• EGFL7
• FFPE tissues
• malignant pleural mesothelioma
• miR-126
• MSP
• Pyrosequencing
• Real time PCR

Many studies have shown that aberrant expression of microRNAs (miRNAs) is involved in the initiation and progression of cancer, and several miRNAs have shown tumor suppressor or oncogenic functions. Restoring the expression of tumor suppressor genes by epigenetic therapy has great potential in cancer treatment and it has been shown that the expression of some miRNAs in cancer cells can be directly regulated by epigenetic changes in their own promoters. However, the majority of miRNAs are located within intronic regions of transcription units and it remains unclear if intronic miRNAs can also be epigenetically regulated. The miR-126 is located within intron 7 of the proangiogenic Epidermal Growth Factor-like domain 7 (EGFL7) gene that is highly expressed in endothelial cells and vascularized tissues. MiR-126 acts as a tumor suppressor gene in certain types of cancers while other reports have described an oncogenic role. These contradictory findings suggest that miR-126 may have several functions specific to each type of malignancy. Mature miR-126 can be generated from three different transcripts of EGFL7 with each one having its own promoter with CpG island, and different studies have shown expression correlation between the miR-126 and its host gene.

Since the Laboratory of Mol. Pathol. has recently found miR-126 down-regulation in Malignant Pleural Mesothelioma (MPM), an aggressive cancer originating from the mesothelial cells lining the pleura, in my study, we used Real-time quantitative RT-PCR (RT-qPCR) to correlate the expression of miR-126 with one of EGFL7 transcripts in patient-matched preoperative diagnostic pleura biopsies and surgical tissue specimens of MPM as well as corresponding non-neoplastic pleura (NNP). Patients diagnosed with MPM typically have a history of long-term exposure to asbestos and poor prognosis with a median survival of 12 months from the time of diagnosis. Our attention was placed also on studying chromatin structural changes, such as DNA methylation, which are important in epigenetic research and clinical diagnostics because the aberrant methylation has been associated with transcriptional inactivation of defined tumor suppressor genes in human cancers. To adress this issue we have used two different methods: Methylation Sensitive PCR (MSP) and pyrosequencing.

The results of RT-qPCR show that the primary transcript of miR-126 corresponds to an alternative transcript of EGFL7 (S2) with a CpG island promoter and that miR-126 and S2 are concomitantly downregulated in MPM as compared to NNP. Our MSP and pyrosequensing data suggest that this could be due to different regulation by DNA methylation of the CpG island in the S2 promoter in MPM and NNP, as MPM shows a greater level of methylation than NNP.
This work has improved our comprehension of the mechanism by which miR-126 is regulated in MPM. It would be interesting in the future to analyze histone modifications in chromatin structure to confirm the importance of the epigenetic mechanisms regulating miRNA expression in MPM.