Figure 3: MEG3/EZH2 functional interaction regulates TGF-β pathway genes. | Nature Communications

Figure 3: MEG3/EZH2 functional interaction regulates TGF-β pathway genes.

From: MEG3 long noncoding RNA regulates the TGF-β pathway genes through formation of RNA–DNA triplex structures

Figure 3

(a–c) MEG3 and EZH2 share common gene targets. (a) Venn diagram showing the number of genes deregulated after downregulation of MEG3 and EZH2 using siRNA in BT-549 and HF cells, and the degree of overlap between the MEG3- and EZH2-dependent genes. The P values were obtained by hypergeometric test using all protein-coding genes as a background. (b) EZH2 protein levels, as determined by western blotting, following EZH2 and MEG3 downregulation in BT-549 and HF cells. Tubulin was used as a loading control. (c) RT–qPCR analysis of EZH2 and MEG3 mRNA expression in Ctrlsi, EZH2si and MEG3si transfected BT-549 and HF cells (±s.d., n=3). (d) RT–qPCR analysis of TGFB2, TGFBR1 and SMAD2 gene expression in Ctrlsi, MEG3si and EZH2si transfected BT-549 cells (±s.d., n=3). (e) Immunoblot showing SMAD2, TGFBR1 and tubulin protein levels following transfection of BT-549 cells with Ctrlsi and MEG3si. (f) Bar graph showing RT–qPCR analysis of TGFB2, TGFBR1 and SMAD2 mRNA levels after overexpression of MEG3 (pREP4MEG3) in BT-549 and MDA-MB-231 cells. The levels in pREP4MEG3 are presented relative to CtrlpREP4 (±s.d., n=3). EZH2 was used as a control showing no change in expression after overexpression of MEG3. The P values were calculated using Student’s t-test (two-tailed, two-sample unequal variance), *P<0.05. (g) Downregulation of MEG3 influences the invasive property of BT-549 cells through regulation of the TGF-β pathway. Images showing Matrigel invasion of the BT-549 cells. The two images in the upper panel show invasion of BT-549 cells infected with Ctrlsh lentivirus, and Ctrlsh infection followed by incubation with TGF-β2 ligand (Ctrlsh-TGFB2). The images in the bottom panel show the cells infected with MEG3Sh and MEG3sh infection followed by incubation with TGF-β inhibitor (MEG3sh+TGF-βin). Scale bar, 10 μm. The bar graph shows quantification (±s.d., n=3) of the matrix invaded cells in MEG3sh relative to the Ctrlsh. The P values were calculated using Student’s t-test (two-tailed, two-sample unequal variance), *P<0.05, **P<0.01.

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