Journal: Frontiers in Oncology

Article Title: The Metalloprotease-Disintegrin ADAM8 Alters the Tumor Suppressor miR-181a-5p Expression Profile in Glioblastoma Thereby Contributing to Its Aggressiveness

doi: 10.3389/fonc.2022.826273

Figure Lengend Snippet: Expression levels of ADAM8 and MMP9 , and miR-181a-5p in GBM tissue samples. (A) RT-qPCR results of GBM tissue samples (n=22, fold change normalized to 1) indicate a higher expression of ADAM8 (p-value: 0.0009) and MMP9 (p-value: 0.0002) than miR-181a-5p. (B) Dividing the RT-qPCR results and patient cohort into two groups (low/high ADAM8 or low/high miR-181a-5p expression) reveals a correlation of ADAM8 with MMP9 (left graph, p-value: 0.001) but no correlation of miR-181a-5p with ADAM8 (middle graph, p-value: 0.577) or MMP9 (right graph, p-value: 0.083) expression. (C) RT-qPCR results for miR-181a-5p expression of each GBM tissue sample. (D) ADAM8 and MMP9 are correlated in GBM tissue samples (p < 0.0001, n=22), whereas the inverse correlations of ADAM8 and miR-181a-5p and of miR-181a-5p and MMP9 are non-significant (p-values: 0.63 and 0.6, respectively). (E) T2-weighted magnetic resonance (MR) image showing a left parietal GBM (segmented in yellow, patient 25) as well as the co-registered choline/N-acetylaspartate (NAA) maps derived from 1H-MR spectroscopy, integrated into the neuronavigation system for navigated extraction of tissue samples (L1: tumor border, L2/L3: tumor, L4: tumor, Cho/NAA hotspot) magnetic resonance (heatmap for choline metabolite). Corresponding molecular analyses are shown in (F, G) (patient 25). RT-qPCR results of ADAM8 (red), MMP9 (tiled red), and miR-181a-5p (blue) in different tissue locations normalized to either L1 (F) or L4 (G) describing the direction of surgery. (H) In a pilot study, three GBM patients (Patient 9, 23, 24) were analyzed for their serum-EV miR-181a-5p expression via RT-qPCR. The serum was collected before and after the first and second surgery. Interestingly, after first surgical resection miR-181a-5p is less expressed in serum-EVs (p-value: 0.042). (I) After second surgery, miR-181a-5p shows a slight increase in serum-EVs (p-value: 0.08). (J) , miR-181a-5p is less detectable in serum-EVs prior to the second surgery compared to pre-first surgery (p-value: 0.02; left graph). Results are shown in mean values +/- SD. Unpaired one-tailed students t test and Wilcoxon signed-rank test were applied to determine significance: ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Soluble ADAM8 (DY1031, R&D Systems, UK) and soluble MMP9 (DY911, R&D Systems, UK) from cell culture supernatants were determined by Sandwich-ELISA method with DuoSet ELISA Kits.

Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, Spectroscopy, Extraction, One-tailed Test

Journal: Frontiers in Oncology

Article Title: The Metalloprotease-Disintegrin ADAM8 Alters the Tumor Suppressor miR-181a-5p Expression Profile in Glioblastoma Thereby Contributing to Its Aggressiveness

doi: 10.3389/fonc.2022.826273

Figure Lengend Snippet: Screening of two representative CRISPR/Cas9 ADAM8 (hA8) KO cell clones reveals an ADAM8-dependent regulation of miR-181a-5p. Confirmation of the CRISPR/Cas9 ADAM8 stable KO in U87 cells by qPCR (A) , Western Blot (B) , and ELISA (C) . (D) A RT-qPCR-based miRNA PCR Array (Human Finder, Qiagen) of U87 CRISPR/Cas9 ADAM8 KO cells enabling the analysis of a total of 84 miRNAs. The legend for the fold-changes in the heat map is given above representing the fold-change values (2 -ΔΔCT ) relative to U87_CTRL cells in both ADAM8 KO clones. Note the variance in fold changes between the two KO clones (E) Confirmation of miR-181a-5p upregulation in U87_KO1 and U87_KO2 cells (p-value: 0.01 and 0.03). The expression of miR-181a-5p (F) and ADAM8 (G) in GBM cell lines (G28, G112, U251), primary patient-derived cell lines (GBM29, 98, and 42), and GBM stem-like cell lines (GSCs) (GSC 2016/240, GSC 2017/74, GSC 2017/151) are shown as relative values to the expression in U87 cells. MiR-181a-5p is mostly expressed in GSCs. The patient-derived cell line GBM42 shows the highest ADAM8 expression. Pearson correlation analysis of miR-181a-5p and ADAM8 in cell lines (H) , primary cell lines (I) , and primary GSCs (J) reveal a negative correlation only observed in GSCs (p-value: 0.01, Pearson r: -0.88). Results are given as mean +/- SD of two to three independent experiments. Unpaired two-tailed students t-test or two way ANOVA for multiple comparison (F) were applied to determine significance: *p < 0.05, **p < 0.01, ***p < 0.001. n.d., not detectable.

