PMC

A A box graph of PTEN expression in the thyroid cancer samples from the TCGA database analyzed using UALCAN. The blue box on the left represents the expression of normal samples, and the red box on the right represents the expression of tumor samples. B The relationship between TUBB3 (TUBB2A) expression and PTEN expression through MEM analysis. C PTEN expression after TUBB3 alteration in TPC-1 cells as determined by RT-qPCR, normalized to GAPDH. *p < 0.05 vs. oe-NC. ^#p < 0.05 vs. sh-NC. D PTEN protein expression after TUBB3 alteration in TPC-1 cells as determined by western blot analysis, normalized to GAPDH. *p < 0.05 vs. oe-NC. ^#p < 0.05 vs. sh-NC. E The effects of TUBB3 on TPC-1 cell viability by regulating PTEN, as detected by MTT. F The effects of TUBB3 on TPC-1 cell colony formation by regulating PTEN, as detected by colony formation assay. G The effects of TUBB3 on TPC-1 cell migration and invasion by regulating PTEN, as detected by Transwell assay (×200). H The effects of TUBB3 on TPC-1 cell apoptosis by regulating PTEN, as detected by flow cytometry. In panel E–H, *p < 0.05 vs. oe-NC. ^#p < 0.05 vs. oe-TUBB3. I The expression of ALDH1A1, TUBB3, and PTEN after treatment with oe-lncRNA ROR + sh-ALDH1A1-1, oe-lncRNA ROR + sh-TUBB3-1, or oe-lncRNA ROR + oe-PTEN in TPC-1 cells as determined by RT-qPCR, normalized to GAPDH. J The effects of lncRNA ROR on TPC-1 cell viability rate by regulating ALDH1A1, TUBB3, and PTEN, as detected by MTT. K The effects of lncRNA ROR on TPC-1 cell colony formation by regulating ALDH1A1, TUBB3, and PTEN, as detected by colony formation assay. L The effects of lncRNA ROR on TPC-1 cell migration and invasion by regulating ALDH1A1, TUBB3, and PTEN, as detected by Transwell assay (×200). M The effects of lncRNA ROR on TPC-1 cell apoptosis by regulating ALDH1A1, TUBB3 and PTEN, as detected by flow cytometry. In panel I–M, *p < 0.05 vs. oe-lncRNA ROR. N Tumor volume growth curve in mice injected with TPC-1 cells upon lncRNA ROR overexpression or combined with PTEN overexpression (n = 12). O Tumor weight of mice injected with TPC-1 cells upon lncRNA ROR overexpression or combined with PTEN overexpression (n = 12). *p < 0.05 vs. oe-NC or oe-lncRNA ROR. Cell experiments were repeated three times independently.

A LncRNA ROR expression in PTC tissue and adjacent tissue samples of patients with PTC as detected by RT-qPCR, normalized to GAPDH (n = 85). *p < 0.05 vs. adjacent tissues. B LncRNA ROR expression in PTC cell lines TPC-1, BCPAP, IHH4, as well as normal thyroid Nthy-ori 3-1 cell line as detected by RT-qPCR, normalized to GAPDH. *p < 0.05 vs. Nthy-ori 3-1. C LncRNA ROR overexpression and silencing efficiency in TPC-1 cells verified by RT-qPCR. D TPC-1 cell viability after alteration of lncRNA ROR as detected by MTT. E Size and number of colonies in TPC-1 cells after alteration of lncRNA ROR, as detected by colony formation assay. F TPC-1 cell migration and invasion after alteration of lncRNA ROR as detected by Transwell assay (×200). G TPC-1 cell apoptosis after alteration of lncRNA ROR as detected by flow cytometry. *p < 0.05 vs. Nthy-ori 3-1 cells or oe-NC, ^#p < 0.05 vs. sh-NC. Cell experiments were repeated three times independently.

ROR competes with G9A at the TESC promoter in vitro. a Schematic diagram showing the first experimental design of purified ROR competing with purified G9A at the TESC promoter. We used 15 μg purified G9A protein and incubated it with 1 μg biotinylated TESC-1 DNA probe. After incubation, 0.1-0.5 μg purified ROR lncRNA was then added to the reaction mixture to compete with the G9A:TESC-1 hybrid. PCR and western blot were used to detect the residual amount of ROR lncRNA and G9A protein after biotin purification and precipitation, respectively. Red helix: TESC DNA; green cycle: G9A protein; small blue cycle: biotin labeled; red arrow: transcriptional direction of TESC. b Expression of purified G9A and purified ROR lncRNA. M: maker. c Different amount of purified ROR competing with the G9A:TESC-1 hybrid. In the presence of 0.4 μg purified ROR, G9A significantly reduced binding with the TESC DNA fragment. CTCF and KCNQ1OT1 were used as a negative control. Input: collected biotinylated TESC-1 fragments before pull-down and PCR with primers aligned with the TESC promoter. d, e The quantitive assay of RoR and G9A interaction. The image J software was used to quantitate the RNA:protein interaction, *P <0.05. f Schematic diagram showing the modified experimental design of total RNA containing ROR competing with purified G9A at the TESC promoter. We used 5 μg purified G9A incubated with 1 μg biotinylated TESC-1 probe. After incubation, different amounts of total RNA extracted from tumor cells were then added to the reaction mixture to compete with the G9A:TESC-1 hybrid. PCR and western blot were used to detect the amount of ROR lncRNA and G9A protein after biotin purification and precipitation, respectively. g, h Different amounts of total RNA containing ROR competed with the G9A:TESC-1 hybrid. In the presence of 30 μg total RNA extracted from tumor cells, the G9A protein significantly reduced binding with the TESC DNA fragment (g). The total RNA of ROR silencing AGS and HT29 tumor cells failed to compete with the G9A:TESC hybrid (h). CTCF and KCNQ1OT1 were used as a negative control. Input: collected biotinylated TESC-1 fragments before pull-down and PCR with primers aligned with the TESC promoter. i, j Competition of total RNA and G9A is shown in i and j. The RNA:protein interaction was quantitated by the image J software,*P <0.05

The boxed section displays the average bias estimates, where available, from the seven meta-epidemiological studies contributing to the BRANDO 2012^a study (however only the BRANDO 2012^a ROR was included in our meta-analysis). The BRANDO 2012^a ROR is based on a multivariable analysis with adjustment for sequence generation and allocation concealment [the corresponding univariable ROR (95% CrI) is 0.87 (0.79, 0.96)]. The BRANDO 2012^b ROR is based on a multivariable analysis with adjustment for sequence generation and allocation concealment [the corresponding univariable ROR (95% CrI) is 0.92 (0.80, 1.04)]. The Unverzagt 2013^c ROR is based on a multivariable analysis with adjustment for sequence generation, allocation concealment, attrition, selective outcome reporting, early stopping, pre-intervention, competing interests, baseline imbalance, switching interventions, sufficient follow-up, and single- versus multi-centre status [the corresponding univariable ROR (95% CI) is 0.84 (0.69, 1.02)]. The BRANDO 2012^d ROR is based on a multivariable analysis with adjustment for sequence generation and allocation concealment [the corresponding univariable ROR (95% CrI) is 0.93 (0.74, 1.18)]. The BRANDO 2012^e ROR is based on a multivariable analysis with adjustment for sequence generation and allocation concealment [the corresponding univariable ROR (95% CrI) is 0.78 (0.65, 0.92)].