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. 2018 Dec 31;14:450–464. doi: 10.1016/j.omtn.2018.12.013

Figure 7.

Figure 7

IR/Src/Bcl-w Signaling Axis Drives to Tumor Progression and Metastasis because of IR-Induced Hyper-methylation of miR-205-5p

(A) Left, miR-205-5p expression levels were measured in H460 and MDA-MB231 cells, which were treated with inhibitors of ERK (PD98059), MEK (U0126), p38 (SB203580), JNK (SP600125), JAK, Src (PP2), STAT3, and PI3K (LY294002) by qRT-PCR. Right, expression levels of Src or Bcl-w protein by Src inhibitor (PP2) were determined by western blot analysis. β-actin was used as loading control. (B) H460 cells were transfected with vector control (pcDNA3.1) and Src-MT (c-SrcF527; active form). Expressions of Src phosphorylation (Tyr416) were shown by western blot analysis. (C) After transfection with Src-MT (left) or small interfering RNA Src (right) in MDA-MB-231 cells, the level of miR-205-5p was determined by qRT-PCR. (D and E) After H460 and MDA-MB-231 cells were exposed to IR (5 Gy) in presence or absence of miR-205-5p, levels of Src protein (D) and mRNA (E) were determined by western blot analysis and qRT-PCR, respectively. (F) Top, structure of reporter constructs containing Src 3′ UTR downstream of the luciferase open reading frame (ORF). pGL3UC-Src vectors containing the WT miR-205-5p binding site or a non-binding MT were constructed. Bottom, luciferase assays were performed with MDA-MB-231 cells, which were co-transfected with negative control, or miR-205-5p and pGL3UC-Src-WT, pGL3UC-Src mutant, or the empty vector for 48 h and were normalized by pRL-CMV-Renilla. (G) Kaplan-Meier analysis of the probability of survival as a function of relative expression of Src in breast adenocarcinoma tumors. (H and I) After H460 and MDA-MB-231 cells were exposed to IR (5 Gy) in presence or absence of siRNA (H) or inhibitor of Src (I), methylation or unmethylation of miR-205-5p CpG island was determined by qRT-PCR. (J) Scheme of IR-induced Src/miR-205-5p/Bcl-w signaling axis. IR induced downregulation of miR-205-5p expression through increasing its methylation by activating Src. As a result, IR-induced Src/miR-205-5p/Bcl-w axis is involved in tumor progression and metastasis of human cancer. Therefore, miR-205-5p may be useful as a genetic Bcl-w/Src-inhibiting therapeutic agent when treated with IR. All data are presented as the mean SD (*p < 0.05, **p < 0.005, ***p < 0.0005, Student’s t test).