Journal: Advanced Science

Article Title: Circular RNA PTPN4 Contributes to Blood‐Brain Barrier Disruption during Early Epileptogenesis

doi: 10.1002/advs.202502250

Figure Lengend Snippet: CircPTPN4 disrupts tight junction by upregulating ECE‐1 expression via miR‐145a‐5p sponging in BMECs. A) Quantitative RT‐PCR analyses of ECE‐1 mRNA levels in BMECs co‐cultured with Control neurons or Mg 2 ⁺‐free neurons, and transduced with either Control‐ShRNA or CircPTPN4‐ShRNA lentivirus ( n = 5; p < 0.0001, ShRNA‐Ctrl‐treated Mg 2 ⁺‐free N BMECs versus ShRNA‐Ctrl‐treated Ctrl N BMECs; p = 0.0004, ShRNA‐CircPTPN4‐treated Mg 2 ⁺‐free N BMECs versus ShRNA‐Ctrl‐treated Mg 2 ⁺‐free N BMECs). B) Western blot analyses of ECE‐1 protein levels in the cortex of Control and SE‐24 h mice infected with Control‐ShRNA or CircPTPN4‐ShRNA lentivirus. C) Bar graph quantifying ECE‐1 protein levels as the intensity ratio of ECE‐1 to GAPDH ( n = 5; p < 0.0001, ShRNA‐Ctrl‐treated SE‐24 h mice versus ShRNA‐Ctrl‐treated Ctrl mice; p < 0.0001, ShRNA‐CircPTPN4‐treated SE‐24 h mice versus ShRNA‐Ctrl‐treated SE‐24 h mice). D) Quantitative RT‐PCR analyses of ECE‐1 mRNA expression in Mg 2+ free N BMECs transduced with ShRNA‐Ctrl or ShRNA‐CircPTPN4 lentivirus, and co‐treated with anti‐miR Control or miR‐145a‐5p inhibitors ( n = 5; p < 0.0001, anti‐miR‐145a‐5p and ShRNA‐Ctrl BMECs versus Control anti‐miRNA and ShRNA‐Ctrl BMECs; p = 0.0048, anti‐miR‐145a‐5p and ShRNA‐CircPTPN4 BMECs versus Control anti‐miRNA and ShRNA‐CircPTPN4 BMECs; p < 0.0001, anti‐miR‐145a‐5p and ShRNA‐CircPTPN4 BMECs versus anti‐miR‐145a‐5p and ShRNA‐Ctrl BMECs). E) Quantitative RT‐PCR analyses of ECE‐1 mRNA expression in Mg 2+ free N BMECs infected with OE‐Ctrl or OE‐CircPTPN4 lentivirus, and co‐treated with miR Control or miR‐145a‐5p mimics ( n = 5; p < 0.0001, miR Control and OE‐Ctrl BMECs versus miR‐145a‐5p mimics and OE‐Ctrl BMECs; p < 0.0001, miR Control and OE‐Ctrl BMECs versus miR Control and OE‐CircPTPN4 BMECs; p < 0.0001, miR Control and OE‐CircPTPN4 BMECs versus miR‐145a‐5p mimics and OE‐CircPTPN4 BMECs). F) Representative immunofluorescence images of Occludin staining in Mg 2+ free N BMECs infected with ShRNA‐Ctrl or ShRNA‐CircPTPN4 lentivirus, and co‐infected with anti‐miR Control or miR‐145a‐5p inhibitors. G) Bar graph showing quantification of Occludin staining intensity in Mg 2+ free N BMECs infected with ShRNA‐Ctrl or ShRNA‐CircPTPN4 lentivirus, and co‐infected with anti‐miR Control or miR‐145a‐5p inhibitors ( n = 5; p = 0.0066, anti‐miR‐145a‐5p and ShRNA‐Ctrl BMECs versus Control anti‐miRNA and ShRNA‐Ctrl BMECs; p = 0.0021, anti‐miR‐145a‐5p and ShRNA‐CircPTPN4 BMECs versus Control anti‐miRNA and ShRNA‐CircPTPN4 BMECs; p = 0.0049, anti‐miR‐145a‐5p and ShRNA‐CircPTPN4 BMECs versus anti‐miR‐145a‐5p and ShRNA‐Ctrl BMECs). H) Bar graph showing the permeability of Mg 2+ free N BMECs transfected with ShRNA‐Ctrl or ShRNA‐CircPTPN4 lentivirus, and co‐treated with anti‐miR Control or miR‐145a‐5p inhibitors ( n = 5; p = 0.0021, anti‐miR‐145a‐5p and ShRNA‐Ctrl BMECs versus Control anti‐miRNA and ShRNA‐Ctrl BMECs; p = 0.0045, anti‐miR‐145a‐5p and ShRNA‐CircPTPN4 BMECs versus Control anti‐miRNA and ShRNA‐CircPTPN4 BMECs; p = 0.0002, anti‐miR‐145a‐5p and ShRNA‐CircPTPN4 BMECs versus anti‐miR‐145a‐5p and ShRNA‐Ctrl BMECs). I) Representative immunofluorescence images of Occludin staining in Mg 2+ free N BMECs co‐cultured with Control or Mg 2 ⁺‐free neurons, and infected with Control‐ShRNA or ECE‐1‐ShRNA lentivirus. J) Bar graph showing quantification of Occludin staining in Ctrl N BMECs or Mg 2 ⁺‐free N BMECs, and infected with Control‐ShRNA or ECE‐1‐ShRNA lentivirus ( n = 5; p < 0.0001, ShRNA‐ECE‐1‐treated Ctrl N BMECs versus ShRNA‐Ctrl‐treated Ctrl N BMECs; p =0 .0108, ShRNA‐ECE‐1‐treated Mg 2 ⁺‐free N BMECs versus ShRNA‐Ctrl‐treated Mg 2 ⁺‐free N BMECs; p = 0.0004, ShRNA‐Ctrl‐treated Mg 2 ⁺‐free N BMECs versus ShRNA‐Ctrl‐treated Ctrl N BMECs; p < 0.0001, ShRNA‐ECE‐1‐treated Mg 2 ⁺‐free N BMECs versus ShRNA‐ECE‐1‐treated Ctrl N BMECs). Values represent means ± S.E.M. Statistical analysis were performed using one‐way ANOVA followed by Tukey's test. Scale bar = 25 µm.

Article Snippet: The miR‐145a‐5p mimics (HY‐ R00282 ), miRNA mimics negative Control (HY‐ R04602 ), miR‐145a‐5p inhibitor (HY‐RI00282), and miRNA inhibitor negative Control (HY‐RI04602) were purchased from Med Chem Express (MCE).

Techniques: Expressing, Quantitative RT-PCR, Cell Culture, Control, Transduction, shRNA, Western Blot, Infection, Immunofluorescence, Staining, Permeability, Transfection