Journal: Oncology Reports

Article Title: METTL3‑mediated N6‑methyladenosine modification of MMP9 mRNA promotes colorectal cancer proliferation and migration

doi: 10.3892/or.2024.8842

Figure Lengend Snippet: METTL3 stabilizes MMP9 mRNA in a m 6 A-dependent manner. MMP9 mRNA m 6 A levels of stable METTL3 knockdown (A) HCT116 and (B) SW480 cell lines was detected by m 6 A-immunoprecipitated qPCR. The (C) protein and (D) mRNA levels of MMP9 in METTL3-knockdown HCT116 and SW480 cells. MMP9 protein expression levels in shNC and shMETTL3 (E) HCT116 and (F) SW480 cell lines treated with cycloheximide (20 µg/ml) for the indicated time points were detected by western blotting. (G) MMP9 mRNA expression levels in shNC and shMETTL3 cells treated with Actinomycin (5 µg/ml) for the indicated time points were detected by qPCR. (H) Western blot analysis of MMP9 expression in oe-MMP9 cells was performed. The viability of (I) HCT116 and (J) SW480 cells with or without METTL3 knockdown after transfection with oe-MMP9 were determined by Cell Counting Kit-8 assay. (K) The invasion ability of HCT116 and SW480 cells with or without METTL3 knockdown after transfection with oe-MMP9 was determined by Transwell assay. Scale bars, 100 µm. Error bars, SD. *P<0.05, **P<0.01, ***P<0.001. m 6 A, N6-methyladenosine; METTL3/M3, methyltransferase-like 3; MMP9, matrix metallopeptidase 9; CRC, colorectal cancer; qPCR, quantitative PCR; sh, short hairpin (RNA); NC, negative control; oe, overexpression.

Article Snippet: Tissue sections were incubated overnight at 4°C with primary antibodies targeting METTL3 (1:50; cat. no. 86132S; Cell Signaling Technology, Inc.), MMP9 (1:1,000; cat. no. ab76003; Abcam) and Ki67 (1:100; cat. no. ab16667; Abcam).

Techniques: Knockdown, Immunoprecipitation, Expressing, Western Blot, Transfection, Cell Counting, Transwell Assay, Real-time Polymerase Chain Reaction, shRNA, Negative Control, Over Expression

Journal: Oncology Reports

Article Title: METTL3‑mediated N6‑methyladenosine modification of MMP9 mRNA promotes colorectal cancer proliferation and migration

doi: 10.3892/or.2024.8842

Figure Lengend Snippet: METTL3 promotes colorectal cancer cell progression in vivo . (A) Western blot analysis of METTL3 was performed on HCT116 cells transfected with oe-METTL3. (B) Flow chart of the in vivo experimental design. (C) Xenograft assay was performed using HCT116 cells transfected with sh-METTL3, oe-METTL or empty vector (pcDNA3; control). (D) Quantitative analysis of the xenograft tumor volume. (E) The METTL3 and MMP9 mRNA levels in tumor tissues expressing sh-METTL3 or oe-METTL3 or the control HCT116 cells. (F) Expression of METTL3, Ki67 and MMP9 was detected by immunohistochemistry of paraffin-embedded tissues. Scale bars, 100 µm. Error bars, SD. *P<0.05, **P<0.01, ***P<0.001. METTL3, methyltransferase-like 3; MMP9, matrix metallopeptidase 9; sh, short hairpin (RNA); NC, negative control; oe, overexpression; s.c., subcutaneous.

Article Snippet: Tissue sections were incubated overnight at 4°C with primary antibodies targeting METTL3 (1:50; cat. no. 86132S; Cell Signaling Technology, Inc.), MMP9 (1:1,000; cat. no. ab76003; Abcam) and Ki67 (1:100; cat. no. ab16667; Abcam).

