Fig 1: LINC00355 promoted GEFT expression and improved GEFT mRNA stability by recruiting RBP LIN28A. (A) Screening of seven CRC differentially expressed RBPs that have the potential to interact with both LINC00355 and GEFT through Venn analysis. Green: Starbase-predicted RBPs with the possibility to interact with LINC00355; blue: Starbase-predicted RBPs with the probability to interact with GEFT; red: Differentially expressed genes in CRC tissues versus normal tissues. (B) GEFT mRNA level enriched by IgG or LIN28A antibody were examined by RIP coupled with RT-qPCR assay in HCT-116 cells. (C) Predicted binding sites between LIN28A and GEFT 3’UTR. (D) RNA pull-down and western blot assays were performed to test LIN28A protein level pulled down by biotinylated sense or antisense GEFT 3’UTR. (E) HCT-116 cells transfected with pcDNA3.1 empty vector or LIN28A overexpression plasmid were co-transfected with pRL-TK Renilla luciferase plasmid and pGL3-Basic luciferase vector/construct, followed by the measurement of luciferase activity at 48 h post transfection. (F) RNA pull-down and western blot assays were performed to test LIN28A protein level pulled down by biotinylated sense or antisense LINC00355. (G) LINC00355 level enriched by IgG or LIN28A antibody was examined by RIP coupled with RT-qPCR assay. (H) HCT-116 cells were transfected with si-con or si- LINC00355#1. At 48 h post transfection, RIP coupled with RT-qPCR assay was performed to test GEFT mRNA level enriched by IgG or LIN28A antibody. (I, J) SW480 and HCT-116 cells were transfected with empty vector, LINC00355 overexpression plasmid, LINC00355 overexpression plasmid + si-con, LINC00355 overexpression plasmid + si-LIN28A. (I) At 48 h upon transfection, GEFT mRNA and protein levels were measured by RT-qPCR and western blot assays, respectively. (J) At 24 h post transfection, cells were treated with actinomycin D. At the indicated time points after actinomycin D treatment, GEFT mRNA level was measured by RT-qPCR assay. **P < 0.01. ***P < 0.001. ## P < 0.01. ### P < 0.001.
Fig 2: Lin28A promoted the proliferation and invasion of OC cells by up-regulating RAN. (A) Transiently transfected siRNA knocked down the expression of RAN at the protein level (right) and the RNA level (left) in A2780 Lin28A cells. (B) The role of knockdown RAN on the expression of the stem cell marker molecules CD44, CD133, ABCG2, OCT4, Nanog and SOX2 were measured with qRT-PCR. (C) CCK-8 analysis showed that siRAN reduced the proliferation rate of A2780 Lin28A (top) and PA-1 (bottom) cells compared to control siNC. (D) Knockdown of RAN by siRNA promoted the apoptosis of A2780 Lin28A and PA-1 cells by flow cytometry analysis. (E) Knockdown of RAN by siRNA promoted the expression of cleavage caspase-7, and reduced the expression of PARP with western blot, actin as an internal reference. (F) Knockdown of RAN decreased the invasion of A2780 Lin28A (top) and PA-1 (bottom) cells. (G) The role of knockdown RAN in the expression of invasion-related molecules was examined with western blot, actin as an internal reference.
Fig 3: A model for Lin28A/RAN/HSBP1 promotes stemness and invasion and inhibits cell apoptosis of OC cells. Lin28A enhanced the stem-like features through up-regulating RAN/HSBP1 and subsequent surface marker molecules CD44, OCT4 and Nanog; Lin28A inhibited the apoptosis of OC cells through up-regulating RAN/HSBP1 and subsequent inhibition of poly ADP ribose polymerase PARP; Lin28A promoted the invasion of OC cells by up-regulating RAN/HSBP1 and subsequent expression of EMT-related molecule.
Fig 4: LIN28A knockdown weakened the promotive effects of LINC00355 on CRC cell proliferation, migration, and invasion. (A–C) SW480 and HCT-116 cells were transfected with empty vector, LINC00355 overexpression plasmid, LINC00355 overexpression plasmid + si-con, LINC00355 overexpression plasmid + si-LIN28A. (A) Cell proliferative ability was assessed by CCK-8 assay at the indicated time points post transfection. (B, C) At 24 h post transfection, cell migratory and invasive abilities were determined by Transwell migration and invasion assays, respectively. **P < 0.01. ***P < 0.001. ## P < 0.01.
Fig 5: Lin28A can up-regulate stem-like properties of OC cells. (A-C) The effects of overexpression of Lin28A on side population cells of A2780 cells were detected by flow cytometry analysis (A2780 Ctrl and Verapamil group as negative control and blank control, respectively). (D-G) The role of Lin28A on the proportion of CD133+, CD44+, ABCG2+ cells were detected by flow cytometry analysis. (H) The effects of Lin28A on the expression of the stem cell marker molecules CD44, CD133, ABCG2, OCT4, Nanog and SOX2 were measured with qRT-PCR.
Supplier Page from Abcam for Anti-Lin28A antibody [EPR4640]