Abstract
BackgroundGain-of-function mutations and overexpression of KIT are characteristic features of gastrointestinal stromal tumor (GIST). Dysregulation in miRNA expression may lead to KIT overexpression and tumorigenesis.MethodsmiRNA microarray analysis and real-time PCR were used to determine the miRNA expression profiles in a cohort of 69 clinical samples including 50 CD117IHC+/KITmutation GISTs and 19 CD117IHC−/wild-type GISTs. GO enrichment and KEGG pathway analyses were performed to reveal the predicted targets of the dysregulated miRNAs. Of the dysregulated miRNAs whose expression was inversely correlated with that of KIT miRNAs were predicted by bioinformatics analysis and confirmed by luciferase reporter assay. Cell counting kit-8 (CCK-8) and flow cytometry were used to measure the cell proliferation, cycle arrest and apoptosis. Wound healing and transwell assays were used to evaluate migration and invasion. A xenograft BALB/c nude mouse model was applied to investigate the tumorigenesis in vivo. Western blot and qRT-PCR were used to investigate the protein and mRNA levels of KIT and its downstream effectors including ERK, AKT and STAT3.ResultsOf the six miRNAs whose expression was inversely correlated with that of KIT, we found that miR-148b-3p was significantly downregulated in the CD117IHC+/KITmutation GIST cohort. This miRNA was subsequently found to inhibit proliferation, migration and invasion of GIST882 cells. Mechanistically, miR-148b-3p was shown to regulate KIT expression through directly binding to the 3’-UTR of the KIT mRNA. Restoration of miR-148b-3p expression in GIST882 cells led to reduced expression of KIT and the downstream effectors proteins ERK, AKT and STAT3. However, overexpression of KIT reversed the inhibitory effect of miR-148b-3p on cell proliferation, migration and invasion. Furthermore, we found that reduced miR-148b-3p expression correlated with poor overall survival (OS) and disease-free survival (DFS) in GIST patients.ConclusionmiR-148b-3p functions as an important regulator of KIT expression and a potential prognostic biomarker for GISTs.
Highlights
Gain-of-function mutations and overexpression of KIT are characteristic features of gastrointestinal stromal tumor (GIST)
Among these miRNAs, miR122-3p, miR-483-3p, miR-101-3p, miR-598 and miR4492 were significantly upregulated, whereas miR-140-5p, miR-148b-3p, miR-1587 and miR-4507 were significantly downregulated in the CD117IHC+/KITmutation GISTs
MiR-148b-3p overexpression did not affect cell cycle arrest and apoptosis in GIST882 cells either alone or in combination with imatinib treatment (Fig. 5c-d). These results indicated that miR-148b-3p acts synergistically with imatinib to suppress cell proliferation and sensitizes GIST882 cells to imatinib treatment
Summary
Gain-of-function mutations and overexpression of KIT are characteristic features of gastrointestinal stromal tumor (GIST). Dysregulation in miRNA expression may lead to KIT overexpression and tumorigenesis. Recent studies have revealed that wt GISTs represent a heterogeneous group in clinical, pathological and biological features that is profoundly different from KIT/ PDGFRA mutant tumors [5]. Several studies have explored the function of miRNAs in the tumorigenesis of GISTs, demonstrating the involvement of miRNA dysregulation in gene mutation, chromosomal changes, tumor risk and metastasis [12,13,14,15]. Since activating mutation in the KIT gene is the main oncogenic trigger in > 80% of GISTs, the aberrant expression of miRNAs that interrelates with KIT-induced tumorigenesis is of particular interest [13]
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