Journal: Oncology Letters

Article Title: RNA methyltransferase NSUN2 enhances vasculogenic mimicry and malignant progression of cervical cancer through upregulation of MMP-9

doi: 10.3892/ol.2026.15518

Figure Lengend Snippet: MMP-9 , a factor that promotes Vasculogenic mimicry, is highly expressed in CC and is associated with poor prognosis. (A) CC database of TCGA was used to analyze key factors associated with VM. (B) Association of Sox2 expression with overall survival in CC (log-rank test). (C) Association of MMP-9 expression with overall survival in CC (log-rank test). (D) Panoramic scans after immunohistochemical detection of MMP-9 and H&E staining in samples from cancerous and paracancerous tissues from subjects with CC. Scale bar, 50 µm. Original magnification, ×20. (E) Protein levels of MMP-9 in 20 paired samples, with the MMP-9 level in CC tissue expressed compared with that in the paired normal tissue. (F) Expression levels of MMP-9 mRNA in 44 paired CC and paracancerous tissues, with MMP-9 expression in CC tissue expressed compared with that in the paired normal tissue. (G) Comparison of the average expression levels of MMP-9 mRNA in CC tissues compared with paracancerous tissues. (H) HeLa and SiHa cells were incubated under hypoxia (0.1% O 2 ) and proteins collected at 24, 48 and 72 h for western blotting of ALDH1, EPHA2, MMP-9 and GAPDH. ImageJ was used to semi-quantify western blotting signals from HeLa (I) and SiHa (J) cells. GAPDH served as an internal reference. *P<0.05, **P<0.01 and ***P<0.001. MMP-9, matrix metalloproteinase 9; VM, vasculogenic mimicry; ALDH1, aldehyde dehydrogenase 1; EPHA2, ephrin type-A receptor 2; TCGA, The Cancer Genome Atlas; Sox2, SRY-box transcription factor 2; CC, cervical cancer; CESC, cervical squamous cell carcinoma.

Article Snippet: The tissue samples were treated with PH9.0 EDTA repair solution for antigen retrieval and then treated with a rabbit polyclonal anti-CD31 antibody (1:2,000; cat. no. AB76533; Abcam), a rabbit polyclonal anti-NSUN2 antibody (1:200; cat. no. AB259941; Abcam) or a rabbit polyclonal anti-MMP-9 antibody (1:200; cat. no. 10375-2-AP; Proteintech Group, Inc.; Wuhan Sanying Biotechnology).

Techniques: Expressing, Immunohistochemical staining, Staining, Comparison, Incubation, Western Blot

Journal: Oncology Letters

Article Title: RNA methyltransferase NSUN2 enhances vasculogenic mimicry and malignant progression of cervical cancer through upregulation of MMP-9

