Human microRNA expression in sporadic and FAP-associated desmoid tumors and correlation with beta-catenin mutations

Abstract

Desmoid tumors (DT) are rare, benign, fibroblastic neoplasm with challenging histological diagnosis. DTs can occur sporadically or associated with the familial adenomatous polyposis coli (FAP). Most sporadic DTs are associated with β-catenin gene (CTNNB1) mutations, while mutated APC gene causes FAP disease. microRNAs (miRNAs) are involved in many human carcinogenesis.The miRNA profile was analyzed by microarray in formalin-fixed, paraffin-embedded (FFPE) specimens of 12 patients (8 sporadic, 4 FAP-associated) and 4 healthy controls. One hundred and one mRNAs resulted dysregulated, of which 98 in sporadic DTs and 8 in FAP-associated DTs, 5 were shared by both tumors. Twenty-six miRNAs were then validated by RT-qPCR in 23 sporadic and 7 FAP-associated DT samples matched with healthy controls. The qPCR method was also used to evaluate the CTNNB1 mutational status in sporadic DTs. The correlation between sporadic DTs and miRNA expression showed that miR-21-3p increased in mutated versus wild-type DTs, while miR-197-3p was decreased. The mRNA expression of Tetraspanin3 and Serpin family A member 3, as miR-21-3p targets, and L1 Cell Adhesion Molecule, as miR-197-3p target, was also evaluate. CTNNB1 mutations associated to miRNA dysregulation could affect the genesis and the progression of this disease and help histological diagnosis of sporadic DTs.

Keywords: CTNNB1 mutations; desmoid tumor; microRNA; serpin family A member 3 (SERPINA3) mRNA; tetraspanin3 (TSPAN3) mRNA.

Figure 1. Validation of microarray analysis

Comparison between microarray (mean) and RT-qPCR (mean ± SD) data (A) Comparison of the miRNA profiling in sporadic desmoid tumors. (B) Comparison of the miRNA profiling in FAP-associated desmoid tumors. The Y axis shows the fold change values between tumor samples and controls obtained by microarray and RT-qPCR experiments, respectively.

Figure 2. miR-21-3p and mR-197-3p expression in sporadic desmoids with and without CNTTB1 gene mutations

Of 26 patients with sporadic desmoids, 14 showed the gene mutated (M), while the remaining 12 had the wild type gene (Wt). The scatter dot blot graph shows the distribution of each miRNA relative to CNTTB1 mutational status. The miRNA levels, generated by qRT-PCR, are expressed as log2 (2^−ΔΔCt) that represents the fold change values of tumor vs. control samples. Differences between two M and Wt groups were evaluated by nonparametric Mann-Whitney test with significant value of p < 0.05.

Figure 3. Expression of the miR-21-3p and miR-197-3p mRNA targets

miR-21-3p promotes the L1CAM expression, while the negative regulation for COL1A1 and MAT2A is not observed in mutated (M) vs. wild type (Wt) sporadic desmoid tumors. Decreased levels of miR-197-3p induce a TSPAN3 and SEPINA3 mRNA over-expression in M vs. Wt sporadic tumors. The gene expression was quantified using RT-qPCR. Bars represent the mean ± SD and the values are significant for p < 0.05.