Article Snippet: Soluble ADAM8 (DY1031, R&D Systems, UK) and soluble MMP9 (DY911, R&D Systems, UK) from cell culture supernatants were determined by Sandwich-ELISA method with DuoSet ELISA Kits.

Techniques: CRISPR, Clone Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Derivative Assay, Two Tailed Test, Comparison

Journal: Frontiers in Oncology

Article Title: The Metalloprotease-Disintegrin ADAM8 Alters the Tumor Suppressor miR-181a-5p Expression Profile in Glioblastoma Thereby Contributing to Its Aggressiveness

doi: 10.3389/fonc.2022.826273

Figure Lengend Snippet: ADAM8 regulates the expression of miR-181a-5p via JAK2/STAT3 signaling. (A) U87_CTRL cells were analyzed for miR-181a-5p expression by RT-qPCR after treatment with the broad-spectrum MMP inhibitor BB-94 (left) and the ADAM8 inhibitor BK-1361 (right). (B) One representative western blot of three independent experiments shows the rescue of either ADAM8 lacking the cytoplasmatic domain (Delta CD) or the full-length ADAM8 (hA8). The quantifications of pEGFR, pSTAT3, and pERK1/2 are depicted on the right side and were normalized to β-Tubulin and total-EGFR/β-Tubulin, total STAT3/β-Tubulin, or total ERK1/2/β-Tubulin. Also, RT-qPCR results show no differences in miR-181a-5p expression after the transfection of ADAM8 Delta CD but a downregulation with the full-length ADAM8 rescue (p-value: 0.002). (C) The expression of ADAM8 mRNA (RT-qPCR, left) and secreted ADAM8 (ELISA, right, n=2) is not affected after miR-181a-5p mimic transfection. U87_CTRL cells (D) and patient-derived GBM42 cells (E) were treated with JAK2/STAT3 inhibitor WP1066 as indicated and analyzed via western blot and RT-qPCR. In (D) , qPCR results are shown as mean values +/- SD of four independent experiments and in (E) , results of miR-181a-5p are described as mean values of three technical replicates. Inhibition of JAK2/STAT3 increases miR-181a-5p expression (U87 p-value: 0.027; GBM42 p-value: 0.004). Results are shown as mean values +/- SD from three independent experiments if not otherwise stated. Unpaired two-tailed students t-test was applied to determine significance: ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Soluble ADAM8 (DY1031, R&D Systems, UK) and soluble MMP9 (DY911, R&D Systems, UK) from cell culture supernatants were determined by Sandwich-ELISA method with DuoSet ELISA Kits.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay, Derivative Assay, Inhibition, Two Tailed Test

Journal: Frontiers in Oncology

Article Title: The Metalloprotease-Disintegrin ADAM8 Alters the Tumor Suppressor miR-181a-5p Expression Profile in Glioblastoma Thereby Contributing to Its Aggressiveness

doi: 10.3389/fonc.2022.826273

Figure Lengend Snippet: ADAM8 affects MMP9 expression and proliferation via miR-181a-5p targeting ERK2 and CREB-1. (A) The upregulation of miR-181a-5p in U87_KO cells (RT-qPCR, n=3, left) causes inhibition of proliferation (CellTiter Glo, n=2, measurement after 48 h, right). (B) Overexpressing miR-181a-5p in U87_CTRL cells via transient transfection (RT-qPCR, n=3, left) causes inhibition of proliferation after 48 h (CellTiter Glo, n=3 technical replicates, right). (C) MMP9 is downregulated in U87_KO cells on mRNA (RT-qPCR, n=3, left). After miR-181a-5p mimic transfection, MMP9 is downregulated in U87 cells on the mRNA (RT-qPCR, n=3, p-value: 0.0009) (C) and on protein level (ELISA, n=2, p-value: 0.0004) analyzed by western blot (D) . (E) Analyses of kinase activation after miR-181a-5p mimic transfection for ERK1/2 (p-value: 0.003) and CREB-1 (p-value: 0.0007). Results are shown as mean values +/- SD of three independent experiments unless otherwise stated. Results are given in mean +/- SD. Unpaired one-tailed students t test was applied to determine significance: *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Soluble ADAM8 (DY1031, R&D Systems, UK) and soluble MMP9 (DY911, R&D Systems, UK) from cell culture supernatants were determined by Sandwich-ELISA method with DuoSet ELISA Kits.

