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. 2017 May 19;8(41):69559–69576. doi: 10.18632/oncotarget.18012

Figure 4. HCaRG suppresses EGFR and ErbB3 by enhancing their promoter methylation.

Figure 4

(A) The expression of total (t) ErbB receptors was demonstrated by Real-Time PCR and western blot. HCaRG overexpression reduced EGFR and ErbB3 expression at the mRNA and protein levels in Renca cells. ErbB2 mRNA and total protein levels were not modified by HCaRG overexpression. †P < 0.005. NS, not significant. (B) Knockdown of HCaRG with siRNA treatment in WT-Renca cells. siRNA-HCaRG treatment reduced HCaRG protein expression compared to a negative control (NC) and led to up-regulation of EGFR and ErbB3. ErbB2 expression was not changed by siRNA against HCaRG. (C) The effect of inhibition of MAPK and PI3K/AKT signals on downstream genes was demonstrated by Real-Time PCR in Renca cells. The decrement of HIF-1α and subsequent VEGFA and PDGF genes was caused by HCaRG overexpression independent of the expression of anti-oncogene of RCC, VHL. ‡P < 0.05, *P < 0.01, †P < 0.005. NS, not significant. (D) Renca lysates were analyzed by western blot using appropriated antibodies. Representative blots show the inactivation of subsequent MAPK and/or PI3K/AKT signaling pathways by HCaRG overexpression. (E) HCaRG inhibits the transcriptional activity of EGFR and ErbB3 at the promoter level. Human EGFR, ErbB2 and ErbB3 5′ flanking regions containing their respective promoters were cloned and transfected into Neo- or HCaRG-Renca cells. HCaRG overexpression repressed the promoter activities of EGFR and ErbB3, but not of ErbB2. Promoter activity was normalized to the activity of pGL4.74-TK control vector. ‡P < 0.05, †P < 0.005. (F) mRNA degradation of ErbB receptors was tested by inhibiting their transcription with α-amanitin. mRNA levels were normalized to polymerase I-dependent transcription of the 28S ribosomal gene. mRNA degradation was presented as percentage of mRNA levels relative to controls without α-amanitin. HCaRG did not cause mRNA degradation after 24 hours. (G) Demethylation of promoter DNA with treatment of 5-aza-2’-deoxycytidine rescued and/or increased mRNA levels of EGFR and ErbB3 in HCaRG-Renca cells in a dose-dependent manner. ‡P < 0.05, †P < 0.005.