Periostin is a mesenchyme specific cell adhesion molecule, which is mainly expressed in periosteum, periodontal ligaments and placenta. Periostin also has a potential role in tooth and bone development and tissue repair. Recent studies have revealed that periostin plays a crucial role in the epithelial to mesenchymal transition (EMT). Moreover, overexpression of periostin has been reported in several types of human cancer, including breast cancer and is associated with enhanced tumour growth and angiogenesis. Periostin was found to be highly expressed in Hs578T cells, a highly metastatic triple-negative breast cancer cell line, but not in normal breast tissues or other cancer cell lines (e.g. HeLa).
In the initial part of this study, the hypothesis that miR-30e-5p might regulate gene expression in Hs578T cell line was investigated. This was done by using high-throughput assays, including RNA-seq and microarrays followed by validation with qPCR. Results demonstrated changes in the levels of hundreds of transcripts. Several target genes were identified and verified that might have a role in cancer progression. However, our result shows that POSTN mRNA level was not affected by miR-30e despite having a predicted miR-30e-5p site in its 3'UTR.
An alternative hypothesis was that the POSTN promoter is unregulated, by mutation or transcription factors in Hs578T cells. This was investigated in both Hs578T and HeLa (control) cell lines by using luciferase reporter assays. Different fragments of the human POSTN promoter (1 kb, 2 kb and 3 kb) were amplified from Hs578T and HeLa genomic DNA and were used to direct the synthesis of a luciferase reporter. From the sequencing data, a few mutations in the POSTN promoter region of Hs578T and HeLa were observed. However, these mutations had no substantial effect in modulating the POSTN promoter activity in both of these cell lines used in the study. Unexpectedly, the POSTN promoter activity in HeLa cells was observed to be significantly high compared to Hs578T cells. Finally, the copy number variations of POSTN gene in Hs578T cell line were analysed using the Cancer Cell Line Encyclopaedia (CCLE) and was observed to be highly amplified in this cell line. In conclusion, the results presented here show that the POSTN gene is amplified in Hs578T cells, which suggests a cause for periostin overexpression in these cells.
