Journal: Molecular Cancer

Article Title: Circular RNA CircEYA3 induces energy production to promote pancreatic ductal adenocarcinoma progression through the miR-1294/c-Myc axis

doi: 10.1186/s12943-021-01400-z

Figure Lengend Snippet: c-Myc is a direct target of miR-1294 in PDAC cells. A . The miRDB, miRWalk, TargetScan, and miRTarBase databases were utilized to predict the target genes of miR-1294. B and C . PANC-1 and MiaPaCa-2 cells were transfected with the miR-1294 mimic, miR-1294 inhibitor or the corresponding negative control, and the expression of c-Myc and SURF4 was evaluated by qRT-PCR. D . Schematic showing the c-Myc-WT and c-Myc-Mut luciferase reporter plasmids. E and F . Relative luciferase activity in PANC-1 and MiaPaCa-2 cells after co-transfection with c-Myc-WT or c-Myc-Mut and the miR-1294 mimic, inhibitor or corresponding negative control. G and H . Western blot analysis was performed to assess the protein levels of c-Myc, E-cadherin, N-cadherin, Vimentin, Snail, Bax, and cleaved caspase-3 after knocking down or overexpressing miR-1294 or circEYA3 in PANC-1 and MiaPaCa-2 cells, as indicated. β-actin was used as the loading control. I . The correlation between the miR-1294 and c-Myc expression levels in 104 paired PDAC patients was analysed by qRT-PCR and Pearson correlation analysis. * P < 0.05, ** P < 0.01, *** P < 0.001; ns indicates no significance

Article Snippet: Subsequently, the membranes were probed with primary antibodies specific for the following proteins: c-Myc (1:1000, ab32072, Abcam), E-cadherin (1:1000, #3195, CST), N-cadherin (1:1000, #13116, CST), Vimentin (1:1000, #5741, CST), Snail (1:1000, #3879, CST), Bax (1:1000, #14796, CST), cleaved caspase-3 (1:1000, #9661, CST), and β-actin (1:10000, 66,009–1-lg, Proteintech).

Techniques: Transfection, Negative Control, Expressing, Quantitative RT-PCR, Luciferase, Activity Assay, Cotransfection, Western Blot, Control

Journal: Molecular Cancer

Article Title: Circular RNA CircEYA3 induces energy production to promote pancreatic ductal adenocarcinoma progression through the miR-1294/c-Myc axis

doi: 10.1186/s12943-021-01400-z

Figure Lengend Snippet: c-Myc is a direct target of miR-1294 in PDAC cells. A . The miRDB, miRWalk, TargetScan, and miRTarBase databases were utilized to predict the target genes of miR-1294. B and C . PANC-1 and MiaPaCa-2 cells were transfected with the miR-1294 mimic, miR-1294 inhibitor or the corresponding negative control, and the expression of c-Myc and SURF4 was evaluated by qRT-PCR. D . Schematic showing the c-Myc-WT and c-Myc-Mut luciferase reporter plasmids. E and F . Relative luciferase activity in PANC-1 and MiaPaCa-2 cells after co-transfection with c-Myc-WT or c-Myc-Mut and the miR-1294 mimic, inhibitor or corresponding negative control. G and H . Western blot analysis was performed to assess the protein levels of c-Myc, E-cadherin, N-cadherin, Vimentin, Snail, Bax, and cleaved caspase-3 after knocking down or overexpressing miR-1294 or circEYA3 in PANC-1 and MiaPaCa-2 cells, as indicated. β-actin was used as the loading control. I . The correlation between the miR-1294 and c-Myc expression levels in 104 paired PDAC patients was analysed by qRT-PCR and Pearson correlation analysis. * P < 0.05, ** P < 0.01, *** P < 0.001; ns indicates no significance

Article Snippet: Subsequently, the membranes were probed with primary antibodies specific for the following proteins: c-Myc (1:1000, ab32072, Abcam), E-cadherin (1:1000, #3195, CST), N-cadherin (1:1000, #13116, CST), Vimentin (1:1000, #5741, CST), Snail (1:1000, #3879, CST), Bax (1:1000, #14796, CST), cleaved caspase-3 (1:1000, #9661, CST), and β-actin (1:10000, 66,009–1-lg, Proteintech).

Techniques: Transfection, Negative Control, Expressing, Quantitative RT-PCR, Luciferase, Activity Assay, Cotransfection, Western Blot, Control

Journal: Molecular Cancer

Article Title: Circular RNA CircEYA3 induces energy production to promote pancreatic ductal adenocarcinoma progression through the miR-1294/c-Myc axis

