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Purpose: To better understand microRNA miR-21 function in carcinogenesis, we analyzed miR-21 expression patterns in different stages of colorectal cancer development using in situ hybridization (ISH). Experimental Design: Locked nucleic acid (LNA)/DNA probes and a biotin-free tyramide signal amplification system were used in ISH analyses of miRNA expression. Conditions for specific detection of miR-21 were determined using human cell lines and miR-21-expressing lentiviral vectors. Expression was determined in 39 surgically excised colorectal tumors and 34 endoscopically resected colorectal polyps. Results: In the surgical samples, miR-21 expression was much higher in colorectal cancers than in normal mucosa. Strong miR-21 expression was also observed in cancer-associated stromal fibroblasts, suggesting miR-21 induction by cancer-secreted cytokines. Protein expression of PDCD4, a miR-21 target, was inversely correlated with miR-21 expression, confirming that miR-21 is indeed a negative regulator of PDCD4 in vivo. In the endoscopic samples, miR-21 expression was very high in malignant adenocarcinomas but was not elevated in nontumorigenic polyps. Precancerous adenomas also frequently showed miR-21 up-regulation. Conclusion: Using the LNA-ISH system for miRNA detection, miR-21 was detectable in precancerous adenomas. The frequency and extent of miR-21 expression increased during the transition from precancerous colorectal adenoma to advanced carcinoma. Expression patterns of miR-21 RNA and its target, tumor suppressor protein PDCD4, were mutually exclusive. This pattern may have clinical application as a biomarker for colorectal cancer development and might be emphasized by self-reinforcing regulatory systems integrated with the miR-21 gene, which has been previously shown in cell culture.

Fos family proteins form stable heterodimers with Jun family proteins, and each heterodimer shows distinctive transactivating potential for regulating cellular growth, differentiation, and development via AP-1 binding sites. However, the molecular mechanism underlying dimer specificity and the molecules that facilitate transactivation remain undefined. Here, we show that BAF60a, a subunit of the SWI⅐SNF chromatin remodeling complex, is a determinant of the transactivation potential of Fos/Jun dimers. BAF60a binds to a specific subset of Fos/Jun heterodimers using two different interfaces for c-Fos and c-Jun, respectively. Only when the functional SWI⅐SNF complex is present, can c-Fos/c-Jun (high affinity to BAF60a) but not Fra-2/JunD (no affinity to BAF60a) induce the endogenous AP-1-regulated genes such as collagenase and c-met. These results indicate that a specific subset of Fos/Jun dimers recruits SWI⅐SNF complex via BAF60a to initiate transcription.Transcription factor AP-1, which plays pivotal roles in cell growth, differentiation, development, and tumor formation, is composed of Fos family proteins (Fos; c-Fos, Fra-1, Fra-2, and FosB) and Jun family proteins (Jun; c-Jun, JunB, and JunD). The members of both families form dimers through a leucine zipper structure; Jun family members can form low-affinity homodimers and high affinity heterodimers with the Fos family (1, 2). However, members of the Fos family do not form stable homodimers. Although these hetero-and homodimers bind to similar sites on DNA (TGAC/GTCA, AP-1 binding sites) through the basic domains of both proteins, each dimer has a distinct transcriptional regulatory function that can be modulated either positively or negatively (3). For example, transcriptional activity of the c-Fos/c-Jun dimer is much higher than the Fra-2/c-Jun dimer. Although functional interactions between some members of the Fos/Jun family of proteins and adaptor proteins such as CREB-binding protein (CBP) or TATA-binding protein (TBP) have been reported (4, 5), crucial proteins that recognize the dimer specificity and/or facilitate the induction of transcriptional initiation were largely unknown. Therefore, we proposed here to isolate such a crucial protein using a yeast two-hybrid system and have demonstrated that BAF60a (6), a component of the SWI⅐SNF chromatin remodeling complex, can select specific Fos/Jun dimers and function as a determinant of transcriptional activation via AP-1 binding sites. EXPERIMENTAL PROCEDURESPlasmid Construction-The Gal4DBD-c-Jun (25-187) fusion construct (pDBLeu-cJ(25-187)) was generated by inserting the 0.49 kilobase pair EheI fragment of the rat c-jun gene into the MscI restriction endonuclease cleavage site within the open reading frame of Gal4DBD. For the construction of template DNA for in vitro translation, we first generated a starter plasmid from pGEM2-475/Jun-D, the translation initiation site of which has a Kozak consensus sequence with a unique NcoI site that is preceded by a fragment 475 sequence for an efficient translational ...