Journal: Scientific Reports

Article Title: MicroRNA-141-regulated KLK10 and TNFSF-15 gene expression in hepatoblastoma cells as a novel mechanism in liver carcinogenesis

doi: 10.1038/s41598-024-63223-4

Figure Lengend Snippet: Microarray analysis of transfected HepG2 cells with miR-141 overexpression and its predicted targets. ( A ) Microarray analysis of gene expression in HepG2 cells transfected with miR-141 overexpression vs. cells transfected with the control vector using a miRNA library from Exiqon. ( B ) Microarray analysis of gene expression in HepG2 cells transfected with miR-141 overexpression vs. cells transfected with the control vector using oligonucleotides from Ambion. The blue color indicates the sustained gene expression, the red color indicates the upregulated genes, and the green color indicates the downregulated genes. ( C ) The expression of the most relevant genes to liver cancer indicates the upregulated genes in red columns and downregulated genes in green. Error bars indicate the STD between Exiqon and Ambion data. ( D ) The potential seeding regions of hsa-miR-141 within the coding sequences and 3-UTR of KLK10 and TNFSF-15 gene sequences that carried out in-silico by miRWalk online tool.

Article Snippet: The cells were then incubated in dark conditions for 2 h. The same instructions was followed for staining the cells with the primary and secondary antibodies against TNFSF-15 using rabbit monoclonal anti- TNFSF-15 (1–500) (Ab 275022, Abcam, USA) and goat anti-rabbit IgG (1–1000) (Alexa Fluor 594, Abcam, USA) .

Techniques: Microarray, Transfection, Over Expression, Expressing, Plasmid Preparation, In Silico

Journal: Scientific Reports

Article Title: MicroRNA-141-regulated KLK10 and TNFSF-15 gene expression in hepatoblastoma cells as a novel mechanism in liver carcinogenesis

doi: 10.1038/s41598-024-63223-4

Figure Lengend Snippet: The correlation between the level of miR-141 and KLK10 and TNFSF-15 on gene expression level and protein level in HepG2 cells. ( A ) The relative gene expression of KLK10 and TNFSF-15 in HepG2 cells transfected with the inhibitor against miR-141 and miR-141 overexpression vector indicated by fold change compared with control-transfected and nontreated cells using qRT-PCR. Error bars indicate the STD of three independent experiments. Student two-tailed t -test used for statistical analysis , (*) indicates P-values ≤ 0.05, and (**) indicates P ≤ 0.01. ( B ) Flow cytometric assay quantifies the kinetic proteins expression profile of KLK10 (the blue dots) and TNFSF-15 (the red dots) in transfected cells compared with control-transfected and nontreated (NT) cells. ( C ) Western blot analysis shows the protein expression of KLK10 and TNF SF15 in transfected cells compared to control-transfected and nontreated cells. β-actin expression severed as an internal control. ( D and E ) Schematic representation of miR-141 binding sites and seeding regions (SR) in KLK10 and TNFSF-15 gene sequence indicated by IntaRNA program. ( F ) In HepG2 cells pre-transfected with miR-141 overexpressing vector or specific inhibitor, the luciferase activities upon cotransfection with luciferase reporter constructs, pGL3-KLK10 or pGL3-TNFSH-15 compared with cells cotransfected with pGL3-control vector. Error bars reveal the STD of three replicates. Student two-tailed t -test used for statistical analysis, (*) indicates P- values ≤ 0.05, and (**) indicates P ≤ 0.01.

Article Snippet: The cells were then incubated in dark conditions for 2 h. The same instructions was followed for staining the cells with the primary and secondary antibodies against TNFSF-15 using rabbit monoclonal anti- TNFSF-15 (1–500) (Ab 275022, Abcam, USA) and goat anti-rabbit IgG (1–1000) (Alexa Fluor 594, Abcam, USA) .

Techniques: Expressing, Transfection, Over Expression, Plasmid Preparation, Quantitative RT-PCR, Two Tailed Test, Flow Cytometry, Western Blot, Binding Assay, Sequencing, Luciferase, Cotransfection, Construct