Techniques: In Vivo, Western Blot, Transfection, Xenograft Assay, Plasmid Preparation, Control, Expressing, Immunohistochemistry, shRNA, Negative Control, Over Expression

Journal: Oncology Reports

Article Title: Compression force promotes glioblastoma progression through the Piezo1‑GDF15‑CTLA4 axis

doi: 10.3892/or.2024.8835

Figure Lengend Snippet: Compressive solid stress increases the expression of the GDF15 and Piezo1 mechanosensors. (A) Comparisons of GDF15 and PIEZO1 gene expression between human GBM and normal brain tissues using data from The Cancer Genome Atlas and GTEx, analyzed with GEPIA. The red box plots represent GBM tissues (n=163) and the gray box plots represent non-GBM tissue (n=207). The figure was obtained from GEPIA. The expression levels of (B) GDF15 and (C) Piezo1 in human GBM tissue were compared with normal brain tissue by immunohistochemistry. The tissues were obtained from the Korea Brain Bank Network. Scale bars represent 50 µm and 10 µm. (D) H4 cells were subjected to compression for 12 h, and the gene expression levels of GDF15, PIEZO1 and MMP9 were evaluated using reverse transcription-quantitative PCR (n=3 independent experiments). (E) Piezo1 and GDF15 protein expression in H4 cells exposed to pressure for 12 and 24 h measured via immunoblotting analysis. *P<0.05, **P<0.01. GBM, glioblastoma multiforme; GDF15, growth differentiation factor 15; MMP9, matrix metalloproteinase 9; N, normal tissue; T, tumor tissue; TPM, transcripts per million.

Article Snippet: Total RNA was extracted from H4 cells using the RNeasy Mini Kit (cat. no. 74004; Qiagen GmbH) according to the manufacturer's instructions. cDNAs were synthesized from 1–2 µg of total RNA using the Maxima First Stand cDNA synthesis kit (cat. no. K1642; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. qPCR was performed using Power SYBR Green PCR Master Mix reagent (cat. no. 4367659; Applied Biosystems; Thermo Fisher Scientific, Inc.) and primers specific to the target genes [ PIEZO1, GDF15 , matrix metalloproteinase 9 ( MMP9), CTLA4 and GAPDH ] in a StepOnePlus ™ Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.).

Techniques: Expressing, Immunohistochemistry, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot

Journal: Oncology Reports

Article Title: Compression force promotes glioblastoma progression through the Piezo1‑GDF15‑CTLA4 axis

doi: 10.3892/or.2024.8835

Figure Lengend Snippet: Knockdown of GDF15 and PIEZO1 using siRNA significantly reduces cell motility under pressure. (A) Motility plots of siCon or siGDF15 transfected cells subjected to pressure for 12 h and then observed for 5 h under a time-lapse imaging microscope. Plots depict the motility of individual cells in 1 representative experiment. (B) Quantification of the migration speed of individual cells (n=200). (C) Motility plots of siCon or siPiezo1 transfected cells subjected to pressure for 12 h and then observed for 5 h under a time-lapse imaging microscope. Plots depict the motility of individual cells in 1 representative experiment. (D) Quantification of the migration speed of individual cells (n=200). Cells with or without PIEZO1 knockdown were subjected to pressure for 12 h, and the gene expression levels of (E) PIEZO1 and (F) GDF15 were evaluated using RT-qPCR (n=3 independent experiments for each gene). Cells were subject to pressure with or without the addition of 10 µM BAPTA-AM for 12 h, and the gene expression levels of (G) PIEZO1 and (H) GDF15 were assessed using RT-qPCR (n=3 independent experiments for each gene). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, determined using one-way ANOVA followed by Tukey's test. Con, control; GDF15, growth differentiation factor 15; ns, not significant; RT-qPCR, reverse transcription-quantitative PCR; siRNA, small interfering RNA.

Article Snippet: Total RNA was extracted from H4 cells using the RNeasy Mini Kit (cat. no. 74004; Qiagen GmbH) according to the manufacturer's instructions. cDNAs were synthesized from 1–2 µg of total RNA using the Maxima First Stand cDNA synthesis kit (cat. no. K1642; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. qPCR was performed using Power SYBR Green PCR Master Mix reagent (cat. no. 4367659; Applied Biosystems; Thermo Fisher Scientific, Inc.) and primers specific to the target genes [ PIEZO1, GDF15 , matrix metalloproteinase 9 ( MMP9), CTLA4 and GAPDH ] in a StepOnePlus ™ Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.).

