Journal: Carcinogenesis

Article Title: Lin28B promotes melanoma growth by mediating a microRNA regulatory circuit.

doi: 10.1093/carcin/bgv085

Figure Lengend Snippet: Figure 2. Lin28B is a direct target and functional effector of miR-125a-5p in melanoma. (A) Heatmap of differentially expressed genes in both melanoma cell lines overexpressing miR-125a-5p compared with control. (B) Alignment of the predicted miR-125a-5p binding site in Lin28B 3′ UTR. (C) Dual luciferase assay on wild-type or mutated Lin28B 3′ UTR in COS-7 cells transfected with control or miR-125a-5p. *P < 0.05, Student’s t-test. (D) Quantitative PCR of Lin28B in LOX-IMVI and A375 cell lines infected with miR-125a-5p or control lentivirus. Each group had three biological replicates. *P < 0.05, Student’s t-test. (E) Western blot analysis of Lin28B in LOX-IMVI and A375 cell lines infected with miR-125a-5p or control lentivirus. Two bands were detected, corresponding to the two Lin28B isoforms. (F) Quantitative PCR of miR- 125a-5p and Lin28B in LOX-IMVI cells expressing control, miR-125a-5p or both miR-125a-5p and Lin28B. Each group had three biological replicates. *P < 0.05, analysis of variance followed by Tukey’s post-test. (G) Tumor volume of LOX-IMVI cells expressing control, miR-125a-5p or both miR-125a-5p and Lin28B, ten mice per group. *P < 0.05, analysis of variance followed by Tukey’s post-test.

Article Snippet: Antibodies against the following proteins were used: Lin28B, pSer-465/467 Smad2, Actin (Cell Signaling Technology). miRNA profiling and quantitative PCR Total cell RNA was extracted with mirVana miRNA isolation kit (Ambion) following the manufacturer’s protocol. miRNA in 500 ng total RNA was reverse transcribed using the Megaplex reverse transcriptase primer pools and the Taqman miRNA reverse transcription kit (Applied Biosystems), and loaded into the Taqman array miRNA cards.

Techniques: Functional Assay, Control, Binding Assay, Luciferase, Transfection, Real-time Polymerase Chain Reaction, Infection, Western Blot, Expressing

Journal: Carcinogenesis

Article Title: Lin28B promotes melanoma growth by mediating a microRNA regulatory circuit.

doi: 10.1093/carcin/bgv085

Figure Lengend Snippet: Figure 3. Lin28B is mutated and overexpressed in a subset of melanoma patients. (A) Mutation spectrum of Lin28B in melanoma patients. (B) Lin28B expression in normal skin tissues, early or late stages of melanoma, as measured by quantitative PCR. *P < 0.05, analysis of variance. (C) Lin28B expression in primary or metastatic melanoma, as measured by RNA-sequencing. *P < 0.05, Student’s t-test. (D) Western blot analysis of Lin28B in a panel of melanoma cell lines. (E) Immunohistochemistry of Lin28B in a tissue microarray containing 191 melanoma samples.

Article Snippet: Antibodies against the following proteins were used: Lin28B, pSer-465/467 Smad2, Actin (Cell Signaling Technology). miRNA profiling and quantitative PCR Total cell RNA was extracted with mirVana miRNA isolation kit (Ambion) following the manufacturer’s protocol. miRNA in 500 ng total RNA was reverse transcribed using the Megaplex reverse transcriptase primer pools and the Taqman miRNA reverse transcription kit (Applied Biosystems), and loaded into the Taqman array miRNA cards.

Techniques: Mutagenesis, Expressing, Real-time Polymerase Chain Reaction, RNA Sequencing, Western Blot, Immunohistochemistry, Microarray

Journal: Carcinogenesis

Article Title: Lin28B promotes melanoma growth by mediating a microRNA regulatory circuit.

doi: 10.1093/carcin/bgv085

Figure Lengend Snippet: Figure 5. Lin28B blocks the let-7 miRNA cluster and activates the TGF-β pathway in melanoma. (A) Quantitative PCR of let-7 family miRNAs in melanoma cells over- expressing Lin28B relative to control. Each group had three biological replicates. *P < 0.05, Student’s t-test. (B) Sylamer analysis of over-represented miRNA seed sites in melanoma cells overexpressing Lin28B. (C) Heatmap of differentially expressed genes regulated by Lin28B in all three models. (D) Western blot analysis of TGF-β1- induced pSmad2 upon Lin28B knockdown or overexpression. (E) A schematic model of Lin28B-regulated miRNA circuit in melanoma.

Article Snippet: Antibodies against the following proteins were used: Lin28B, pSer-465/467 Smad2, Actin (Cell Signaling Technology). miRNA profiling and quantitative PCR Total cell RNA was extracted with mirVana miRNA isolation kit (Ambion) following the manufacturer’s protocol. miRNA in 500 ng total RNA was reverse transcribed using the Megaplex reverse transcriptase primer pools and the Taqman miRNA reverse transcription kit (Applied Biosystems), and loaded into the Taqman array miRNA cards.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control, Western Blot, Knockdown, Over Expression