutations are mostly detected in the maiority of spindle phenotype GISTs,while PDGFRA oncogenic mutations are most likely existed in epithelioid or mixed phenotype GISTs.The mutations of C-KIT gene in GISTs showed a significant correlation with histological type,tumor recurrence and metastasis.The deletion mutation of exon 11 showed a high degree of malignancy and a poor prognosis.Part ?:MiRNA expression profiles in gastrointestinal stromal tumorsObjective:By comparing the globe expression profiles of microRNA(miRNA)between the CD 117-positive/C-KIT mutation type GISTs and CD 117-negative/wild-type GISTs to identify the miRNA possibly associated with the expression and mutation of C-KIT gene in the development of GISTs.Methods:MiRNA expression profiles were performed in 5 cases of CD117-positive/C-KIT mutation type GISTs and 5 cases of CD117-negative/wild type GISTs using miRNA microarray.Furthermore,some of the differentially expressed miRNAs were chosen to determine in 45 cases of CD117-positive/C-KIT mutation type GISTs and 14 cases of CD117-negative/wild type GISTs by real-time RT-PCR analysis.Then the aberrant expressed miRNAs were evaluated for possible target genes using bioinformatic tools.The known and predicted target genes were mapped to signaling pathway of tumorigenesis and progession using GO and KEGG database.Results:As demonstrated in miRNA microarray,ten differentially expressed miRNAs were found between CD117-positive/KIT mutation type group and CD 117-negative/wild-type group.Of which,6 miRNAs including miR-140-5p,miR-598,miR-122-3p,miR-101-3p,miR-148b-3p and miR-483-3p were significantly up-regulated,while miR-1587,miR-4492,miR-4507 and miR-6510-5p were significantly down-regulated in CD 117-positive group.Among them,six differentially expressed miRNAs were confirmed with good concordance between qRT-PCR and miRNA microarray,which including three up-regulated miRNAs(miR-140-5p,miR-148b-3p and miR-483-3p)and three down-regulated miRNAs(miR-1587,miR-4507and miR-6510-5p).Furthermore,miR-140-5p and miR-148b-3p were found significantlly correlated with recurrence and metastasis of GISTs(p<0.05),but not with age,agenda,sizes of tumor,histological type,primary tumor site,as well as risk stratification.The expression of miR-140-5p,miR-483-3p were found to be increased gradually with the increasing of risk stratification in CD 117-positive group.However,the expression of miR-148b-3p and miR-1587 was found to be decreased gradually with the increasing of risk stratification.The results of bioinformatic study demonstrated that the differential expression of miR-148b-3p and miR-140-5p may be contributed to the malignant progression in GISTs.Their target genes included:KIT,PDGFRA,MAPK,SMAD,VEGF and PTEN.Differential expression of miR-140-5p and miR-483-3p may contribute to the imatinib-resistance in GISTs.Their target genes included:ABCB?BCL2.Conclusions:Six differentially expressed miRNAs were confirmed with good concordance between qRT-PCR and miRNA microarray in CD 117-positive group.The expression level of miR-140-5p,miR-483-3p and miR-148b-3p were correlated with the biological behavior and prognosis of GISTs.The differential expression of miR-148b-3p and miR-140-5p may be involved in the KIT/PDGFRA signal transduction and contribute to the malignant progression in GISTs,while the differential expression of miR-140-5p and miR-483-3p may contribute to the imatinib-resistance in GISTs.Conclusion:Taken together,we propose that activating mutations in either the KIT or PDGFRA gene are the most important molecular events in GISTs.The differential expression of miR-148b-3p and miR-140-5p may be involved in the KIT/PDGFRA signal transduction and contribute to the malignant progression in GISTs.Further study was needed to clarify the molecular mechanism.