Techniques: Expressing, Quantitative RT-PCR, Inhibition, Transfection, Enzyme-linked Immunosorbent Assay, Western Blot, Activation Assay, One-tailed Test

Journal: Frontiers in Oncology

Article Title: The Metalloprotease-Disintegrin ADAM8 Alters the Tumor Suppressor miR-181a-5p Expression Profile in Glioblastoma Thereby Contributing to Its Aggressiveness

doi: 10.3389/fonc.2022.826273

Figure Lengend Snippet: ADAM8 affects miRNA181a-5p levels associated with EVs in U87 cells. (A) U87_CTRL and U87_KO derived EVs were characterized regarding their concentration (upper graph) and size (lower graph). Measurements were taken with the NanoFCM device and results are shown as mean values +/- SEM of four independent experiments. (B) Representative size distributions of U87_CTRL and U87_KO derived EVs (NanoFCM) are shown in bar graphs. (C) Via western blot, the presence of EVs was confirmed. CALNEXIN was used as negative control and FLOTILLIN-1 as well as CD81 as EV markers. (D) MiR-181a-5p is detectable in U87 derived EVs. RT-qPCR results show a non-significant tendency of miR-181a-5p upregulation in U87_ KO derived EVs. Mean values +/- SD of three independent experiments if not otherwise stated. Unpaired two-tailed students t-test was applied to determine significance: ns, not significant, *p < 0.05.

Article Snippet: Soluble ADAM8 (DY1031, R&D Systems, UK) and soluble MMP9 (DY911, R&D Systems, UK) from cell culture supernatants were determined by Sandwich-ELISA method with DuoSet ELISA Kits.

Techniques: Derivative Assay, Concentration Assay, Western Blot, Negative Control, Quantitative RT-PCR, Two Tailed Test

Journal: Frontiers in Oncology

Article Title: The Metalloprotease-Disintegrin ADAM8 Alters the Tumor Suppressor miR-181a-5p Expression Profile in Glioblastoma Thereby Contributing to Its Aggressiveness

doi: 10.3389/fonc.2022.826273

Figure Lengend Snippet: Sketch of ADAM8-dependent effects caused by regulation of miR-181a-5p in GBM cells. ADAM8 with homologous domains including the metalloprotease domain (MP), the disintegrin/cysteine-rich domain (DC), the EGF-like domain (EGF), the transmembrane (TM), and the cytoplasmic domain (CD). ADAM8 activates intracellular signaling cascades by STAT3 and MAPK in the presence of the cytoplasmic domain. ADAM8/STAT3/miR-181a/ SPP1 axis: ADAM8 dependent STAT3 activation downregulates miR-181a-5p, as miR-181a-5p targets SPP1 , disinhibition of SPP1 leads to several tumor progressing effects such as induction of angiogenesis and enhanced immune cell recruitment. ADAM8/MAPK/MMP9 axis: ADAM8 activates the MAPK pathway, the transcription factor CREB-1 induces MMP9 transcription and inhibits miR-181a-5p transcription. MMP9 promotes tumor cell proliferation and invasion. By targeting CREB-1, ERK2, and MEK1, miR-181a-5p downregulates MMP9 expression most likely by an indirect mechanism. Created with BioRender.com .

Article Snippet: Soluble ADAM8 (DY1031, R&D Systems, UK) and soluble MMP9 (DY911, R&D Systems, UK) from cell culture supernatants were determined by Sandwich-ELISA method with DuoSet ELISA Kits.