doi: 10.3892/ol.2026.15518

Figure Lengend Snippet: NSUN2 promotes Vasculogenic mimicry, invasion and migration of CC cells under hypoxic conditions. (A) Expression levels of NSUN2 in CC cells lines (HeLa, SiHa, CaSki, C33A and HT-3) and in a normal cervical cell line (HaCaT). (B) RT-qPCR was used to determine relative expression levels of NSUN2 mRNA in HeLa and SiHa cells after transfection of shRNAs and incubation under hypoxia for 24 h. (C) Relative expression levels of MMP-9 mRNA in HeLa and SiHa cells transfected with the NSUN2 -interfering plasmid and incubated under hypoxia for 24 h. (D) Dot blot assay analysis of m 5 C expression levels in HeLa and SiHa cells after 48 h of hypoxia culture following transfection with NSUN2 knockdown plasmids. (E) Semi-quantitative analysis of dot blot results in HeLa cells. (F) Semi-quantitative analysis of dot blot results in SiHa cells. (G) Western blotting was used to investigate NSUN2 and MMP-9 protein levels in HeLa and SiHa cells transfected with the NSUN2 -interfering plasmid and incubated under hypoxia for 48 h. ImageJ was used to semi-quantify western blotting bands for (H) NSUN2 and (I) MMP-9 protein levels. (J) 2D tube-forming assays of HeLa and SiHa cells transfected with an NSUN2 -interfering plasmid and incubated under hypoxia. Scale bar, 100 µm. Original magnification, ×4. (K) Assay displayed in panel (J) was quantified using Image Pro. (L) Invasion assay of HeLa and SiHa cells transfected with a control plasmid, shNSUN2-2 or shNSUN2-3 or with shNSUN2 and pcDNA MMP-9. Scale bar, 200 µm. Original magnification, ×10. (M) Assay displayed in panel was quantified using Image Pro. (N) Migration assay of HeLa and SiHa cells transfected with a control plasmid, shNSUN2-2 or shNSUN2-3 or with shNSUN2 and pcDNA MMP-9. Scale bar, 200 µm. Original magnification, ×10. (O) Assay displayed in panel (N) was quantified using Image Pro. *P<0.05, **P<0.01 and ***P<0.001. VM, Vasculogenic mimicry; CC, cervical cancer; IHC, immunohistochemistry; NSUN2, NOP2/Sun RNA methyltransferase 2; m 5 C, 5-methylcytidine; RT-qPCR, reverse transcription-quantitative PCR; IHC, immunohistochemistry; CaSki, human cervical cancer cell line with intestinal metastasis; C33A, human cervical cancer cell line; HaCaT, human skin keratinocytes cell line; HeLa, human cervical cancer cell line; HT-3, human cervical cancer cell line; MMP9, matrix metalloproteinase 9; SiHa, human cervical squamous cell line; pcDNA, plasmid cloning DNA; shRNA, short hairpin RNA; NC, negative control.

Article Snippet: The tissue samples were treated with PH9.0 EDTA repair solution for antigen retrieval and then treated with a rabbit polyclonal anti-CD31 antibody (1:2,000; cat. no. AB76533; Abcam), a rabbit polyclonal anti-NSUN2 antibody (1:200; cat. no. AB259941; Abcam) or a rabbit polyclonal anti-MMP-9 antibody (1:200; cat. no. 10375-2-AP; Proteintech Group, Inc.; Wuhan Sanying Biotechnology).

Techniques: Migration, Expressing, Quantitative RT-PCR, Transfection, Incubation, Plasmid Preparation, Dot Blot, Knockdown, Quantitative Dot Blot, Western Blot, Invasion Assay, Control, Immunohistochemistry, Reverse Transcription, Real-time Polymerase Chain Reaction, Cloning, shRNA, Negative Control

Journal: Oncology Letters

Article Title: RNA methyltransferase NSUN2 enhances vasculogenic mimicry and malignant progression of cervical cancer through upregulation of MMP-9

doi: 10.3892/ol.2026.15518

Figure Lengend Snippet: NSUN2 increases the stability of MMP-9 mRNA. (A) A positive correlation was observed between NSUN2 and MMP-9 mRNA expression levels in 44 pairs of samples from subjects with CC. Enrichment of the m 5 C modification of MMP-9 mRNA in HeLa (B) and SiHa (C) Cells were measured with anti-m 5 C methylated-RNA IP assays. Interaction of NSUN2 with MMP-9 mRNA in (D) HeLa and (E) SiHa cells was measured with anti- NSUN2 RNA IP assays. Stability of MMP-9 mRNA after interference with and overexpression of NSUN2 was measured in (F) HeLa and (G) SiHa cells. (H) A model illustrating the proposed mechanism by which NSUN2-mediated stabilization of MMP-9 mRNA promotes Vasculogenic mimicry in CC. NSUN2, NOP2/Sun RNA methyltransferase 2; m 5 C, 5-methylcytidine; pcDNA, plasmid cloning DNA; shRNA, short hairpin RNA; HeLa, human cervical cancer cell line; MMP9, matrix metalloproteinase 9; SiHa, human cervical squamous cell line; IP, immunoprecipitation; CC, cervical cancer; NC, negative control.

Article Snippet: The tissue samples were treated with PH9.0 EDTA repair solution for antigen retrieval and then treated with a rabbit polyclonal anti-CD31 antibody (1:2,000; cat. no. AB76533; Abcam), a rabbit polyclonal anti-NSUN2 antibody (1:200; cat. no. AB259941; Abcam) or a rabbit polyclonal anti-MMP-9 antibody (1:200; cat. no. 10375-2-AP; Proteintech Group, Inc.; Wuhan Sanying Biotechnology).

