Ll lines using the Invitrogen SYBR green qRT-PCR kit. Untransfected cells were also included in the analysis (WT). With down-regulated miR-21 in both MCF-7 and Hs578T cells, MSH2 and SMAD7 mRNA expression was up-regulated by ,1.67 and ,3.6 fold, respectively (Fig. 6A and 6B), while the protein level was increased by ,35?3 for MSH2 and ,80?33 for SMAD7 (Fig. 6C). doi:10.1371/journal.pone.0054213.gthat miR-200 members enhance cell colonization to form distant metastases [40]. Additionally, miR-200c was actually observed to be up-regulated to stimulate proliferation 11967625 in human pancreatic cancer [26]. The function of miR-200 family remains to be elucidated in pre-invasive breast cancer. Similarly, another well studied miRNA, miR-21 was observed to have displayed an increasing expression trend during breast cancer progression. Target prediction and pathway analysis on the potential downstream targets of miR-21 indicated that miR-21 might promote tumor progression by targeting the TGF-b pathway. MiR-21 can regulate the TGF-b pathway by silencing its inhibitors, such as SMAD7. An up-regulated TGF-b pathway can expedite the generation of mature miR-21 in a feed-forward manner [41,42]. The nature of TGF-b signaling is controversial, as it can play both a tumor suppressive role by inhibiting cell proliferation and inducing apoptosis in normal epithelial cells, as well as a more aggressive role by promoting tumor growth andinvasion. Active TGF-b pathway correlates with poor prognostic and survival rates of breast cancer in the clinic, while suppression of the TGF-b pathway is also reported to be lethal for mice. Therefore it is important to develop strategies to selectively block the cancer-promoting Title Loaded From File branch but maintain the anti-mitogenic branch of the TGF-b pathway for developing therapeutic drugs. It was recently reported that miR-21 mediates TGF-b bidirectional regulation on MSH2, a central component of DNA mismatch repair (MMR), to contribute chemo-resistance in breast cancer [30]. In the normal cells with intact p53 function and a lower level of miR-21, TGF-b predominantly promotes MSH2 expression, contributing to DNA repair and maintenance of genomic stability. On the other hand, overexpression of miR-21 is often coupled with p53 inactivation in cancerous context, and silences MSH2 by directly binding on its 39-UTR, resulting in genomic instability and resistance to DNA-damaging chemotherapy agents. MiR-Deregulated miRNAs in Breast Cancermight be a mediator for the TGF-b pathway and thus can be a potential target for breast cancer therapy. In conclusion, deregulation of miRNA expression during tumorigenesis might be an early event as it Title Loaded From File occurs significantly during normal to ADH transition. Target prediction and pathway analysis revealed that miR-21 has a pivotal role on selective utilization of the TGF-b pathway in breast cancer initiation. Importantly, we have demonstrated the feasibility of miRNA expression profiling analysis using archived FFPE tissues, rich with clinical information, as a means towards miRNA biomarker discovery.Feature Extraction and Pre-processing of miRNA Microarray DataProbe level data was extracted from the microarray image by using the Agilent Feature Extraction Software (v10.5). QC reports were automatically generated for each array. All the raw data from Feature Extraction were logarithmically transformed to base 2, with quantile algorithm normalization as described [45].Statistical AnalysisFor the microdissected.Ll lines using the Invitrogen SYBR green qRT-PCR kit. Untransfected cells were also included in the analysis (WT). With down-regulated miR-21 in both MCF-7 and Hs578T cells, MSH2 and SMAD7 mRNA expression was up-regulated by ,1.67 and ,3.6 fold, respectively (Fig. 6A and 6B), while the protein level was increased by ,35?3 for MSH2 and ,80?33 for SMAD7 (Fig. 6C). doi:10.1371/journal.pone.0054213.gthat miR-200 members enhance cell colonization to form distant metastases [40]. Additionally, miR-200c was actually observed to be up-regulated to stimulate proliferation 11967625 in human pancreatic cancer [26]. The function of miR-200 family remains to be elucidated in pre-invasive breast cancer. Similarly, another well studied miRNA, miR-21 was observed to have displayed an increasing expression trend during breast cancer progression. Target prediction and pathway analysis on the potential downstream targets of miR-21 indicated that miR-21 might promote tumor progression by targeting the TGF-b pathway. MiR-21 can regulate the TGF-b pathway by silencing its inhibitors, such as SMAD7. An up-regulated TGF-b pathway can expedite the generation of mature miR-21 in a feed-forward manner [41,42]. The nature of TGF-b signaling is controversial, as it can play both a tumor suppressive role by inhibiting cell proliferation and inducing apoptosis in normal epithelial cells, as well as a more aggressive role by promoting tumor growth andinvasion. Active TGF-b pathway correlates with poor prognostic and survival rates of breast cancer in the clinic, while suppression of the TGF-b pathway is also reported to be lethal for mice. Therefore it is important to develop strategies to selectively block the cancer-promoting branch but maintain the anti-mitogenic branch of the TGF-b pathway for developing therapeutic drugs. It was recently reported that miR-21 mediates TGF-b bidirectional regulation on MSH2, a central component of DNA mismatch repair (MMR), to contribute chemo-resistance in breast cancer [30]. In the normal cells with intact p53 function and a lower level of miR-21, TGF-b predominantly promotes MSH2 expression, contributing to DNA repair and maintenance of genomic stability. On the other hand, overexpression of miR-21 is often coupled with p53 inactivation in cancerous context, and silences MSH2 by directly binding on its 39-UTR, resulting in genomic instability and resistance to DNA-damaging chemotherapy agents. MiR-Deregulated miRNAs in Breast Cancermight be a mediator for the TGF-b pathway and thus can be a potential target for breast cancer therapy. In conclusion, deregulation of miRNA expression during tumorigenesis might be an early event as it occurs significantly during normal to ADH transition. Target prediction and pathway analysis revealed that miR-21 has a pivotal role on selective utilization of the TGF-b pathway in breast cancer initiation. Importantly, we have demonstrated the feasibility of miRNA expression profiling analysis using archived FFPE tissues, rich with clinical information, as a means towards miRNA biomarker discovery.Feature Extraction and Pre-processing of miRNA Microarray DataProbe level data was extracted from the microarray image by using the Agilent Feature Extraction Software (v10.5). QC reports were automatically generated for each array. All the raw data from Feature Extraction were logarithmically transformed to base 2, with quantile algorithm normalization as described [45].Statistical AnalysisFor the microdissected.