Techniques: Activation Assay, Expressing

Journal: Frontiers in oncology

Article Title: The Metalloprotease-Disintegrin ADAM8 Alters the Tumor Suppressor miR-181a-5p Expression Profile in Glioblastoma Thereby Contributing to Its Aggressiveness.

doi: 10.3389/fonc.2022.826273

Figure Lengend Snippet: FIGURE 1 | Screening of two representative CRISPR/Cas9 ADAM8 (hA8) KO cell clones reveals an ADAM8-dependent regulation of miR-181a-5p. Confirmation of the CRISPR/Cas9 ADAM8 stable KO in U87 cells by qPCR (A), Western Blot (B), and ELISA (C). (D) A RT-qPCR-based miRNA PCR Array (Human Finder, Qiagen) of U87 CRISPR/Cas9 ADAM8 KO cells enabling the analysis of a total of 84 miRNAs. The legend for the fold- changes in the heat map is given above representing the fold-change values (2-DDCT) relative to U87_CTRL cells in both ADAM8 KO clones. Note the variance in fold changes between the two KO clones (E) Confirmation of miR- 181a-5p upregulation in U87_KO1 and U87_KO2 cells (p-value: 0.01 and 0.03). The expression of miR-181a-5p (F) and ADAM8 (G) in GBM cell lines (G28, G112, U251), primary patient-derived cell lines (GBM29, 98, and 42), and GBM stem-like cell lines (GSCs) (GSC 2016/240, GSC 2017/74, GSC 2017/151) are shown as relative values to the expression in U87 cells. MiR- 181a-5p is mostly expressed in GSCs. The patient-derived cell line GBM42 shows the highest ADAM8 expression. Pearson correlation analysis of miR- 181a-5p and ADAM8 in cell lines (H), primary cell lines (I), and primary GSCs (J) reveal a negative correlation only observed in GSCs (p-value: 0.01, Pearson r: -0.88). Results are given as mean +/- SD of two to three independent experiments. Unpaired two-tailed students t-test or two way ANOVA for multiple comparison (F) were applied to determine significance: *p < 0.05, **p < 0.01, ***p < 0.001. n.d., not detectable.

Article Snippet: Enzyme-Linked Immunosorbent Assay (ELISA) Soluble ADAM8 (DY1031, R&D Systems, UK) and soluble MMP9 (DY911, R&D Systems, UK) from cell culture supernatants were determined by Sandwich-ELISA method with DuoSet ELISA Kits.

Techniques: CRISPR, Clone Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Derivative Assay, Two Tailed Test, Comparison

Journal: Frontiers in oncology

Article Title: The Metalloprotease-Disintegrin ADAM8 Alters the Tumor Suppressor miR-181a-5p Expression Profile in Glioblastoma Thereby Contributing to Its Aggressiveness.

doi: 10.3389/fonc.2022.826273

Figure Lengend Snippet: FIGURE 2 | ADAM8 regulates the expression of miR-181a-5p via JAK2/STAT3 signaling. (A) U87_CTRL cells were analyzed for miR-181a-5p expression by RT- qPCR after treatment with the broad-spectrum MMP inhibitor BB-94 (left) and the ADAM8 inhibitor BK-1361 (right). (B) One representative western blot of three independent experiments shows the rescue of either ADAM8 lacking the cytoplasmatic domain (Delta CD) or the full-length ADAM8 (hA8). The quantifications of pEGFR, pSTAT3, and pERK1/2 are depicted on the right side and were normalized to b-Tubulin and total-EGFR/b-Tubulin, total STAT3/b-Tubulin, or total ERK1/2/b- Tubulin. Also, RT-qPCR results show no differences in miR-181a-5p expression after the transfection of ADAM8 Delta CD but a downregulation with the full-length ADAM8 rescue (p-value: 0.002). (C) The expression of ADAM8 mRNA (RT-qPCR, left) and secreted ADAM8 (ELISA, right, n=2) is not affected after miR-181a-5p mimic transfection. U87_CTRL cells (D) and patient-derived GBM42 cells (E) were treated with JAK2/STAT3 inhibitor WP1066 as indicated and analyzed via western blot and RT-qPCR. In (D), qPCR results are shown as mean values +/- SD of four independent experiments and in (E), results of miR-181a-5p are described as mean values of three technical replicates. Inhibition of JAK2/STAT3 increases miR-181a-5p expression (U87 p-value: 0.027; GBM42 p-value: 0.004). Results are shown as mean values +/- SD from three independent experiments if not otherwise stated. Unpaired two-tailed students t-test was applied to determine significance: ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Enzyme-Linked Immunosorbent Assay (ELISA) Soluble ADAM8 (DY1031, R&D Systems, UK) and soluble MMP9 (DY911, R&D Systems, UK) from cell culture supernatants were determined by Sandwich-ELISA method with DuoSet ELISA Kits.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay, Derivative Assay, Inhibition, Two Tailed Test