Techniques: Expressing, Modification, Methylation, Over Expression, Plasmid Preparation, Cloning, shRNA, Immunoprecipitation, Negative Control

Journal: Journal of Advanced Research

Article Title: Hyodeoxycholic acid relieves neuropathic pain by activating farnesoid X receptor signaling

doi: 10.1016/j.jare.2025.07.017

Figure Lengend Snippet: HDCA upregulates the intestinal PPAR-γ and downregulates the MMP-9/2 expression. (A) The degree of the node between the HDCA and the intersection target. (B) Molecular docking analysis between HDCA and PPAR-γ. (C) Molecular docking analysis between HDCA and MMP-9. (D) Molecular docking analysis between HDCA and MMP-2. (E-G) Relative expression of mRNA of ppar-γ, mmp9 and mmp2 in the distal ileum. (n = 6) . (H) Representative protein immunoblots in distal ileum. (I-K) Relative expression of PPAR-γ, MMP-9, MMP-2 (n = 3–4). (L) Representative immunohistochemical staining and quantitative analysis of MMP-2+ (M), PPAR-γ+ (N) and MMP-9+ (O) cells in the distal ileum (scale bar, 100 μm, n = 6). * P < 0.05, ** P < 0.01, *** P < 0.001 by One-way ANOVA with post hoc Tukey's test (E–G, I–K, M–O). Data is presented as mean ± SEM.

Article Snippet: Tissue sections of each group were blocked with 1 % bovine serum albumin and 10 % donkey serum at room temperature for 1 h and then incubated at 4 °C overnight with primary antibodies for neuronal nuclear protein (NeuN) (1:500, Abcam, ab104224), ionized calcium-binding adapter molecule 1 (IBA-1) (1:500, Abcam, ab5076), glial fibrillary acidic protein (GFAP) (1:500, Millipore, MAB360), FXR (1:200, Proteintech, 25055–1-AP), MMP-2 (1:200, Proteintech, 10373-2-AP), MMP-9 (1:200, Proteintech, 10375-2-AP), PPAR-γ (1:200, Proteintech, 16643–1-AP), NLRP3 (1:200, Immunoway, YT5382), and cluster of differentiation 86 (CD86) (1:200, CST, #91882).

Techniques: Expressing, Western Blot, Immunohistochemical staining, Staining

Journal: Journal of Advanced Research

Article Title: Hyodeoxycholic acid relieves neuropathic pain by activating farnesoid X receptor signaling

doi: 10.1016/j.jare.2025.07.017

Figure Lengend Snippet: HDCA upregulates PPAR-γ expression and reduces MMP-9/2 expression in the spinal cord. (A–C) The relative expression of mRNA in spinal cord of ppar-γ, mmp9 and mmp2 . (n = 6) . (D–G) Representative immunoblots of proteins and relative expression of PPAR-γ, MMP-2, MMP-9 (n = 4). (H–J) Immunofluorescence staining of PPAR-γ, MMP-9, MMP-2 in spinal cord (scale, 100 μm). (n = 3). (K) Heatmap of Correlation Analysis. * P < 0.05, ** P < 0.01, *** P < 0.001 by One-way ANOVA with post hoc Tukey's test (A–D, F, G). Data is presented as mean ± SEM.

Article Snippet: Tissue sections of each group were blocked with 1 % bovine serum albumin and 10 % donkey serum at room temperature for 1 h and then incubated at 4 °C overnight with primary antibodies for neuronal nuclear protein (NeuN) (1:500, Abcam, ab104224), ionized calcium-binding adapter molecule 1 (IBA-1) (1:500, Abcam, ab5076), glial fibrillary acidic protein (GFAP) (1:500, Millipore, MAB360), FXR (1:200, Proteintech, 25055–1-AP), MMP-2 (1:200, Proteintech, 10373-2-AP), MMP-9 (1:200, Proteintech, 10375-2-AP), PPAR-γ (1:200, Proteintech, 16643–1-AP), NLRP3 (1:200, Immunoway, YT5382), and cluster of differentiation 86 (CD86) (1:200, CST, #91882).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining

