ABSTRACT
BACKGROUND: MicroRNAs (miRNAs) regulate the translation of mRNA during gene expression and investigations have highlighted their importance in pathophysiology. qRT-PCR is currently the gold standard method for detecting changes in miRNA expression. However, when used on heterogeneous samples, it cannot identify individual cell types harbouring miRNAs. For this, in situ hybridisation (ISH) can be used. ISH methods using locked nucleic acid (LNA) probes give reliable results in formalin fixed paraffin-embedded (FFPE) samples. In this study their use has been directly compared with conventional oligonucleotide probes (COP) for ISH. METHODS: FFPE samples of colorectal adenocarcinoma, squamous carcinoma of lung and cases of invasive breast carcinoma were used to evaluate COP and LNA methods for the demonstration of miR-126 and miR-205. To demonstrate the utility of the COP method demonstration of miR-21 in 19 Gleason stage 7 prostate biopsy FFPE tissues was also undertaken. The demonstration of miR-21 by ISH in high and low expressing prostate cancer cell lines was also compared with qRT-PCR. RESULTS: Similar results were obtained using the COP and LNA ISH methods for the demonstration of miR-126 and miR-205. miR-21 was successfully demonstrated in the prostate cancer samples by COP ISH and expression levels of the miRNA demonstrated in the cell lines corresponded with qRT-PCR. CONCLUSION: This study has shown that simplification of ISH protocols by the use of COPs provides equivalent results to the use of LNA methods and it can be used to precisely identify cells in which miRNAs are expressed.
Subject(s)
MicroRNAs/genetics , Oligonucleotide Probes/genetics , Oligonucleotides/genetics , Cell Line, Tumor , Formaldehyde/chemistry , Humans , In Situ Hybridization/methods , Neoplasms/genetics , PC-3 Cells , Paraffin/chemistry , Paraffin Embedding/methodsABSTRACT
Genetic studies of Wnt11 have revealed many insights into the roles and regulation of Wnt11, particularly during development. New tools to study Wnt11 have recently become available, making it timely to review the literature regarding this unique Wnt family member. In this study, we focus on mammalian Wnt11, describing its main sites of expression during development, and how the Wnt11 gene is regulated. We highlight an emerging theme in which canonical Wnt signals regulate Wnt11 expression through transcription factors in addition to, or other than, Tcf/LEF family members. We also discuss the frizzled family and other receptors that bind to Wnt11, the intracellular kinases and small GTPases that act downstream of Wnt11, and the effects of Wnt11 on Wnt/ß-catenin signalling. Finally, we elaborate on the relevance of Wnt11 to human cancer, where it appears to be important both for proliferation and/or survival during normal differentiation and for migration/invasion.
Subject(s)
Protein Isoforms/metabolism , Wnt Proteins/metabolism , Wnt Signaling Pathway/physiology , Animals , Cell Adhesion , Cell Differentiation/physiology , Cell Movement , Frizzled Receptors/genetics , Frizzled Receptors/metabolism , Gene Expression Regulation , Humans , Neoplasms/physiopathology , Protein Isoforms/genetics , Wnt Proteins/genetics , beta Catenin/metabolismABSTRACT
Secreted Frizzled-related protein-1 (sFRP1) associates with Wnt proteins and its loss can lead to activation of Wnt/beta-catenin signalling. It is frequently downregulated in cancer, including prostate cancer, but its function in prostate cancer is unclear because it can increase proliferation of prostate epithelial cells. We investigated the function of sFRP1 in androgen-dependent prostate cancer and found that sFRP1 inhibited androgen receptor (AR) transcriptional activity. In addition, sFRP1 inhibited the proliferation of androgen-dependent LNCaP cells but not of an androgen-independent subline LNCaP-r, suggesting a role in androgen-dependent growth. The inhibition of AR by sFRP1 was unaffected by co-expression of Wnt3a, stabilised beta-catenin or beta-catenin shRNA, suggesting it does not involve Wnt/beta-catenin signalling. Wnt5a also inhibited AR and expression of Wnt5a and sFRP1 together did not further inhibit AR, suggesting that Wnt5a and sFRP1 activate the same signal(s) to inhibit AR. However, sFRP1 inhibition of AR was unaffected by inhibitors of kinases involved in Wnt/Ca(2+) and Wnt/planar cell polarity non-canonical Wnt signalling. Interestingly, the cysteine-rich domain of sFRP1 interacted with Frizzled receptors expressed in prostate cancer cells, suggesting that sFRP1/Frizzled complexes activate a signal that leads to repression of AR. Taken together, these observations highlight the function of beta-catenin-independent Wnt signalling in the control of AR activity and provide one explanation for sFRP1 downregulation in prostate cancer.