Journal: Frontiers in oncology

Article Title: The Metalloprotease-Disintegrin ADAM8 Alters the Tumor Suppressor miR-181a-5p Expression Profile in Glioblastoma Thereby Contributing to Its Aggressiveness.

doi: 10.3389/fonc.2022.826273

Figure Lengend Snippet: FIGURE 3 | ADAM8 affects MMP9 expression and proliferation via miR- 181a-5p targeting ERK2 and CREB-1. (A) The upregulation of miR-181a-5p in U87_KO cells (RT-qPCR, n=3, left) causes inhibition of proliferation (CellTiter Glo, n=2, measurement after 48 h, right). (B) Overexpressing miR- 181a-5p in U87_CTRL cells via transient transfection (RT-qPCR, n=3, left) causes inhibition of proliferation after 48 h (CellTiter Glo, n=3 technical replicates, right). (C) MMP9 is downregulated in U87_KO cells on mRNA (RT- qPCR, n=3, left). After miR-181a-5p mimic transfection, MMP9 is downregulated in U87 cells on the mRNA (RT-qPCR, n=3, p-value: 0.0009) (C) and on protein level (ELISA, n=2, p-value: 0.0004) analyzed by western blot (D). (E) Analyses of kinase activation after miR-181a-5p mimic transfection for ERK1/2 (p-value: 0.003) and CREB-1 (p-value: 0.0007). Results are shown as mean values +/- SD of three independent experiments unless otherwise stated. Results are given in mean +/- SD. Unpaired one- tailed students t test was applied to determine significance: *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Enzyme-Linked Immunosorbent Assay (ELISA) Soluble ADAM8 (DY1031, R&D Systems, UK) and soluble MMP9 (DY911, R&D Systems, UK) from cell culture supernatants were determined by Sandwich-ELISA method with DuoSet ELISA Kits.

Techniques: Expressing, Quantitative RT-PCR, Inhibition, Transfection, Enzyme-linked Immunosorbent Assay, Western Blot, Activation Assay, One-tailed Test

Journal: Frontiers in oncology

Article Title: The Metalloprotease-Disintegrin ADAM8 Alters the Tumor Suppressor miR-181a-5p Expression Profile in Glioblastoma Thereby Contributing to Its Aggressiveness.

doi: 10.3389/fonc.2022.826273

Figure Lengend Snippet: FIGURE 4 | ADAM8 affects miRNA181a-5p levels associated with EVs in U87 cells. (A) U87_CTRL and U87_KO derived EVs were characterized regarding their concentration (upper graph) and size (lower graph). Measurements were taken with the NanoFCM device and results are shown as mean values +/- SEM of four independent experiments. (B) Representative size distributions of U87_CTRL and U87_KO derived EVs (NanoFCM) are shown in bar graphs. (C) Via western blot, the presence of EVs was confirmed. CALNEXIN was used as negative control and FLOTILLIN-1 as well as CD81 as EV markers. (D) MiR-181a-5p is detectable in U87 derived EVs. RT-qPCR results show a non-significant tendency of miR-181a-5p upregulation in U87_ KO derived EVs. Mean values +/- SD of three independent experiments if not otherwise stated. Unpaired two-tailed students t-test was applied to determine significance: ns, not significant, *p < 0.05.

Article Snippet: Enzyme-Linked Immunosorbent Assay (ELISA) Soluble ADAM8 (DY1031, R&D Systems, UK) and soluble MMP9 (DY911, R&D Systems, UK) from cell culture supernatants were determined by Sandwich-ELISA method with DuoSet ELISA Kits.

