Journal: Signal Transduction and Targeted Therapy

Article Title: Semaphorin 3C (Sema3C) reshapes stromal microenvironment to promote hepatocellular carcinoma progression

doi: 10.1038/s41392-024-01887-0

Figure Lengend Snippet: Sema3C is involved in the ECM remodeling and induces the HSCs activation. a GO analysis based on intersection proteins binding to Sema3C and NRP1. b Representative images of collagen gel contraction assays with MHCC-97L cells in either MHCC-97L supernatants or OE-Sema3C-MHCC-97L supernatants. Graph quantifying the change in the percentages of gel contraction by MHCC-97L cells in different conditions. Scale bar, 5 mm. c Representative images showing H&E, Masson’s trichrome and picrosirius red staining, and IHC staining for α-SMA and collagen I expression in serial sections of orthotopic liver xenograft tumors of BALB/C nude mice injected with MHCC-97L-Vector or MHCC-97L-OE-Sema3C cells. Gross tissue image scale bar, 1 cm, microscope image scale bar, 200 μm. d Percent of collagen fibers and α-SMA per field were counted in at least three random fields per animal, (n = 5). e Representative immunofluorescence images for α-SMA and collagen I in HSCs treated with dosage rhSema3C or supernatants of different Sema3C expression levels. Scale bar, 50 μm. LX-2 cells were treated with a gradient dose of rhSema3C or supernatants from Sema3C-overexpressed Hep3B and MHCC-97L cells. The α-SMA expression levels were detected by western blotting analysis ( f ). The TGF-β1 secretion was examined by ELISA assay ( g ). The chemoresistance ability was reflected by an MTT assay ( h ). The ability of migration and invasion was performed by Transwell assays, scale bar, 200 μm ( i ). 5 × 10 5 Hep3B cells or OE-Sema3C Hep3B cells were injected subcutaneously into nude mice alone or mixed with LX-2 cells in a 1:1 ratio. The mice were sacrificed, and the xenograft tumors were excised 32 days after inoculation ( j ). The bar chart showed the tumor weight in each treatment group ( k ). The tumor volumes were monitored for 32 days ( l ). n = 5 per group. Data are presented as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: To verify the effect of TGF-β1 on Sema3C expression in HCC, 1 μg/mL anti-TGF-β1 antibody or an IgG control (Proteintech, China) was added to the CM of CAFs and incubated at room temperature for 1 h. The treated CM was then used to co-culture with the corresponding HCC cells.

Techniques: Activation Assay, Binding Assay, Staining, Immunohistochemistry, Expressing, Injection, Plasmid Preparation, Microscopy, Immunofluorescence, Western Blot, Enzyme-linked Immunosorbent Assay, MTT Assay, Migration

Journal: Signal Transduction and Targeted Therapy

Article Title: Semaphorin 3C (Sema3C) reshapes stromal microenvironment to promote hepatocellular carcinoma progression

doi: 10.1038/s41392-024-01887-0

Figure Lengend Snippet: IL-6 and cholesterol biosynthesis are responsible for Sema3C-mediated HSCs activation. a The volcano map showed the differentially expressed genes of LX-2 cells treated with rhSema3C or PBS in vitro. HSCs were treated with rhSema3C or supernatants of MHCC-97L cells with different Sema3C expression levels. mRNA levels of IL6 and IL8 were detected using qRT-PCR ( b ). ELISA was used to examine the IL6 secretion level ( c ). d GSEA identified an enrichment of genes involved in the NF-κB pathway in rhSema3C-treated LX-2 cells. e Western blotting showed phosphorylated and total p65 protein expression levels in LX-2 cells treated with rhSema3C. LX-2 cells were pre-treated with Bay 11-7082 and subsequently stimulated with rhSema3C, the levels of phosphorylated p65, total p65, and α-SMA expression were determined by western blotting ( f ), and IL6 secretion levels were examined by ELISA assay ( g ). h LX-2 cells were treated with OE-Sema3C-Hep3B cell-derived supernatants, Co-IP assay was used to detect the interaction between ITGB1 and NRP1. LX-2 cells were transfected with siNRP1 and subsequently stimulated with rhSema3C, IL6 secretion levels were detected by ELISA ( i ). The phosphorylated p65, total p65, α-SMA, and HMGCR expression levels were determined by using western blotting ( j ). k LX-2 cells were treated with a dosage of rhSema3C or supernatants of Sema3C-overexpressed HCC cells, and ITGB1 protein expression was determined by western blotting. LX-2 cells were transfected with siITGB1 and subsequently stimulated with rhSema3C, and the IL6 secretion levels were detected by ELISA assay ( l ), the phosphorylated p65, total p65, and α-SMA expression levels were determined by using Western blotting ( m ). LX-2 cells were treated with siNRP1, siITGB1 or rhSema3C as indicated, the secretion of TGF-β1 in each group was detected by ELISA ( n ), the chemotherapy resistance of LX-2 cells was evaluated by MTT assay ( o ), and migration and invasion were performed by Transwell assay, scale bar, 200 μm ( p ). rhSema3C recombinant human Sema3C; Data are presented as means ± SD. ns not significantly; *P < 0.05, **P < 0.01, and ***P < 0.001

