Increased microRNA-34b and -34c predominantly expressed in stromal tissues is associated with poor prognosis in human colon cancer

Abstract

The microRNA-34 family (miR-34a, -34b and -34c) have been reported to be tumor suppressor microRNAs (miRNAs) that are regulated by the TP53 and DNA hypermethylation. However, the expression, regulation, and prognostic value of the miR-34 family have not been systematically studied in colon cancer. To elucidate the roles of miR-34 family in colon carcinogenesis, miR-34a/b/c were measured in tumors and adjacent noncancerous tissues from 159 American and 113 Chinese colon cancer patients using quantitative RT-PCR, and we examined associations between miR-34a/b/c expression with TNM staging, cancer-specific mortality, TP53 mutation status and Affymetrix microarray data. All miR-34 family members were significantly increased in colon tumors, counter to the proposed tumor suppressor role for these miRNAs. Increased miR-34b/c were observed in more advanced tumors in two independent cohorts and increased expression of miR-34b/c was associated with poor cancer-specific mortality. While the expression of miR-34 family was not associated with TP53 mutation status, TP53 transcriptional activity was associated with miR-34a/b/c expression that is consistent with the proposed regulation of miR-34a/b/c by TP53. To examine where the miR-34 family is expressed, the expression of miR-34 family was compared between epitheliums and stromal tissues using laser microdissection technique. The expression of miR-34b/c was increased significantly in stromal tissues, especially in cancer stroma, compared with epithelial tissue. In conclusion, increased miR-34b/c predominantly expressed in stromal tissues is associated with poor prognosis in colon cancer. MiR-34 may contribute to cancer-stromal interaction associated with colon cancer progression.

Fig 1. The miR-34 family members significantly increase in colon tumors compared to adjacent non-tumor tissues.

(A, B) Dot plots represent miR-34a/b/c threshold cycle values from TaqMan qRT-PCR normalized to U66 in American cohort (A) and Chinese cohort (B). Horizontal bars indicate median expression value. Wilcoxon matched-pairs test was used. MiR-34a was not detectable in one patient and this patient was excluded from all analyses.

Fig 2. The high expression of miR-34b/c is associated with advanced colon tumor and poor prognosis.

(A, B) Tissues were ordered from TNM stage I to IV tumors in the American cohort (A) and the Chinese cohort (B). Dot plots represent miR-34a/b/c threshold cycle values from TaqMan qRT-PCR normalized to U66. Horizontal bars indicate median expression value. The Cuzick nonparametric test for trend was used to evaluate trends. (C) Kaplan-Meier survival analysis of all stage cases in the American and Chinese combined cohorts stratified by median miR-34a expression and combined miR-34b/c expression. *Cases with low miR-34b and/or low miR-34c. **Cases with both high miR-34b and high miR-34c.

Fig 3. TP53 mutation is not associated with the expression of miR-34 family.

(A, B) TP53 mutational status was evaluated by immunohistochemistry using Formalin-Fixed Paraffin-Embedded (FFPE) tissues. Dot plots represent miR-34a/b/c threshold cycle values of tumor tissue from TaqMan qRT-PCR normalized to U66 in American cohort (A) and Chinese cohort (B). Horizontal bars indicate median expression value. Mann-Whitney test was used. WT: wild-type p53, Mut: mutant p53.

Fig 4. miR-34 family tends to be expressed predominantly in stromal tissue.

RNA samples from cancer epithelium, cancer stroma, normal adjacent epithelium and normal adjacent stroma were extracted separately using laser microdissection technique from 5 paired samples in the American cohort. Dot plots represent miR-34a/b/c threshold cycle values of tumor tissue from TaqMan qRT-PCR normalized to U48. Horizontal bars indicate median expression value. Un-paired t test was used.