Reconstituted TGFBR2 expression alters the microRNA profiles of extracellular vesicles and parental colorectal cancer cells

Background: MicroRNAs (miRNAs) are important posttranscriptional modulators of gene expression that impact various biological and pathological processes, including colorectal carcinogenesis. More than 90 % of microsatellite unstable (MSI) colorectal cancers (CRC) are affected by inactivating frameshift mutations of Transforming Growth Factor Beta Receptor Type 2 (TGFBR2), leading to abrogated TGF-ß signaling and tumor progression. TGF-ß signaling can alter the expression of miRNAs. Functional miRNAs have been found in the cargo of small extracellular vesicles (EVs). In the present study, we sought to determine whether TGFBR2 expression and downstream signaling alter the miRNA profile of EVs derived from MSI tumor cells. Methods: The MSI cell line HCT116-TGFBR2 was used as a model system that enables doxycycline-inducible reconstituted expression of TGFBR2. RNA was extracted from four biological replicates of TGFBR2-proficient and TGFBR2-deficient cells and their EVs. cDNA libraries were generated and analyzed by small RNA sequencing. TGFBR2-dependent differential miRNA expression was assessed by DESeq2. Results: Exploratory data analysis of identified miRNA profiles revealed clear cluster separation between small EVs and parental MSI tumor cells in a TGFBR2-dependent manner. Most miRNAs (263/471; 56 %) remained unaffected by TGFBR2 expression and are shared between EVs and parental cells. Differential expression analysis identified TGFBR2-regulated miRNA candidates in EVs (n=10) and MSI cells (n=15). Among all differentially expressed candidates, miR-381-3p, -323a-3p, and -889-3p were found to be downregulated in both MSI cells lacking TGFBR2 expression and small EVs derived thereof. From these shared TGFBR2-dependent candidates, miR-381 and two additional TGFBR2-regulated miRNAs were validated by RT-qPCR. Conclusions: The present study emphasizes the general overlap of miRNA profiles in MSI tumor cells and their small EVs, but also highlights the impact of the MSI tumor driver mutation affecting TGFBR2 on altering miRNA expression as exemplified by the downregulation of miR-381-3p in MSI tumor cells that lost TGFBR2 expression and their small EVs.