hsa-miR-29c* is linked to the prognosis of malignant pleural mesothelioma

Abstract

The inability to forecast outcomes for malignant mesothelioma prevents clinicians from providing aggressive multimodality therapy to the most appropriate individuals who may benefit from such an approach. We investigated whether specific microRNAs (miR) could segregate a largely surgically treated group of mesotheliomas into good or bad prognosis categories. A training set of 44 and a test set of 98 mesothelioma tumors were analyzed by a custom miR platform, along with 9 mesothelioma cell lines and 3 normal mesothelial lines. Functional implications as well as downstream targets of potential prognostic miRs were investigated. In both the training and test sets, hsa-miR-29c* was an independent prognostic factor for time to progression as well as survival after surgical cytoreduction. The miR was expressed at higher levels in epithelial mesothelioma, and the level of this miR could segregate patients with this histology into groups with differing prognosis. Increased expression of hsa-miR-29c* predicted a more favorable prognosis, and overexpression of the miR in mesothelioma cell lines resulted in significantly decreased proliferation, migration, invasion, and colony formation. Moreover, major epigenetic regulation of mesothelioma is mediated by hsa-miR-29c* and was shown through downregulation of DNA methyltransferases as well as upregulation of demethylating genes. A single miR has the potential to be a prognostic biomarker in mesothelioma, and validation of these findings as well as investigation of its downstream targets may give insight for potential therapies in the future.

Figure 1

Elevated hsa-miR-29c* was associated with a significantly longer time to progression and survival time (A) Training set: time to progression (B) Training set: survival time. (C) Test set: time to progression (D) Test set: survival time

Figure 2

Histology and mesothelioma prognosis influenced by mir-29c*. (A) hsa-miR-29c* was differentially expressed between epithelial and biphasic or sarcomatoid cases with higher levels in the former (B) hsa-mir-29c* could subclassify epithelial mesotheliomas into those with short versus long time to progression. (C) hsa-mir-29c* could subclassify epithelial mesotheliomas into those with short versus long survival times.

Figure 3

Validation of prognostic potential of hsa-miR-29c* using qRT-PCR (A) left panel reveals microarray expression values for hsa-mir-29c* in 16 mesotheliomas compared to expression measured by qRT-PCR (right panel). In both cases, elevated hsa-mir-29c* was associated with good prognosis. (B) qRT-PCR for hsa-mir-29c* separating the validation individuals significantly by their survival at a cut-off comparable to the consistent cutoff used for the microarray data.

Figure 4

Functional consequences of hsa-miR-29c* overexpression in mesothelioma cell lines compared to lipofectamine alone or irrelevant (scrambled) miR. (A) hsa-miR-29c* overexpression resulted in a significant decrease in cell proliferation (B) hsa-miR-29c* overexpression resulted in a significant decrease in cellular migration (C) hsa-miR-29c* overexpression resulted in a significant decrease in invasion (D) hsa-miR-29c* overexpression resulted in a significant decrease in soft agar colony formation

Figure 5

hsa-miR-29c* regulates epigenetic gene expression in mesothelioma (A) DNA methyltransferase 1 expression measured by U133Plus 2 Affymetrix microarray platform is elevated in mesothelioma compared to normal mesothelium from the peritoneum (B) RT-PCR validates overexpression of all of the DNMTs in mesothelioma compared to matching normal peritoneum. 18/21 specimen revealed overexpression of a DNA methyltransferase in the tumor. (C) Transfection of hsa-miR-29c* resulted in decreased expression of all DNA methyltransferases in the two cell lines.. H2595 did not have expression of DNMT3B (D) Transfection of hsa-miR-29c* upregulated Adiponectin, C1QTNF1 and C1QTNF8, three genes associated with active gene demethylation. C1QTNF8 was thus validated as a target of hsa-miR-29c*.