doi: 10.1186/s12943-021-01400-z

Figure Lengend Snippet: CircEYA3 performs its oncogenic functions by increasing ATP production through the miR-1294/c-Myc axis. A and B . PANC-1 cells were transfected with NC, si-circ-1 and the miR-1294 inhibitor, and MiaPaCa-2 cells were transfected with NC, pLCDH-circEYA3 and the miR-1294 mimics, as indicated. qRT-PCR and western blot analysis were performed to assess the expression of c-Myc. C , D and E . The cell proliferation ability was evaluated by CCK-8 ( C ), colony formation ( D ), and EdU incorporation assays ( E ). Scale bars, 100 μm. The miR-1294 inhibitor significantly reversed the reduction in cell proliferation caused by circEYA3 knockdown in PANC-1 cells, while the miR-1294 mimic further enhanced the proliferation of MiaPaCa-2 cells. F . The effects of si-circ-1 and miR-1294 or pLCDH-circEYA3 and the miR-1294 mimic on the migratory and invasive capabilities of PDAC cells were evaluated by Transwell assays. Scale bars, 100 μm. G . The effects of si-circ-1 and miR-1294 or pLCDH-circEYA3 and the miR-1294 mimic on apoptosis were evaluated by Annexin V-PE/7-AAD staining. H and I . Western blot analysis was performed to determine the protein levels of c-Myc, E-cadherin, N-cadherin, Vimentin, Snail, Bax, and cleaved caspase-3 in order to evaluate the effects of si-circ-1 and miR-1294 or pLCDH-circEYA3 and the miR-1294 mimic on PANC-1 ( H ) or MiaPaCa-2 ( I ) cells, respectively. L . CircEYA3 knockdown decreased and circEYA3 overexpression increased ATP production. M . The miR-1294 mimic decreased but the miR-1294 inhibitor induced ATP production. N . The effects of si-circ-1 and the miR-1294 inhibitor or pLCDH-circEYA3 and the miR-1294 mimic on ATP production in PANC-1 and MiaPaCa-2 cells. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Subsequently, the membranes were probed with primary antibodies specific for the following proteins: c-Myc (1:1000, ab32072, Abcam), E-cadherin (1:1000, #3195, CST), N-cadherin (1:1000, #13116, CST), Vimentin (1:1000, #5741, CST), Snail (1:1000, #3879, CST), Bax (1:1000, #14796, CST), cleaved caspase-3 (1:1000, #9661, CST), and β-actin (1:10000, 66,009–1-lg, Proteintech).

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Expressing, CCK-8 Assay, Knockdown, Staining, Over Expression

Journal: Molecular Cancer

Article Title: Circular RNA CircEYA3 induces energy production to promote pancreatic ductal adenocarcinoma progression through the miR-1294/c-Myc axis

doi: 10.1186/s12943-021-01400-z

Figure Lengend Snippet: Knockdown of circEYA3 suppresses tumor growth in vivo. A. Stably transfected PANC-1 cells from different groups were inoculated into BALB/c nude mice to establish subcutaneous xenograft tumors ( n = 5 mice/group). Representative images of tumor-bearing mice. In addition, tumor volumes were monitored weekly. B . The excised tumors were photographed and weighed. C . The relative expression levels of circEYA3, miR-1294 and c-Myc in subcutaneous tumor tissues were determined by qRT-PCR. D . The c-Myc, E-cadherin, N-cadherin, Vimentin, Snail, Bax, cleaved caspase-3 protein levels in tumours from different groups were determined by western blot analysis. E . IHC staining showed the relative levels of c-Myc, E-cadherin, N-cadherin, Bax, cleaved caspase-3, and Ki-67 in tumours from different groups. Scale bar, 50 μm. * P < 0.05, ** P < 0.01, *** P < 0.001; ns indicates no significance

Article Snippet: Subsequently, the membranes were probed with primary antibodies specific for the following proteins: c-Myc (1:1000, ab32072, Abcam), E-cadherin (1:1000, #3195, CST), N-cadherin (1:1000, #13116, CST), Vimentin (1:1000, #5741, CST), Snail (1:1000, #3879, CST), Bax (1:1000, #14796, CST), cleaved caspase-3 (1:1000, #9661, CST), and β-actin (1:10000, 66,009–1-lg, Proteintech).

Techniques: Knockdown, In Vivo, Stable Transfection, Transfection, Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry

Journal: International Journal of Oncology

Article Title: Downregulated RRS1 inhibits invasion and metastasis of BT549 through RPL11-c-Myc-SNAIL axis

doi: 10.3892/ijo.2022.5323

Figure Lengend Snippet: RPL11 reversed EMT which inhibited by RRS1 reduction. After knockdown of RRS1, cells were lysed to extract total protein and western blotting assays using indicated antibodies. (A) Depletion of RRS1 inhibited SNAIL and EMT processes. (B) RRS1 knockdown and used siRNA to interfere with RPL11 expression, reversing the inhibition of RRS1 depletion on SNAIL and EMT processes, * P<0.05, ** P<0.01, *** P<0.001. RPL11, ribosome protein L11; EMT, epithelial mesenchymal transition; RRS1, regulator of ribosome synthesis 1; VIM, vimentin; Con, control; si, short interfering; sh, short hairpin.

Article Snippet: After blocking with 5% BSA (Beijing Solarbio Science & Technology Co., Ltd., A8020) for 2 h at room temperature, the membranes were incubated overnight with primary antibodies against RRS1 (1:1,000; Abcam; cat. no. ab188161), c-Myc (1:1,000; Abcam; cat. no. ab32072), NPM1 (1:1,000; Proteintech; cat. no. 60096-1-Ig), Lamin B1 (1:1,000; Proteintech; cat. no. 12987-1-AP), N-cadherin (1:1,000; Proteintech; cat. no. 22018-1-AP), E-cadherin (1:1,000; Proteintech; cat. no. 20874-1-AP), c-Myc (1:1,000; Proteintech; cat. no. 67447-1-Ig), RPL11 (1:1,000; Proteintech; cat. no. 16277-1-AP), Vimentin (1:1,000; Proteintech; cat. no. 10366-1-AP), RPL23 (1:1,000; ABclona; cat. no. A4292) and Snail (1:1,000; ABclona; cat. no. A11794) at 4°C, followed by the secondary antibodies at room temperature (1:3,000, Bioss; cat. nos. bs-40296G-HRP and bs-40295G-HRP) for 1 h. The positive bands were visualized using enhanced chemiluminescence (ECL) kit and the bands were measured using ImageJ (v1.53, National Institutes of Health).

Techniques: Knockdown, Western Blot, Expressing, Inhibition, Control