Fig 2: miR-30a acts in a feedback loop to inhibit YY1. a Predicted binding sites for miR-30a in the YY1 3'UTR. The engineered mutant sequence is also shown. b The luciferase activities of BXPC-3 and PANC-1 cells were measured after co-transfection with the indicated YY1 3'UTR constructs and miR-30a for 24 h. c Western blot and qRT-PCR analysis of YY1 protein or mRNA levels in BXPC-3 and PANC-1 cells transfected with miR-30a-overexpressing or -inhibiting lentivirus. **p < 0.01, ***p < 0.001

Fig 3: YY1 knockdown upregulates ITGAV and ITGB1 expression in both DLD-1 and SW48 cells. (A and B) The microarray analysis revealed that YY1 knockdown increased the mRNA expression of (A) ITGAV and (B) ITGB1. (C and D) Western blot analysis demonstrated that knockdown of YY1 significantly increased the expression of (C) ITGAV and (D) ITGB1. **P<0.01 and ***P<0.001. ITGAV, integrin alpha V; ITGB1, integrin beta 1; YY1, Yin Yang 1; si, small interfering.

Fig 4: YY1 suppresses the transcription of VSMC genes encoding contractile proteins in PAC1 VSMCs. (A) PAC1 cells were transfected with YY1 expression plasmid (YY1) or the empty vector control (Ctr). The mRNA expression level of YY1, SMa-actin and SM22a was determined by qRT-PCR assays. (B) Knocking down YY1 by YY1 siRNA significantly increased the transcription of SMC-specific genes by qRT-PCR assays. Scr: the scramble siRNA as the control. (C) YY1 overexpression in PAC1 cells inhibited the promoter activity of SMa-actin-Luc, SM22a-Luc, SMMHC-Luc, but not ACLP-Luc by the luciferase assay. Values are mean ± SEM (n = 3) *P < 0.05, **P < 0.01 versus the control (Ctr).

Fig 5: YY1 reintroduction abrogates the effects of GACAT1. (A) The transfection of pcDNA-YY1 was confirmed using western blot analysis. siRNA-GACAT1 combined with pcDNA-vector or pcDNA-YY1 was co-transfected into both A549 and H1299 cells. The expression level of GACAT1 and YY1 was determined using (B) RT-qPCR (three replicates) and (C) western blot analysis. The reduction in cell proliferation following GCAGT1 knockdown was abrogated with the transfection of YY1 in (D) A549 and (E) H1299 cells. Data was obtained from three independent experiments and analyzed using one-way ANOVA. *P<0.05, **P<0.01 and ***P<0.001. si, short inhibiting; YY1, YY1 transcription factor; GACAT1, gastric cancer-associated transcript 1; OD, optical density.