Inhibition of the lncRNA Coded within Transglutaminase 2 Gene Impacts Several Relevant Networks in MCF-7 Breast Cancer Cells

Figures

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  Figure 1. Silencing of TG2-lncRNA. The data show RT-qPCR analysis after 48 h of treatment with siRNAs employed at a concentration of 50 nM. Black, siRNA targeting TG2-lncRNA; White, siRNA directed against GATA3; Grey, control treatment with siRNA neg. In (A,B), the histograms indicate Fold Change obtained using 2−∆∆CT method, referred to HPRT1 as housekeeping gene to quantify target genes with respect to the set of samples treated with siRNA neg. Error bars represent standard deviation (n = 3 biological replicates). * p < 0.05 by two-tailed unpaired t test.

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  Figure 2. Expression heatmap of dysregulated genes in response to TG2-lncRNA silencing. “MT” group represents the treated class with siRNA against the TG2-lncRNA, whereas “WT” represents the siRNA neg control. The level of expression of the genes was obtained through DESeq2 R package, and is represented as Log2FC, filtered out based on p-adj < 0.01. The red/blue colour bar highlights the Log2FC rate variation across six samples for each gene. The upregulated are showed as red, whereas the downregulated genes are labelled in blue.

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  Figure 3. Functional classification of the most represented dysregulated pathways. The top 9 highly dysregulated pathways obtained using PANTHERdb classification system (http://www.pantherdb.org/, accessed on 21 June 2021) are indicated with the relative functional classification number, which is depicted below the bar charts, where Red and Green bars represent respectively upregulated and downregulated pathways. This analysis derives from Table S2 data (p-adj < 0.05). The height of each bar is proportional to the total number of dysregulated genes within the related pathway, as further labelled on the top of them.

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  Figure 4. Networks of the genes upregulated by silencing of TG2-lncRNA involved in the apoptosis and PDGF signalling pathways. Each node represents one of the differentially expressed genes. In the left are depicted 2 networks belonging to apoptosis, while in the right panel the only one relevant for PDGF signalling. Red color indicates that all genes are upregulated by silencing. The node size is proportional with the degree of association with the altered gene expression value based on the Log2FC after siRNA treatment, while intensity of the node color scales the weight of connections. Gene correlated to each other are linked by lines.

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  Figure 5. Networks of the genes downregulated by silencing of TG2-lncRNA involved in the angiogenesis and integrin signalling pathways. Each node represents one of the differentially expressed genes. In the left chart are depicted 3 networks belonging to angiogenesis, while in the right panel is reported the only one of integrin signalling. Green color indicates that all genes are downregulated by silencing. The node size is proportional with the degree of association with the modified gene expression value based on the Log2FC after siRNA treatment, while intensity of the node color scales the weight of connections. Gene correlated to each other are linked by lines.

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  Figure 6. Network of the genes dysregulated by the silencing of TG2-lncRNA belonging to inflammation pathways mediated by chemokine and cytokine signalling. Each node represents one of the differentially expressed genes. In red depicted are upregulated genes, while in green, those downregulated. The node size is proportional with the degree of association with the modified gene expression value based on the Log2FC after siRNA treatment, while intensity of the node colour scales the weight of connections. Gene correlated to each other are linked by lines.

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  Figure 7. Sequences recognized by transcription factors within promoters of genes dysregulated by silencing and common to TG2-lncRNA. The left panel lists the sites within the genes downregulated and upregulated by siRNA that are also present in the sequence of TG2-lncRNA. In the right panel they are divided in three groups: dark blue, transcription factor sites common to downregulated genes also identified in TG2-lncRNA; medium blue, those common to all dysregulated genes and TG2-lncRNA; light blue, sites common to upregulated genes also identified in TG2-lncRNA.

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  Figure 8. In vitro binding of RXRα, TP53 and YY1 to biotinylated single strand DNA molecule mimic of TG2-lncRNA, as detected by Western blotting of the associated protein complexes. Experimental details are reported in Material and Methods. The top picture (INPUT) refers to nuclear extracts (10 μL of input) from the mentioned cell lines used in each reaction of in vitro binding assay. NB4 cells represent positive control of RXRα. MCF-7 NC and MDA-MB-231 NC are control reactions in which the extracts were incubated with beads without biotinylated mimic molecule. Hybridization was carried out with specific antibody against RXRα, TP53 and YY1. In the bottom panel, we have compared (OUTPUT) reactions performed using the nuclear extracts after binding with the biotinylated probe. In addition, extracts from NBA cells untreated or induced with 1 and 3 μM ATRA as positive control.