time atcc 1st  (ATCC)

Journal: Cell Adhesion & Migration

Article Title: MiR-646 inhibited cell proliferation and migration by targeting P62 in glioma

doi: 10.1080/19336918.2025.2515763

Figure Lengend Snippet: MiR-646 overexpression attenuates GBM cell tumorigenesis in vitro . (a) RT-qPCR analysis of miRNA-646 expression in A172 and U87 cells treated with 30 nM miRNA-646 mimics, 30 nM miRNA-646 inhibitor, or 30 nM scrambled negative control RNA. (b) MTT assay detected proliferation rates of A172 and U87 cells transfected with 30 nM miR-646 mimics or 30 nM inhibitor-646 after 24, 48, 72 and 96 h. (c) Colony formation detected long-term cell viability of A172 and U87 cells treated with 30 nM miRNA-646 mimics, 30 nM miRNA-646 inhibitor, or 30 nM scrambled negative control RNA. (d) Western blot assay showing CyclinD1 protein levels in A172 and U87 cells transfected with 30 nM miRNA-646 mimics, 30 nM miRNA-646 inhibitor, or 30 nM scrambled negative control RNA. (e) Transwell assay revealing cell invasion in A172 and U87 cells transfected with 30 nM miRNA-646 mimics, 30 nM miRNA-646 inhibitor, or 30 nM scrambled negative control RNA. (f) Wounded healing assays showing cell migration in A172 and U87 cells treated with 30 nM miRNA-646 mimics, 30 nM miRNA-646 inhibitor, or 30 nM scrambled negative control RNA. Scale bar = 100 μm. (g) Western blot analysis of major migration and invasion proteins in A172 and U87 cells transfected with 30 nM miRNA-646 mimics, 30 nM miRNA-646 inhibitor, or 30 nM scrambled negative control RNA. Representative images were shown and analyzed as mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs miR-nc mimics. # p < 0.05, ## p < 0.01, ### p < 0.001 vs inhibitor-nc.

Article Snippet: MiR-646 was quantified using a stem-loop real-time PCR miRNA kit (Vazyme, China).

Techniques: Over Expression, In Vitro, Quantitative RT-PCR, Expressing, Negative Control, MTT Assay, Transfection, Western Blot, Transwell Assay, Migration

Journal: Cell Adhesion & Migration

Article Title: MiR-646 inhibited cell proliferation and migration by targeting P62 in glioma

doi: 10.1080/19336918.2025.2515763

Figure Lengend Snippet: MiR-646 targets p62 in 3’UTR and negatively regulates its expression. (a) The Venn diagram of miR-646 targeting p62 mRNA intersecting three prediction databases: miRtaebase, miRwalk and Targetscan. (b) The expression of p62 protein were detected by western blotting in A172 and U87 cells transfected with 30 nM miRNA-646 mimics or negative control RNA; ** p < 0.01. (c) Western blot assay showing p62 protein levels in NHA, A172 and U87 cells.; * p < 0.05, *** p < 0.001. (d) Relative expression of p62 in 226 WHO 2 cases, 244 WHO 3 cases, and 150 WHO 4 cases were analyzed using the TCGA database; ** p < 0.01, *** p < 0.001, *** p < 0.001. (e) The Kaplan – Meier curve of overall survival with log‐rank test according to the p62 level (high and low expression divided by the median expression level) in all grades pf gliomas in the CGGA database; *** p < 0.001. (f) p62 protein expression were determined by immunohistochemistry in adjacent and glioma samples; scale bar = 50 μm, *** p < 0.001. (g) Algorithms predicted the miR-646 target sequences in the p62 mRNA 3’UTR. (h) Dual-luciferase reporter assays were used to assess GBM cells transfected with luciferase vectors carrying WT or MUT 3’-UTR of p62, in response to miR-646(30 nM) or miR-nc(30 nM); ** p < 0.01, *** p < 0.001.

Article Snippet: MiR-646 was quantified using a stem-loop real-time PCR miRNA kit (Vazyme, China).

Techniques: Expressing, Western Blot, Transfection, Negative Control, Immunohistochemistry, Luciferase

Journal: Cell Adhesion & Migration

Article Title: MiR-646 inhibited cell proliferation and migration by targeting P62 in glioma

doi: 10.1080/19336918.2025.2515763

Figure Lengend Snippet: Restoration of p62 abrogates the inhibitory effects of miR-646 on GBM cell proliferation and migration. (a) MTT assay showing proliferation rates of A172 and U87 cells transfected with 30 nM miR-646 mimics or negative control RNA plus p62-overexpression plasmid or vector after 24, 48, 72 and 96 h. (b) Colony formation assays demonstrating the long- term cell viability of A172 and U87 cells transfected with 30 nM miR-646 mimics or negative control RNA, plus p62-overexpression plasmid or vector. (c) Western blot assay showing CyclinD1 and p62 protein levels in A172 and U87 cells transfected with 30 nM miR-646 mimics or negative control RNA plus p62-overexpression plasmid or vector. (d) Transwell assay revealing cell invasion in A172 and U87 cells transfected with 30 nM miRNA-646 mimics or negative control RNA, plus p62-overexpression plasmid or vector. (e) Wounded healing assays showing cell migration in A172 and U87 cells treated with 0 nM miRNA-646 mimics or negative control RNA, plus p62-overexpression plasmid or vector. Scale bar = 100 μm. (f) Western blot analysis of major migration and invasion proteins in A172 and U87 cells transfected with 30 nM miRNA-646 mimics or negative control RNA, plus p62-overexpression plasmid or vector. Data are presented as the mean ± SEM of three independent experiments. Significant results are presented as * p < 0.05, ** p < 0.01, *** p < 0.001 versus the miR-nc+p62-nc group; # p < 0.05, ## p < 0.01, ### p < 0.001 versus the miR-nc+p62 group.

Article Snippet: MiR-646 was quantified using a stem-loop real-time PCR miRNA kit (Vazyme, China).

Techniques: Migration, MTT Assay, Transfection, Negative Control, Over Expression, Plasmid Preparation, Western Blot, Transwell Assay

Journal: Cell Adhesion & Migration

Article Title: MiR-646 inhibited cell proliferation and migration by targeting P62 in glioma

doi: 10.1080/19336918.2025.2515763

Figure Lengend Snippet: MiR-646 exerts inhibitory effects via keap1/Nrf2 pathway by p62. (a) Western blot analysis of LC3 II/I and Beclin-1 in A172 and U87 cells transfected with 30 nM miRNA-646 mimics or negative control RNA. (b) Immunofluorescence assays were applied to analyze autophagy flux in A172 and U87 transfected with 30 nM miRNA-646 mimics or negative control RNA. Cell nuclei were stained by DAPI. Scale bar = 20 μm. (c) Western blot analysis of Keap1 and Nrf2 in A172 and U87 cells transfected with 30 nM miRNA-646 mimics or negative control RNA. (d) The expressions of Nrf2 in A172 and U87 cells transfected with 30 nM miR-646 mimics in cytoplasmic and nuclear fraction were detected by western blotting. (e) The expression of HO-1 mRNA in A172 and U87 cells transfected with miR-646 mimics were detected by RT-qPCR. Data are presented as the mean ± SEM of three independent experiments. n.S., no significance; ** p < 0.01, *** p < 0.001.

Article Snippet: MiR-646 was quantified using a stem-loop real-time PCR miRNA kit (Vazyme, China).

Techniques: Western Blot, Transfection, Negative Control, Immunofluorescence, Staining, Expressing, Quantitative RT-PCR