Fig 2: The regulatory roles of NF90 on miR-548k. (A) After transfection of NF90 overexpression or control plasmids into KYSE30 cells, NF90 expression were measured by western blot. (B) After transfection of NF90 overexpression or control plasmids into KYSE30 cells, miR-548k expression were measured by qRT-PCR. (C) After transfection of NF90 specific or control shRNAs into Eca-109 cells, NF90 expression were measured by western blot. (D) After transfection of NF90 specific or control shRNAs into Eca-109 cells, miR-548k expression were measured by qRT-PCR. (E) RIP assay followed by qRT-PCR was performed to detect the specific enrichment of pri-miR-548k with NF90 specific antibody compared with nonspecific IgG. pri-miR-21 was used as negative control. (F) After transfection of NF90 overexpression or control plasmids into KYSE30 cells, the stability of pri-miR-548k transcript over time was evaluated by qRT-PCR relative to time 0 after blocking new RNA synthesis with α-amanitin and normalized to 18S rRNA (transcribed by RNA polymerase I and not influenced by α-amanitin). (G) After transfection of NF90 specific or control shRNAs into Eca-109 cells, the stability of pri-miR-548k transcript over time was evaluated by qRT-PCR relative to time 0 after blocking new RNA synthesis with α-amanitin and normalized to 18S rRNA. (H) After transfection of NF90 overexpression or control plasmids into KYSE30 cells, pri-miR-548k expression was measured by qRT-PCR. (I) After transfection of NF90 specific or control shRNAs into Eca-109 cells, pri-miR-548k expression was measured by qRT-PCR. Results are presented as mean ± S.D. (n = 3). **P < 0.01, ***P < 0.001, ns, not significant, by Student's t-test (A, B, E, F and H) or one-way ANOVA followed by Dunnett's multiple comparison test (C, D, G and I).

Fig 3: LincIN interacts with NF90 in the translation regulation of p21 expression. a Left panel: Western blot of p21 and beta-actin in vector and LincIN overexpressed MCF10A cells. Right panel: Western blot of p21, beta-actin or NF90, in SC and LincIN knockdown groups of HeLa cells. b RT-qPCR results of p21 in LincIN overexpression and vector control groups in MCF10A cells (left). RT-qPCR analysis of LincIN knockdown versus scrambled control in HeLa cells (right). (c) and (d) Cell cycle profiles and quantification of MDA-MB-231 cells treated with SC or LincIN siRNAs for 48 h (n = 3). Data was analyzed using one-way ANOVA and t test (** P < 0.01, and * P < 0.05). e Right panel: MCF10A cells (vector or LincIN) were stably transfected with siRNA targeting NF90 or scrambled control. Cell lysates were collected at 48 h after transfection. Left panel: ratios of p21expression in LincIN-overexpression to vector control cells (right panel) (* P < 0.05; t test). f Right panel: Western blot of NF90, NF45, and p21 in HeLa cells treated with SC, LincIN or NF90 siRNA alone or in combination. Cell lysates were collected at 48 h after transfection, repeated in triplicate. Left panel: ratios of p21expression in siLincIN-transfected to SC control cells (* P < 0.05; t test)

Fig 4: LincIN interacts with the NF90/NF45 complex. a Schematic flow of RNA pull-down experiments. b RNA pull-down was performed using the RNA-protein Pull Down Kit. Bands with arrows were submitted for mass spectrometric identification, and the most abundant band was identified as NF90/ILF3. Positive control (PC) and negative control (NC) were HuR and polyA as provided by the RNA pull-down kit, n = 4. c Western blot analysis was used to validate the specific association of NF90 with LincIN in pull-down lysates, repeated experiment. d Left panel: schematic diagram of LincIN fragments; right panel: Western blot analysis of NF90 in eluted protein samples pulled down by in vitro transcribed LincIN fragments. e RT-qPCR analysis of RNP samples (right) enriched by NF90 antibodies. RNP: RNA immunoprecipitation. Data was analyzed using t test (** P < 0.01, and * P < 0.05)

Fig 5: Activation of the NF90/miR-548k/lncRNA-LET feedback loop significantly promotes ESCC progression. (A) NF90 expressions in NF90 and miR-548k simultaneously overexpressed and lncRNA-LET simultaneously depleted KYSE30 cells were measured by western blot. (B) miR-548k and lncRNA-LET expressions in NF90 and miR-548k simultaneously overexpressed and lncRNA-LET simultaneously depleted KYSE30 cells were measured by qRT-PCR. (C) Glo cell viability assay was performed to detect cell viability of NF90 and miR-548k simultaneously overexpressed and lncRNA-LET simultaneously depleted KYSE30 cells. (D) EdU incorporation assay was performed to detect cell proliferation of NF90 and miR-548k simultaneously overexpressed and lncRNA-LET simultaneously depleted KYSE30 cells. The red color represents EdU-positive and proliferation active cells. Scale bars, 100 μm. (E) Transwell assay was performed to detect cell migration of NF90 and miR-548k simultaneously overexpressed and lncRNA-LET simultaneously depleted KYSE30 cells. Scale bars, 100 μm. For A-E, results are presented as mean ± S.D. (n = 3). ***P < 0.001 by Student's t-test. (F) NF90 and miR-548k simultaneously overexpressed and lncRNA-LET simultaneously depleted KYSE30 cells were subcutaneously injected into nude mice. Xenograft tumor volumes were detected every four days. (G) Xenograft tumor weights were detected at the 20th days after injection. For F-G, results are presented as mean ± S.D. (n = 5 mice). **P < 0.01 by Mann-Whitney U test.