Journal: iScience

Article Title: MicroRNA-31 induced by Fusobacterium nucleatum infection promotes colorectal cancer tumorigenesis

doi: 10.1016/j.isci.2023.106770

Figure Lengend Snippet: STX12, eIF4EBP1, and eIF4EBP2 are direct targets of miR-31 (A) The differentially expressed mRNAs among HCT116 cells transfected with Lv-miR-31, Lv-NC, Lv-anti-miR-31, or Lv-anti-NC, as identified by microarray. (B) eIF4EBP1/2 and STX12 were identified as the direct targets of miR-31 as detected and verified by the TargetScan prediction, MiRnada, miRDB, GO annotation, and target of microarray, respectively. (C) The region of the human eIF4EBP1/2 and STX12 mRNA 3′UTR was predicted to be targeted by miR-31 (TargetScan 7.2). (D) Luciferase reporter plasmids were generated with either WT or MT miR-31 binding sites in 3′-UTR of eIF4EBP1/2 and STX12. The activity of the luciferase constructs was evaluated and normalized to the activity of Renilla luciferase after transfecting HEK293T cells with a miR-31 mimic, an inhibitor, or a control. (E and F) HCT116 cells were transfected with the mimic or the inhibitor of miR-31. After co-culture with F. nucleatum for 4 h, the mRNA and protein expression of eIF4EBP1/2 and STX12 was detected by RT-qPCR and Western blotting, respectively.

Article Snippet: STX12 shRNA (human) lentivirus , Santa Cruz , Cat# sc-88621-V.

Techniques: Transfection, Microarray, Luciferase, Generated, Binding Assay, Activity Assay, Construct, Control, Co-Culture Assay, Expressing, Quantitative RT-PCR, Western Blot

Journal: iScience

Article Title: MicroRNA-31 induced by Fusobacterium nucleatum infection promotes colorectal cancer tumorigenesis

doi: 10.1016/j.isci.2023.106770

Figure Lengend Snippet: miR-31 inhibits STX12 to block the fusion of autophagosomes with lysosomes during Fusobacterium nucleatum infection (A) The protein expression of STX12 in miR-31 overexpressed or inhibited HCT116 cells infected with F. nucleatum (MOI = 100:1) for 6 h. (B) Western blotting detected the protein expression of STX12 in F. nucleatum -infected miR-31 wt or miR-31 −/− mice tissues (n = 5 per group). (C) The protein expression of STX12 was detected by Western blotting in F. nucleatum- positive CRC tissues (Fn + CRC, n = 5) and F. nucleatum- negative CRC tissues (Fn − CRC, n = 5). (D and E) HCT116 cells were treated with Lv-sh-NC or Lv-sh-STX12, and then infected with F. nucleatum (MOI = 100). The expression of MAP1LC3B-I/II and SQSTM1 was detected by Western blotting, and the mRFP-EGFP-LC3 fusion protein was detected by laser scanning confocal microscopy (LSCM). (F) After pretreatment with Lv-sh-NC or Lv-sh-STX12, HCT116 cells were infected with F. nucleatum (MOI = 100:1) for 6 h. Then the intracellular survival of F. nucleatum in HCT116 cells were detected by TEM. (G) HCT116 cells treated with Lv-sh-STX12 were transiently transfected with a miR-31 mimic or an inhibitor, and then infected with F. nucleatum, the intracellular survival of F. nucleatum was detected 6 h post-infection. (H) Both miR-31 wt and miR-31 −/− mice were initially injected with Av-sh-NC or Av-sh-STX12 via the caudal vein and then infected with F. nucleatum for 20 weeks. (I) Representative images of H&E staining of the colonic epithelium from miR-31 wt and miR-31 −/− mice. (J) The colonization of F. nucleatum in colorectal tissues of miR-31 wt and miR-31 −/− mice. ∗p < 0.05.

Article Snippet: STX12 shRNA (human) lentivirus , Santa Cruz , Cat# sc-88621-V.

Techniques: Blocking Assay, Infection, Expressing, Western Blot, Confocal Microscopy, Transfection, Injection, Staining

Journal: iScience

Article Title: MicroRNA-31 induced by Fusobacterium nucleatum infection promotes colorectal cancer tumorigenesis

doi: 10.1016/j.isci.2023.106770

Figure Lengend Snippet: NF-κB is a transcription factor for miR-31 A) KEGG pathway analysis of a total of 1074 mRNAs involved in F. nucleatum -associated CRC. (B) Western blotting was performed to measure the protein levels of phosphorylated p65 in the cytoplasm and nucleus of HCT116 cells after F. nucleatum infection. (C and D) miR-31 and pri-miR-31 expression were detected by RT-qPCR in HCT116 or LoVo cells transfected with Lv-sh p65. (E) HCT116 cells transfected with Lv-sh p65 were infected with F. nucleatum for 2 h, and the primary transcript (pri-miRNA) of miR-31 was quantified by real-time PCR. ∗p < 0.05. (F) pGL3 luciferase reporter plasmids that either have the wild-type (WT) or mutant (MT) p65 binding site in the miR-31 promoter. (G) Luciferase constructs (pGL3-WT or pGL3-MT) or Lv-sh p65 were co-transfected into HEK293T cells prior to F. nucleatum infection. The activity of luciferase was adjusted to match that of Renilla luciferase. ∗p < 0.05. (H) miR-31 promoter was detected in the chromatin sample immunoprecipitated from HCT116 (left) and LoVo (right) cells using an antibody against p65. A schematic representation of the mechanism by which F. nucleatum induces intestinal inflammation and colorectal tumorigenesis via targeting NF-κB, miR-31, STX12, and the autophagy pathway.

Article Snippet: STX12 shRNA (human) lentivirus , Santa Cruz , Cat# sc-88621-V.

Techniques: Western Blot, Infection, Expressing, Quantitative RT-PCR, Transfection, Real-time Polymerase Chain Reaction, Luciferase, Mutagenesis, Binding Assay, Construct, Activity Assay, Immunoprecipitation

Journal: iScience

Article Title: MicroRNA-31 induced by Fusobacterium nucleatum infection promotes colorectal cancer tumorigenesis

doi: 10.1016/j.isci.2023.106770

Figure Lengend Snippet:

Article Snippet: STX12 shRNA (human) lentivirus , Santa Cruz , Cat# sc-88621-V.

Techniques: Control, Virus, Luciferase, Plasmid Preparation, shRNA, Recombinant, Protein Extraction, BIA-KA, CCK-8 Assay, Reverse Transcription, Knock-Out, Software, Microscopy