Fig 2: POU6F1 binds RORA and enhances the stabilization and activity of RORA.A Real-time qRT-PCR assay indicating the expression of RORA in A549 cells stably transfected with empty vector (mock) or POU6F1. B Co-IP and western blotting assays indicating the endogenous interaction between POU6F1 and RORA in A549 cells. The immunoglobulin G (IgG)-bound protein was taken as the negative control. C Mass spectrometry detection showing the spectrogram of RORA in A549 and HEK293T cells pulled down by POU6F1 antibody. D Immunofluorescence assay confirming the colocalization of POU6F1 and RORA in A549 cells transfected with empty vector (pCMV-HA) or pCMV-HA-POU6F1. Scale bars:10 µm. E Confocal images of BiFC showing the direct visualization of POU6F1 and RORA in A549 cells co-transfected with pBiFC-VC155-POU6F1 and pBiFC-VN173-RORA. Scale bars:10 µm. F Western blotting assay showing the expression of RORA in A549 and NCI-H1299 cells transfected with mock, POU6F1, CRISPRi-Scb, CRISPRi-POU6F1 #1, or CRISPRi-POU6F1 #1. G Co-IP and western blotting assays indicating the interaction between POU6F1 and RORA in A549 cells transfected with mock or POU6F1. H Western blotting assay showing the expression of POU6F1 and RORA in A549 cells transfected with mock, RORA, scramble shRNA (sh-Scb), sh-RORA #1, or sh-RORA #2. I Western blotting assay showing the expression of RORA in A549 cells transfected with CRISPRi-Scb or CRISPRi-POU6F1 #1, and those treated with MG132 (5 µmol/l) for 6 h. J Dual-luciferase assay indicating relative promoter activity of RORA in A549 and NCI-H1299 cells transfected with mock or POU6F1. Student t-test compared the difference in A and J. *P < 0.05, **P < 0.01.

Fig 3: Expression of lncRNA 430945 and ROR2 are high in human AS tissues. (A) Hematoxylin and eosin-stained representative sections of aortas from human AS tissues and Con tissues. Scale bar=50 µm. Subset of the differentially expressed (B) lncRNAs and (C) mRNAs detected in the AS tissues (n=3) and Con tissues (n=3) using microarray analysis. (D) Six lncRNAs with differential expression in AS tissues and Con tissues were validated by qPCR. (E) Expression levels of ROR2 in AS tissues and Con tissues were analyzed using qPCR. *P<0.05, vs. Con (n=3). (F) Levels of ROR2 in AS tissues and Con tissues were analyzed using western blotting. The band intensities relative to ß-actin are presented as the mean ± standard deviation; n=3; *P<0.05, vs. Con. ROR2, receptor tyrosine kinase-like orphan receptor 2; lncRNAs, long non-coding RNAs; qPCR, quantitative-polymerase chain reaction; AS, atherosclerosis; Con, control (normal aortic tissues).

Fig 4: lncRNA 430945 mediates AngII-induced VSMC proliferation, migration and invasion. (A) VSMCs were stimulated with AngII for 24 h and the expression of lncRNA 430945 was analyzed using qPCR. *P<0.05, vs. control (n=3). (B) VSMCs were transfected with non-specific siRNA (si-NC) or lncRNA 430945-specifc siRNA (si-lncRNA 430945) for 24 h and the expression of lncRNA 430945 was analyzed using qPCR. *P<0.05, vs. control (n=3). (C) VSMCs were pre-treated with si-NC or si-lncRNA 430945 for 24 h prior to being stimulated with AngII for an additional 0, 12, 24, 36 or 48 h. Cell growth was assessed using the cell viability Cell Counting Kit-8 assay. *P<0.05 and ***P<0.001, vs. si-NC at different time (n=3). (D) VSMC monolayers were pre-treated with si-NC or si-lncRNA 430945 for 24 h prior to being wounded and then stimulated with or without AngII for 0 and 24 h, and cell migration was assessed under phase-contrast light microscopy (magnification, ×100). (E) Representative photomicrographs of a Transwell assay show the quantification of VSMC invasion (magnification, ×400). The number of invaded cells was measured in three separate experiments and data represent the mean ± standard deviation (n=3). *P<0.05 and **P<0.01, vs. si-NC. (F) VSMCs were transfected with si-NC or si-lncRNA 430945 for 24 h and total cell lysates were analyzed by western blotting with anti-ROR2 antibody. *P<0.05, vs. si-NC (n=3). VSMCs, vascular smooth muscle cells; ROR2, receptor tyrosine kinase-like orphan receptor 2; lncRNAs, long non-coding RNAs; si-, small interfering RNA; NC, negative control; AngII, angiotensin II; qPCR, quantitative polymerase chain reaction.

Fig 5: lncRNA 430945 promotes VSMC proliferation and migration. (A) VSMCs were transfected with pcDNA or pcDNA-lncRNA430945 for 24 h and the expression of lncRNA 430945 was analyzed by quantitative polymerase chain reaction analysis. *P<0.05, vs. control (n=3). (B) VSMC monolayers were pre-treated with pcDNA or pcDNA-lncRNA430945 for 24 h prior to being wounded, and cells migrating to the scratch gap were quantified (magnification, ×100). The data are presented as the mean ± standard deviation from three independent experiments. *P<0.05, vs. pcDNA. (C) Cell growth was assessed using a cell viability Cell Counting Kit-8 assay following transfection with pcDNA or pcDNA-lncRNA430945 for 0, 12, 24, 36 and 48 h. *P<0.05 and **P<0.01 vs. pcDNA at different times (n=3). (D) Representative photomicrographs of a Transwell assay showing the quantification of VSMC invasion (magnification, ×400). The number of invaded cells was measured in three separate experiments and data represent the mean ± standard deviation (n=3). *P<0.05, vs. pcDNA group. (E) VSMCs were transfected with pcDNA or pcDNA-lncRNA430945 for 24 h and total cell lysates were analyzed by western blotting with anti-ROR2 antibody. *P<0.05, vs. pcDNA (n=3). VSMCs, vascular smooth muscle cells; ROR2, receptor tyrosine kinase-like orphan receptor 2; lncRNAs, long non-coding RNAs.