Techniques: Derivative Assay, Concentration Assay, Western Blot, Negative Control, Quantitative RT-PCR, Two Tailed Test

Journal: Frontiers in oncology

Article Title: The Metalloprotease-Disintegrin ADAM8 Alters the Tumor Suppressor miR-181a-5p Expression Profile in Glioblastoma Thereby Contributing to Its Aggressiveness.

doi: 10.3389/fonc.2022.826273

Figure Lengend Snippet: FIGURE 5 | Expression levels of ADAM8 and MMP9, and miR-181a-5p in GBM tissue samples. (A) RT-qPCR results of GBM tissue samples (n=22, fold change normalized to 1) indicate a higher expression of ADAM8 (p-value: 0.0009) and MMP9 (p-value: 0.0002) than miR-181a-5p. (B) Dividing the RT-qPCR results and patient cohort into two groups (low/high ADAM8 or low/high miR-181a-5p expression) reveals a correlation of ADAM8 with MMP9 (left graph, p-value: 0.001) but no correlation of miR-181a-5p with ADAM8 (middle graph, p-value: 0.577) or MMP9 (right graph, p-value: 0.083) expression. (C) RT-qPCR results for miR-181a-5p expression of each GBM tissue sample. (D) ADAM8 and MMP9 are correlated in GBM tissue samples (p < 0.0001, n=22), whereas the inverse correlations of ADAM8 and miR-181a-5p and of miR-181a-5p and MMP9 are non-significant (p-values: 0.63 and 0.6, respectively). (E) T2-weighted magnetic resonance (MR) image showing a left parietal GBM (segmented in yellow, patient 25) as well as the co-registered choline/N-acetylaspartate (NAA) maps derived from 1H-MR spectroscopy, integrated into the neuronavigation system for navigated extraction of tissue samples (L1: tumor border, L2/L3: tumor, L4: tumor, Cho/NAA hotspot) magnetic resonance (heatmap for choline metabolite). Corresponding molecular analyses are shown in (F, G) (patient 25). RT-qPCR results of ADAM8 (red), MMP9 (tiled red), and miR-181a-5p (blue) in different tissue locations normalized to either L1 (F) or L4 (G) describing the direction of surgery. (H) In a pilot study, three GBM patients (Patient 9, 23, 24) were analyzed for their serum-EV miR-181a-5p expression via RT-qPCR. The serum was collected before and after the first and second surgery. Interestingly, after first surgical resection miR-181a-5p is less expressed in serum-EVs (p-value: 0.042). (I) After second surgery, miR-181a-5p shows a slight increase in serum-EVs (p-value: 0.08). (J), miR-181a-5p is less detectable in serum-EVs prior to the second surgery compared to pre-first surgery (p-value: 0.02; left graph). Results are shown in mean values +/- SD. Unpaired one-tailed students t test and Wilcoxon signed-rank test were applied to determine significance: ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Enzyme-Linked Immunosorbent Assay (ELISA) Soluble ADAM8 (DY1031, R&D Systems, UK) and soluble MMP9 (DY911, R&D Systems, UK) from cell culture supernatants were determined by Sandwich-ELISA method with DuoSet ELISA Kits.

Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, Spectroscopy, Extraction, One-tailed Test

Journal: Frontiers in oncology

Article Title: The Metalloprotease-Disintegrin ADAM8 Alters the Tumor Suppressor miR-181a-5p Expression Profile in Glioblastoma Thereby Contributing to Its Aggressiveness.

doi: 10.3389/fonc.2022.826273

Figure Lengend Snippet: FIGURE 6 | Sketch of ADAM8-dependent effects caused by regulation of miR-181a-5p in GBM cells. ADAM8 with homologous domains including the metalloprotease domain (MP), the disintegrin/cysteine-rich domain (DC), the EGF-like domain (EGF), the transmembrane (TM), and the cytoplasmic domain (CD). ADAM8 activates intracellular signaling cascades by STAT3 and MAPK in the presence of the cytoplasmic domain. ADAM8/STAT3/miR-181a/SPP1 axis: ADAM8 dependent STAT3 activation downregulates miR-181a-5p, as miR-181a-5p targets SPP1, disinhibition of SPP1 leads to several tumor progressing effects such as induction of angiogenesis and enhanced immune cell recruitment. ADAM8/MAPK/MMP9 axis: ADAM8 activates the MAPK pathway, the transcription factor CREB-1 induces MMP9 transcription and inhibits miR-181a-5p transcription. MMP9 promotes tumor cell proliferation and invasion. By targeting CREB-1, ERK2, and MEK1, miR-181a-5p downregulates MMP9 expression most likely by an indirect mechanism. Created with BioRender.com.

Article Snippet: Enzyme-Linked Immunosorbent Assay (ELISA) Soluble ADAM8 (DY1031, R&D Systems, UK) and soluble MMP9 (DY911, R&D Systems, UK) from cell culture supernatants were determined by Sandwich-ELISA method with DuoSet ELISA Kits.

Techniques: Activation Assay, Expressing