Techniques: Knockdown, Transfection, Imaging, Microscopy, Migration, Expressing, Quantitative RT-PCR, Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: Oncology Reports

Article Title: Compression force promotes glioblastoma progression through the Piezo1‑GDF15‑CTLA4 axis

doi: 10.3892/or.2024.8835

Figure Lengend Snippet: Compressive stimuli regulate the expression of CTLA4 in neuroglioma cells. (A) H4 cells were subjected to 12 h of compressive stimuli and the expression of CTLA4 was analyzed using RT-qPCR. n=6 independent experiments; **P<0.01, determined by unpaired t-test. Cells transfected with (B) siCon or siGDF15 and (C) siCon or siPiezo1 were subjected to compressive stimulation for 12 h, and expression of CTLA4 was analyzed using RT-qPCR. n=3 independent experiments for each transfection procedure; *P<0.05, **P<0.01, ****P<0.0001 determined by one-way ANOVA followed by Tukey's test. (D) A proposed mechanism that promotes glioma progression through a mechanosensor that detects mechanical pressure in the brain tumor microenvironment. As GBM grows in a limited space, pressure builds between cells and surrounding tissues. The expression of Piezo1, a mechanosensor present in the cell membrane, increases, followed by the expression of GDF15. Subsequently, the immune checkpoint protein, CTLA4, is upregulated, enhancing the poor prognosis of glioma. Con, control; GBM, glioblastoma; GDF15, growth differentiation factor 15; ns, not significant; RT-qPCR, reverse transcription-quantitative PCR; siRNA, small interfering RNA.

Article Snippet: Total RNA was extracted from H4 cells using the RNeasy Mini Kit (cat. no. 74004; Qiagen GmbH) according to the manufacturer's instructions. cDNAs were synthesized from 1–2 µg of total RNA using the Maxima First Stand cDNA synthesis kit (cat. no. K1642; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. qPCR was performed using Power SYBR Green PCR Master Mix reagent (cat. no. 4367659; Applied Biosystems; Thermo Fisher Scientific, Inc.) and primers specific to the target genes [ PIEZO1, GDF15 , matrix metalloproteinase 9 ( MMP9), CTLA4 and GAPDH ] in a StepOnePlus ™ Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Membrane, Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: Oncology Reports

Article Title: METTL3‑mediated N6‑methyladenosine modification of MMP9 mRNA promotes colorectal cancer proliferation and migration

doi: 10.3892/or.2024.8842

Figure Lengend Snippet: METTL3 stabilizes MMP9 mRNA in a m 6 A-dependent manner. MMP9 mRNA m 6 A levels of stable METTL3 knockdown (A) HCT116 and (B) SW480 cell lines was detected by m 6 A-immunoprecipitated qPCR. The (C) protein and (D) mRNA levels of MMP9 in METTL3-knockdown HCT116 and SW480 cells. MMP9 protein expression levels in shNC and shMETTL3 (E) HCT116 and (F) SW480 cell lines treated with cycloheximide (20 µg/ml) for the indicated time points were detected by western blotting. (G) MMP9 mRNA expression levels in shNC and shMETTL3 cells treated with Actinomycin (5 µg/ml) for the indicated time points were detected by qPCR. (H) Western blot analysis of MMP9 expression in oe-MMP9 cells was performed. The viability of (I) HCT116 and (J) SW480 cells with or without METTL3 knockdown after transfection with oe-MMP9 were determined by Cell Counting Kit-8 assay. (K) The invasion ability of HCT116 and SW480 cells with or without METTL3 knockdown after transfection with oe-MMP9 was determined by Transwell assay. Scale bars, 100 µm. Error bars, SD. *P<0.05, **P<0.01, ***P<0.001. m 6 A, N6-methyladenosine; METTL3/M3, methyltransferase-like 3; MMP9, matrix metallopeptidase 9; CRC, colorectal cancer; qPCR, quantitative PCR; sh, short hairpin (RNA); NC, negative control; oe, overexpression.

Article Snippet: To assess the degradation rate of MMP9 mRNA, cells were treated with Actinomycin-D (Act-D; Sigma-Aldrich; Merck KGaA) at a final concentration of 3 µg/ml at 37°C to inhibit new RNA synthesis.

