Supplementary Figure 6: PMTRIP FISH-PLA can quantify COX-2-TIAR interactions in lung tissue. A) 20 nM of V5 tagged PMTRIPs targeting COX-2 mRNA were delivered as FISH probes to colon cancer samples. PLA (green) was performed between the V5 tag and TIAR. 20x cropped image is indicated by the white box. Extended focus images are shown, with a 1 mm scale bar for full tissue. B) Extended focus images of FISH-PLA (green) controls are shown. Scale bar is 1 mm. C) Extended focus cropped images of full tissues in (A) are indicated by white boxes. Scale bars are 100 μm. D) Extended focus image of cancer tissue FISH-PLA at 63x is also shown. Scale bars for (D) are 10 μm. E) Extended focus cropped images of full tissues in (B) are indicated by white boxes. Extended focus images of tissue FISH-PLA at 63x are also shown. COX-2 FISH shown in red. Scale bars are 100 μm for 20x and 10 μm for 63x images. F) Quantification of PLA signal in (A,B). Statistics were performed with a one-way ANOVA with a Tukey's multiple comparisons test, where n=3 and * p < 0.015 and ** p < 0.0077. Standard deviations are shown in red.
ARTICLE ABSTRACT
Abnormal post-transcriptional regulation induced by alterations of mRNA–protein interactions is critical during tumorigenesis and cancer progression and is a hallmark of cancer cells. A more thorough understanding is needed to develop treatments and foresee outcomes. Cellular and mouse tumor models are insufficient for vigorous investigation as they lack consistency and translatability to humans. Moreover, to date, studies in human tumor tissue are predominately limited to expression analysis of proteins and mRNA, which do not necessarily provide information about the frequency of mRNA–protein interactions. Here, we demonstrate novel optimization of a method that is based on FISH and proximity ligation techniques to quantify mRNA interactions with RNA-binding proteins relevant for tumorigenesis and cancer progression in archival patient-derived tumor tissue. This method was validated for multiple mRNA-protein pairs in several cellular models and in multiple types of archival human tumor samples. Furthermore, this approach allowed high-throughput analysis of mRNA–protein interactions across a wide range of tumor types and stages through tumor microarrays. This method is quantitative, specific, and sensitive for detecting interactions and their localization at both the individual cell and whole-tissue scales with single interaction sensitivity. This work presents an important tool in investigating post-transcriptional regulation in cancer on a high-throughput scale, with great potential for translatability into any applications where mRNA–protein interactions are of interest.
This work presents an approach to sensitively, specifically, and quantitatively detect and localize native mRNA and protein interactions for analysis of abnormal post-transcriptional regulation in patient-derived archival tumor samples.
