Fig 2: Expression of the transcription factors YAP1 and Twist is positively regulated with each other. (a, d) The binding motif of YAP1/TEAD (CATTCCTC) in the promoter of human Twist (a) and Twist (CCAGATGT) in the promoter of YAP1 (d) predicted by “JASPAR” (http://jaspar.genereg.net/). (b) Scheme of the predicted YAP1 binding site of the Twist promoter. (c) A promoter luciferase assay was used to measure Twist promoter activity on YAP1 protein. (e) Scheme of the predicted Twist binding site of the YAP1 promoter. (f) To measure YAP1 promoter activity for Twist protein, promoter luciferase activity was measured. (g–h) After overexpression of Twist, binding of Twist protein to site (−1643 ∼ −1637) of the YAP1 promoter was confirmed by ChIP assay (g) and ChIP-qPCR analysis (h). The band is the PCR product electrophoresed on an agarose gel. (NC, Negative control; O/E, overexpression group) Data are presented as mean ± S.D. *P < 0.05 and **P < 0.01 by a Student's t-test.

Fig 3: LDR increases fibrotic properties by activating YAP1 and Twist. (a–h) WI-38 and MLg were transfected with siRNA against YAP1 and Twist, and then exposed with 0.1 Gy to confirm the characteristics of fibrosis induced by LDR. Protein (a, b) and mRNA (c, d) expression levels of fibrosis-related factors in each indicated cell were confirmed by Western blot and qRT-PCR analysis, respectively. In Western blot analysis, β-actin was used as a loading control. For qRT-PCR analysis, each value was normalized to U6. (e) To confirm cell proliferation, which is a characteristic of fibrosis, two cells were stained with Hoechst 33342 and counted (Scale bar, 100 ㎛). (f) These results are shown graphically. (g) Cell migration was confirmed using a wound healing assay (Scale bar, 100 ㎛). (h) This picture was graphed. Data are presented as mean ± S.D. *P < 0.05, **P < 0.01, and ***P < 0.001 by a Student's t-test.

Fig 4: miR-765 directly down-regulated YAP1, COL1A1, and α-SMA expression. (a) Using three algorithms such as miRWalk, DIANA, and miRanda to find genomic targets for miRNAs, miR-423-5p, miR-211-3p, miR-5006-5p, miR-5194, miR-5682, and miR-765 targeting YAP1, COL1A1, and α-SMA were screened. (b) After overexpression of each miRNA mimic, the mRNA expression levels of three targets, YAP1, COL1A1, and α-SMA, were examined by qRT-PCR analysis. (c) To investigate the expression of its target proteins by the selected miR-765, the miR-765 mimic was overexpressed in two cells, and then the expression of YAP1, COL1A1, and α-SMA proteins was measured by Western blot analysis. (d) Structure of the 3′UTR reporter constructs of YAP1, COL1A1, and α-SMA includes the direct binding site of miR-765 in downstream of the luciferase ORF, respectively. (e) Dual luciferase assay was performed to reconfirm that YAP1, COL1A1, and α-SMA was direct target factors of miR-765. Data are presented as mean ± S.D. *P < 0.05, **P < 0.01, and ***P < 0.001 by a Student's t-test.

Fig 5: miR-765 attenuates LDR-induced fibrotic phenotype. (a–f) To investigate the mechanism of miR-765 on LDR-induced fibrosis, WI-38 and MLg were transfected with negative control (NC) or miR-765 and exposed with or without LDR (0.1 Gy). (a–b) The expression of protein (a) and mRNA (b) of fibrosis-related markers (YAP1, COL1A1, α-SMA, Twist, and Vimentin) was confirmed by Western blot and qRT-PCR analysis, respectively. β-actin and U6 were used as loading controls in Western blot and qRT-PCR analysis, respectively. (c) The cell proliferation capacity was counted after staining the cells of the indicated group with Hoechst 33342 (left) and was shown as a graph (right) (Scale bar, 100 ㎛). (d) Cell migration, one of phenotypes of fibrosis, was confirmed using a wound healing assay (Scale bar, 100 ㎛) (left). This result was graphed (right). Data are presented as mean ± S.D. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to control group. ‡P < 0.05, ‡ ‡P < 0.01, and ‡ ‡ ‡P < 0.001 compared with IR group by a Student's t-test.