Techniques: Knockdown, Immunoprecipitation, Expressing, Western Blot, Transfection, Cell Counting, Transwell Assay, Real-time Polymerase Chain Reaction, shRNA, Negative Control, Over Expression

Journal: Oncology Reports

Article Title: METTL3‑mediated N6‑methyladenosine modification of MMP9 mRNA promotes colorectal cancer proliferation and migration

doi: 10.3892/or.2024.8842

Figure Lengend Snippet: METTL3 promotes colorectal cancer cell progression in vivo . (A) Western blot analysis of METTL3 was performed on HCT116 cells transfected with oe-METTL3. (B) Flow chart of the in vivo experimental design. (C) Xenograft assay was performed using HCT116 cells transfected with sh-METTL3, oe-METTL or empty vector (pcDNA3; control). (D) Quantitative analysis of the xenograft tumor volume. (E) The METTL3 and MMP9 mRNA levels in tumor tissues expressing sh-METTL3 or oe-METTL3 or the control HCT116 cells. (F) Expression of METTL3, Ki67 and MMP9 was detected by immunohistochemistry of paraffin-embedded tissues. Scale bars, 100 µm. Error bars, SD. *P<0.05, **P<0.01, ***P<0.001. METTL3, methyltransferase-like 3; MMP9, matrix metallopeptidase 9; sh, short hairpin (RNA); NC, negative control; oe, overexpression; s.c., subcutaneous.

Article Snippet: To assess the degradation rate of MMP9 mRNA, cells were treated with Actinomycin-D (Act-D; Sigma-Aldrich; Merck KGaA) at a final concentration of 3 µg/ml at 37°C to inhibit new RNA synthesis.

Techniques: In Vivo, Western Blot, Transfection, Xenograft Assay, Plasmid Preparation, Control, Expressing, Immunohistochemistry, shRNA, Negative Control, Over Expression

Journal: International Journal of Molecular Medicine

Article Title: Enocyanin promotes osteogenesis and bone regeneration by inhibiting MMP9

doi: 10.3892/ijmm.2024.5450

Figure Lengend Snippet: ENO inhibits MMP9 in osteogenesis in vitro and bone formation in vivo . (A) Heatmap of the differences in the expression of proteinase-related genes in cells with or without ENO treatment in osteogenic medium (n=2). (B) Reverse transcription-quantitative PCR analysis of the mRNA expression levels of Mmp9 after ENO treatment (n=4). (C) Western blot analysis showed that the protein levels of MMP9 decreased after ENO treatment in a dose-dependent manner (n=3). (D) Relative semi-quantification of the expression of MMP9 normalized to β-actin as determined by Image Lab (n=3). (E) Immunohistochemical staining of MMP9 expression at the callus sites in the femur. Scale bars, 2,000 μ m (left), 500 μ m (middle) and 100 μ m (right). Semi-quantification of the positive staining of MMP9 was performed using ImageJ software (n=4). * P<0.05, ** P<0.01, *** P <0.001, **** P <0.0001 (n=3). ENO, enocyanin; MMP9, matrix metalloproteinase 9; ns, not significant; OM, osteogenic medium.

Article Snippet: The primers used were listed in and Mmp9 primers were purchased from Sino Biological, Inc. (cat. no. MP200552).

Techniques: In Vitro, In Vivo, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemical staining, Staining, Software

Journal: International Journal of Molecular Medicine

Article Title: Enocyanin promotes osteogenesis and bone regeneration by inhibiting MMP9

doi: 10.3892/ijmm.2024.5450

Figure Lengend Snippet: Recombinant MMP9 protein suppresses the osteogenesis of KusaO cells in the presence of ENO. (A) Mineralization after treatment with 30 μ M ENO and MMP9 protein (n=4). Scale bar, 500 μ m. (B) Percentage of calcium nodules was assessed by Alizarin red S staining and was semi-quantified (n=4). (C) mRNA expression levels of several osteoblastic genes, Alpl, Spp1, Sp7, Runx2, Bglap and Bsp, were downregulated by MMP9 treatment (n=4). (D) Western blot analysis showed that the expression of osteoblast-related proteins, Runx2 and OPN, was dose-dependently decreased by MMP9 treatment (n=3). (E) Relative semi-quantification of the normalized expression of OPN and Runx2 to β-actin was determined by Image Lab (n=3). * P< 0.05, ** P <0.01, *** P<0.001, **** P<0.0001. ENO, enocyanin; MMP9, matrix metalloproteinase 9; ns, not significant; OPN, osteopontin.

Article Snippet: The primers used were listed in and Mmp9 primers were purchased from Sino Biological, Inc. (cat. no. MP200552).