Journal: Journal of Advanced Research

Article Title: Hyodeoxycholic acid relieves neuropathic pain by activating farnesoid X receptor signaling

doi: 10.1016/j.jare.2025.07.017

Figure Lengend Snippet: Fxr knock down abolished the protective effect of HDCA in neuropathic pain. (A) Immunofluorescence staining showing FXR co-localization in spinal cord. (scale, 25 μm). (B) Immunofluorescence staining demonstrating MMP-2 colocalization in spinal cord. (scale, 25 μm). (C) Immunofluorescence staining showing PPAR-γ colocalization in spinal cord. (scale, 25 μm). (D) Immunofluorescence staining showing MMP-9 colocalization in spinal cord. (scale, 25 μm). (E) Immunofluorescence staining of FXR and MMP-2 colocalization in spinal cord. (scale, 25 μm). (F) Representative immunoblots of protein expression in the spinal cord from Fxr -/- mice. (G) Relative expression of PPAR-γ, MMP-2 and MMP-9 (n = 4). (H) Representative immunoblots for the proteins in spinal cord following INT-747 treatment. (I) PWT of Fxr -/- mice following HDCA or INT-747 treatment. (n = 6). (J) PWL of Fxr -/- mice following HDCA or INT-747 treatment. (n = 6). (K) Relative expression of PPAR-γ, MMP-2 and MMP-9 following INT-747 treatment. (n = 4). (L, M) Representative immunoblots for the proteins in spinal cord and relative expression of PPAR-γ, MMP-2 and MMP-9 in Fxr -/- mice following HDCA treatment. (n = 4). * P < 0.05, ** P < 0.01, ns by unpaired Student's t‐test (G, M). ns by Two-way repeated ANOVA with post hoc Bonferroni’s test (I and J), * P < 0.05, ** P < 0.01, *** P < 0.001 by One-way ANOVA with post hoc Tukey's test (K). Data are represented as mean ± SEM.

Article Snippet: Tissue sections of each group were blocked with 1 % bovine serum albumin and 10 % donkey serum at room temperature for 1 h and then incubated at 4 °C overnight with primary antibodies for neuronal nuclear protein (NeuN) (1:500, Abcam, ab104224), ionized calcium-binding adapter molecule 1 (IBA-1) (1:500, Abcam, ab5076), glial fibrillary acidic protein (GFAP) (1:500, Millipore, MAB360), FXR (1:200, Proteintech, 25055–1-AP), MMP-2 (1:200, Proteintech, 10373-2-AP), MMP-9 (1:200, Proteintech, 10375-2-AP), PPAR-γ (1:200, Proteintech, 16643–1-AP), NLRP3 (1:200, Immunoway, YT5382), and cluster of differentiation 86 (CD86) (1:200, CST, #91882).

Techniques: Knockdown, Immunofluorescence, Staining, Western Blot, Expressing

Journal: Science Progress

Article Title: Low-dose urapidil mitigates renal ischemia-reperfusion injury through matrix metalloproteinase-9 inhibition and anti-inflammatory effects

doi: 10.1177/00368504261438886

Figure Lengend Snippet: Comparison of MMP-9 IHC between the kidney samples from each group. Details of MMP-9 marking were observed with IHC. MMP-9, matrix metalloproteinase-9; IHC, immunohistochemistry; SH, sham group; CR, control group; I/R, ischemia-reperfusion injury; UP, urapidil.

Article Snippet: Following the completion of the blocking procedure, the samples were treated with primary antibodies, including eNOS (Boster Bio., catalog number: A01604-2, diluted at a ratio of 1:250), caspase 3 (Thermo Scıentıfıc, catalog number: RB-1197-P0, diluted at a ratio of 1:100), TNF-α (Proteintech, catalog number:60291-1-Ig, diluted at a ratio of 1:300), IL-1β (Bioss, catalog number:bs-6319R, diluted at a ratio of 1:150), IL-6 (ST John’s, catalog number:STJ1110424, diluted at a ratio of 1:100), and MMP-9 (Boster Bio, catalog number: PA2140-2, diluted at a ratio of 1:100).

Techniques: Comparison, Immunohistochemistry, Control