Figure S1. The promoter CpG island of TRPM3/miR-204 is hypermethylation in CRC cells. A, Target sequences in the promote CpG islands of miR-638 gene used for the DNA methylation analysis. The underlined sequences are regions corresponding to the primers of bisulfite-sequencing PCR (BSP) used. B, Schematic representation of the methylation status of the CpG island (including 42 CpG sites) in the TRPM3 promoter as detected in the indicated CRC cell lines. Circles mark different CpG sites in the analyzed sequence. C, The relative mRNA levels of TRPM3 in CRC cell lines. Normal, a pooled sample of noncancerous tissues (NCTs). D, The relative expression of miR-204-5p in CRC cell lines. E, The TRPM3 mRNA levels in CRC cell lines were positively correlated with those of miR-204-5p. F, Quantitative analysis of miR-204-5p expression in CRC cells treated with DNA methyltransferase inhibitor (5-aza-dC). Following treatment of HCT116 and LoVo cells with 5-aza-dC for 72 hours, the miR-204-5p expression in the treated CRC cells was significantly increased compared with untreated control cells (***P < 0.001).
ARTICLE ABSTRACT
Purpose: miR-204-5p was found to be downregulated in colorectal cancer tissues in our preliminary microarray analyses. However, the function of miR-204-5p in colorectal cancer remains unknown. We therefore investigated the role, mechanism, and clinical significance of miR-204-5p in colorectal cancer development and progression.Experimental Design: We measured the expression of miR-204-5p and determined its correlation with patient prognoses. Ectopic expression in colorectal cancer cells, xenografts, and pulmonary metastasis models was used to evaluate the effects of miR-204-5p on proliferation, migration, and chemotherapy sensitivity. Luciferase assay and Western blotting were performed to validate the potential targets of miR-204-5p after the preliminary screening by a microarray analysis and computer-aided algorithms.Results: miR-204-5p is frequently downregulated in colorectal cancer tissues, and survival analysis showed that the downregulation of miR-204-5p in colorectal cancer was associated with poor prognoses. Ectopic miR-204-5p expression repressed colorectal cancer cell growth both in vitro and in vivo. Moreover, restoring miR-204-5p expression inhibited colorectal cancer migration and invasion and promoted tumor sensitivity to chemotherapy. Mechanistic investigations revealed that RAB22A, a member of the RAS oncogene family, is a direct functional target of miR-204-5p in colorectal cancer. Furthermore, RAB22A protein levels in colorectal cancer tissues were frequently increased and negatively associated with miR-204-5p levels and survival time.Conclusions: Our results demonstrate for the first time that miR-204-5p acts as a tumor suppressor in colorectal cancer through inhibiting RAB22A and reveal RAB22A to be a new oncogene and prognostic factor for colorectal cancer. Clin Cancer Res; 20(23); 6187–99. ©2014 AACR.