Techniques: Recombinant, Staining, Expressing, Western Blot

Journal: Biomedical Reports

Article Title: Rifaximin prophylaxis in MASLD‑hepatocellular carcinoma: Lessons from a negative animal model

doi: 10.3892/br.2024.1882

Figure Lengend Snippet: Gene expression of liver inflammation markers involved in steatohepatitis pathogenesis.

Article Snippet: To assess the gene expression of interleukin (IL)-1β, IL-6, IL-10, tumoral necrosis factor-α (TNF-α), lipopolysaccharide-binding protein (LBP), myeloid differentiation primary response 88, toll-like receptor (TLR) 4, TLR2, transforming growth factor-β1 (TGF-β1), metalloproteinase (MMP)2 and MMP9 in the liver, a quantitative polymerase chain reaction with the TaqMan assay (Applied Biosystems; Thermo Fisher Scientific, Inc.) was performed according to the manufacturer's instructions.

Techniques: Expressing, Control

Journal: Oncology Letters

Article Title: Anticancer effect of the oncolytic Newcastle disease virus harboring the PTEN gene on glioblastoma

doi: 10.3892/ol.2024.14752

Figure Lengend Snippet: Effect of rNDV-PTEN infection on apoptotic cell death through imbalance of Akt/mTOR pathway. U87-MG cells were infected with rNDV or rNDV-PTEN at an MOI of 1 for 12, 24 or 36 h. (A) Cell proliferation markers PCNA and MMP9, (B) mTOR signaling-related proteins and autophagy-related proteins and (C) pre-apoptotic cell death-related proteins were assessed using immunoblotting analysis in U87-MG cells. GAPDH was used as an internal control. *P<0.05 vs. CON. rNDV, recombinant Newcastle disease virus; PTEN, phosphatase and tensin homolog; PCNA, proliferating cell nuclear antigen; MMP9, matrix metallopeptidase 9; MOI, multiplicity of infection; CON, control; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein.

Article Snippet: The sections were blocked with 1% bovine serum albumin (cat. no. 4378; MilliporeSigma) in PBS for 1 h at room temperature, and then stained with the following primary antibodies: Anti-MMP9 (1:100; cat. no. MA5-15886; Thermo Fisher Scientific, Inc.), anti-PTEN (1:200; cat. no. 9559S; Cell Signaling Technology, Inc.) anti-NDV hemagglutinin-neuraminidase protein (1:200; HN; cat. no. sc-53562; Santa Cruz Biotechnology, Inc.) and anti-Ki-67 (1:100; cat. no. MA5-14520; Thermo Fisher Scientific, Inc.) overnight at 4°C.

Techniques: Infection, Western Blot, Control, Recombinant, Virus

Journal: Oncology Letters

Article Title: Anticancer effect of the oncolytic Newcastle disease virus harboring the PTEN gene on glioblastoma

doi: 10.3892/ol.2024.14752

Figure Lengend Snippet: PTEN restoration induces apoptotic cell death by impacting mTOR signaling and autophagy in the orthotopic GBM mouse model. (A) Immunohistochemical staining of Ki-67 (tumor cell proliferation marker) and MMP9 in the GBM orthotopic mouse tissue. Scale bar, 50 µm. (B) PCNA and MMP9 levels, (C) proteins related to the mTOR signaling pathway and autophagy, and (D) apoptosis markers were assessed using immunoblotting in GBM tissue. These data are expressed as the fold change in expression compared with PBS injected mice. GAPDH was used as an internal control. *P<0.05 vs. PBS-injected mice. PTEN, phosphatase and tensin homolog; MMP9, matrix metallopeptidase 9; PCNA, proliferating cell nuclear antigen; GBM, glioblastoma; CON, control.

Article Snippet: The sections were blocked with 1% bovine serum albumin (cat. no. 4378; MilliporeSigma) in PBS for 1 h at room temperature, and then stained with the following primary antibodies: Anti-MMP9 (1:100; cat. no. MA5-15886; Thermo Fisher Scientific, Inc.), anti-PTEN (1:200; cat. no. 9559S; Cell Signaling Technology, Inc.) anti-NDV hemagglutinin-neuraminidase protein (1:200; HN; cat. no. sc-53562; Santa Cruz Biotechnology, Inc.) and anti-Ki-67 (1:100; cat. no. MA5-14520; Thermo Fisher Scientific, Inc.) overnight at 4°C.

Techniques: Immunohistochemical staining, Staining, Marker, Western Blot, Expressing, Injection, Control