Fig 2: MiR-146a conducted NUMB degradation was blocked by TUSC7 in a sponge combination manner. A The predicated connection sites between TUSC7 and its binding partners showed that miR-146a shared the common untranslated regions. B Recombined miR-146a mimics decreased the Luc-activity of TUSC7 in 293 T cells. MiR-146a decreased the Luc-activity of TUSC7 in PC9ER cells (C) and HCC827ER cells (D). E Blotting results referring to RNA pull-down test showed the connection between TUSC7 and NUMB in PC9ER and HCC827ER cells. F RNA immunoprecipitation revealed that TUSC7 was enriched with NUMB expression in PC9ER cells (Left) and HCC827ER cells (Right), and the IgG was set as the immunoprecipitation control, the MALAT1 was set as the primer control. G Informatic screening of the potential miRNAs’ targets suggested that miR-146a may bind to NUMB. H The expression level of NUMB with 526 cancer and 59 normal samples in LUAD, and the expressions with 501 cancer and 49 normal samples in LUSC were evaluated, and NUMB decreased greatly in cancer group. I The co-transfection of miR-146a mimics and the wide-type reporter plasmid strongly reduced the EGFP intensity, and miR-146a mimics reduced nearly 40% of the TUSC7 luciferase intensity, but not happened in mutant-type reporter plasmid. J TUSC7 alone did not reduce the EGFP activity of NUMB. K MiR-146a decreased the NUMB expression, which could be rescued by TUSC7, and the TUSC7 inhibition (TUSC7-in) also decreased the NUMB level

Fig 3: Aberrant Notch signaling activation in Lung adenocarcinoma. Notch signaling of NOTCH1, NOTCH2, NOTCH3, NOTCH4, DVL1, NUMB, NCSTN, APH1A, SNW1, DTX2, DTX3, DTX1, NCOR2, CTBP2, CTBP1, HDAC1, HDAC2, CIR1, RBPJ, RBPJL, CREBBP, KRAS, MAPK1, MAPK1 were applied for expression level detection, and a total of 507 lung adenocarcinoma samples with mutation and CNA data (TCGA, Pan-Cancer Atlas) were collected. A Expression plots showed the key members of Notch signaling were amplified, together with EGFR overexpression, and KRAS mutations. B Heat map results revealed the universal overexpression of Notch signaling participants, and the TUSC7 result was limited due to the mRNA expression screening system restrictions. Specifically, expression level of TUSC7 showed the reverse consistency with the Notch signaling activation patterns. The TCGA samples data with mutation and CAN from Pan-Lung Cancer (Nat Genet 2016) indicated the grouped enrichment of the Notch signaling factors in Volcano Plots (C), and the changing frequency of each member was consistent with groups alternations (D). E-F The Overall survival and Disease/Progression-free estimates with using Kaplan-Meier analysis showed that Notch signaling activation decreased the survival time, and patients tended to bear relapse or resistance in shorter follow-up periods. G The relative higher level of TUSC7 indicated longer survival time of all lung carcinoma patients significantly, comparting to the lower expressed groups. Sourced data of Star-Base (v3.0 Project) were collected and analyzed, and the results were calculated with using RPM/Log manner, and a total of 512 lung adenocarcinoma samples were enrolled and applied for analysis. H The positive relationship between expression level of miR-146a and level of EGFR was found in total of 512 lung adenocarcinoma samples. Both Notch 1 (I) and Notch 2 (J) were positively correlated to miR-146a expression, pointing to the oncogenic functions of miR-146a