Fig 1: miRNA-143-3p andITM2Bcould be biomarkers for lung cancer. (A and B) Proliferation rate of LTEP-a-2 (A) and SPC-A-1 (B) cells were assessed using CCK-8 assays every 24 hours following treatment with OE-miRNA-143-3p lentivirus and/or OE-ITM2B lentivirus treatment compared with control group; (C and D) Ki67 levels in LTEP-a-2 (C) and SPC-A-1 (D) cells were analyzed using RT-qPCR following OE-miRNA-143-3p lentivirus and/or OE-ITM2B lentivirus treatment compared with control group; (E) Tumor growth rate of lung cancer xenograft with OE-miRNA-143-3p lentivirus and/or OE-ITM2B lentivirus treatment compared with control group; (F) Kaplan–Meier overall survival curve in mice with OE-miRNA-143-3p lentivirus and/or OE-ITM2B lentivirus treatment compared with control group; (G) Correlation of miRNA-143-3p and ITM2B in lung cancer tissues; (H) ROC analysis of the individual miRNA-143-3p in the lung cancer tissues and normal lung tissues; (I) ROC analysis of the individual ITM2B in the lung cancer tissues and normal lung tissues. Mean±SEM, *P<0.05, **P<0.01, ***P<0.005, ****P<0.001.
Fig 2: Schematic model. G-MDSC-derived miRNA-143-3p to promote proliferation of lung cancer cells by targeting ITM2B via PI3K/Akt pathway activation.
Fig 3: ITM2B was decreased in lung cancer tissues and inhibited tumor progression. (A) ITM2B levels in lung cancer tissues compared to normal lung tissues according to TCGA database; (B)ITM2B levels in different stages of lung cancer and normal lung tissues analyzed using RT-qPCR. (C)ITM2B transcription levels in 10 lung cancer cell lines; (D) ITM2B level in LTEP-a-2 cells after mock, empty vector lentivirus, and OE-ITM2B lentivirus treatment for 24 hours; (E) Proliferation rate of LTEP-a-2 cells were assessed using CCK-8 assays every 24 hours following treatment with OE-ITM2B lentivirus compared with empty vector lentivirus treatment; (F) Ki67 levels in LTEP-a-2 cells were assessed using RT-qPCR following OE-ITM2B lentivirus treatment compared with empty vector lentivirus treatment; (G) Tumor growth rate of lung cancer xenograft following OE-ITM2B lentivirus treatment compared with empty vector lentivirus treatment; (H) Kaplan–Meier overall survival curve in mice with OE-ITM2B lentivirus treatment compared with empty vector lentivirus treatment. Mean±SEM, *P<0.05, ****P<0.001, NS, no statistical significance.
Fig 4: ITM2B is a target of miRNA-143-3p in lung cancer cells. (A) RNA sequencing results of downregulated genes in LTEP-a-2, SPC-A-1, and A549 cells following miRNA-143-3p overexpression; (B) Venn diagram of intersection of target genes of miRNA-143-3p predicted using several bioinformatics databases and RNA sequencing results; (C) Levels of four downregulated genes in 10 lung cancer cells following transfection with OE-miRNA-143-3p lentivirus; (D) Representative images of Western blot analysis ofITM2B proteins in lung cancer cells following OE-miRNA-143-3p lentivirus treatment for 48 hours; (E) Schematic view of putative miRNA-143-3p targeting site in the WT and Mut3'-UTR of ITM2B; (F and G) Luciferase activity assay in LTEP-a-2 (F) and SPC-A-1 (G) cells transfected with luciferase report plasmids containing ITM2B3'-UTR (WT or Mut), and control miRNA or miRNA-143-3p. Mean±SEM, ****P<0.001, NS, no statistical significance.
Fig 5: Silencing ITM2B was signi?cantly active PI3K/Akt signaling pathway in lung cancer cells. (A) Hierarchical clustering and heatmap analysis of RNA sequencing results of ITM2B knockdown LTEP-a-2 cells and control group; (B) Pathway analysis of the differentially expressed genes using the KEGG database; (C and D) The phosphorylation and total expression levels of PI3K and Akt in LTEP-a-2 cells (C) and SPC-A-1 cells (D). Cells infected with SH-ITM2B lentivirus and/or Deguelin. The expression levels of the phosphorylated proteins were normalized to those of the respective total protein; (E) Proliferation rate of LTEP-a-2 cells was analyzed using CCK-8 assays every 24 hours following treatment with SH-ITM2B lentivirus and/or Deguelin treatment compared with the control group; (F) Ki67 levels of LTEP-a-2 cells were analyzed using RT-qPCR following SH-ITM2B lentivirus and/or Deguelin treatment compared with the control group; (G) Tumor growth rate of lung cancer xenograft with SH-ITM2B lentivirus and/or Deguelin treatment compared with the control group; (H) Kaplan–Meier overall survival curve in mice with SH-ITM2B lentivirus and/or Deguelin treated compared with the control group. Mean±SEM, *P<0.05, **P<0.01, ***P<0.005, ****P<0.001.
Supplier Page from Abcam for Anti-ITM2B antibody