Article Snippet: To verify the effect of TGF-β1 on Sema3C expression in HCC, 1 μg/mL anti-TGF-β1 antibody or an IgG control (Proteintech, China) was added to the CM of CAFs and incubated at room temperature for 1 h. The treated CM was then used to co-culture with the corresponding HCC cells.

Techniques: Activation Assay, In Vitro, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Co-Immunoprecipitation Assay, Transfection, MTT Assay, Migration, Transwell Assay, Recombinant

Journal: Signal Transduction and Targeted Therapy

Article Title: Semaphorin 3C (Sema3C) reshapes stromal microenvironment to promote hepatocellular carcinoma progression

doi: 10.1038/s41392-024-01887-0

Figure Lengend Snippet: Sema3C expression is closely correlated with CAFs in HCC and is mediated by CAFs-derived TGF-β1. a Correlation between Sema3C expression and CAFs-associated genes ( ACTA2, VIM, FAP, PDGFRB , and S100A4 ) and collagen-associated genes ( COL1A1, COL1A2, FLNA, ELN , and LOX ) in HCC using the HCCDB datasets. The dots in the figure indicate p < 0.05. The color intensity and size of the circle are proportional to the correlation coefficients. b Representative images of IF staining for low or high Sema3C expression (red) with different degrees of stromal infiltration (α-SMA-green) in HCC tissues or DEN+CCl 4 -induced mouse liver tissues. Cell nuclei were stained with DAPI (blue). c The correlation analysis revealed a positive correlation between high Sema3C expression and the stromal percentage in patients with HCC (total n = 27). d TCGA-LIHC analysis of the correlation between patients with T stage or stage and FAP high /Sema3C low or FAP high /Sema3C high in HCC. e Western blotting showed that treatment with CAFs-conditioned media (CAFs-CM) significantly increased Sema3C expression compared with the control in HCC cells. f AP1 (including c-Jun/c-Fos) transcriptional binding sites in the Sema3C gene promoter. g , h MHCC-97L and Huh7 cells were transfected with si-c-Fos or si-c-Jun as indicated and the levels of Sema3C, c-Fos, and c-Jun were validated by western blotting. i ChIP-qPCR analysis showed that TGF-β1 stimulation facilitated the binding of phosphorylated c-Jun (p-c-Jun) and phosphorylated c-Fos (p-c-Fos) to the Sema3C gene promoter. j The levels of Sema3C, phosphorylated c-Jun (p-c-Jun), total c-Jun, p-c-Fos, and total c-Fos in MHCC-97L and Huh7 cells treated with different concentrations of TGF-β1 were determined by using western blotting. k Western blotting indicated that TGF-β1-induced Sema3C expression and AP1 signal activation could be inhibited by galunisertib (TGF-β1 receptor type I Inhibitor). l , m Western blotting confirmed that galunisertib and the TGF-β1 neutralizing antibody (anti-TGF-β1) inhibited the Sema3C upregulation stimulated by treatment with CAFs-CM. Data are presented as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: To verify the effect of TGF-β1 on Sema3C expression in HCC, 1 μg/mL anti-TGF-β1 antibody or an IgG control (Proteintech, China) was added to the CM of CAFs and incubated at room temperature for 1 h. The treated CM was then used to co-culture with the corresponding HCC cells.

Techniques: Expressing, Derivative Assay, Staining, Western Blot, Control, Binding Assay, Transfection, ChIP-qPCR, Activation Assay