Down-Regulation of Neogenin Accelerated Glioma Progression through Promoter Methylation and Its Overexpression in SHG-44 Induced Apoptosis

Dependence receptors have been proved to act as tumor suppressors in tumorigenesis. Neogenin, a DCC homologue, well known for its fundamental role in axon guidance and cellular differentiation, is also a dependence receptor functioning to control apoptosis. However, loss of neogenin has been reported in several kinds of cancers, but its role in glioma remains to be further investigated.Western blot analysis showed that neogenin level was lower in glioma tissues than in their matching surrounding non-neoplastic tissues (n = 13, p<0.01). By immunohistochemical analysis of 69 primary and 16 paired initial and recurrent glioma sections, we found that the loss of neogenin did not only correlate negatively with glioma malignancy (n = 69, p<0.01), but also glioma recurrence (n = 16, p<0.05). Kaplan-Meier plot and Cox proportional hazards modelling showed that over-expressive neogenin could prolong the tumor latency (n = 69, p<0.001, 1187.6 ± 162.6 days versus 687.4 ± 254.2 days) and restrain high-grade glioma development (n = 69, p<0.01, HR: 0.264, 95% CI: 0.102 to 0.687). By Methylation specific polymerase chain reaction (MSP), we reported that neogenin promoter was methylated in 31.0% (9/29) gliomas, but absent in 3 kinds of glioma cell lines. Interestingly, the prevalence of methylation in high-grade gliomas was higher than low-grade gliomas and non-neoplastic brain tissues (n = 33, p<0.05) and overall methylation rate increased as glioma malignancy advanced. Furthermore, when cells were over-expressed by neogenin, the apoptotic rate in SHG-44 was increased to 39.7% compared with 8.1% in the blank control (p<0.01) and 9.3% in the negative control (p<0.01).These observations recapitulated the proposed role of neogenin as a tumor suppressor in gliomas and we suggest its down-regulation owing to promoter methylation is a selective advantage for glioma genesis, progression and recurrence. Furthermore, the induction of apoptosis in SHG-44 cells after overexpression of neogenin, indicated that neogenin could be a novel target for glioma therapy

Inhibition of CD44 suppresses the formation of fibrotic scar after spinal cord injury via the JAK2/STAT3 signaling pathway

Summary: Fibrotic scar is one of the main impediments to axon regeneration following spinal cord injury (SCI). In this study, we found that CD44 was upregulated during the formation of fibrotic scar, and blocking CD44 by IM7 caused downregulation of fibrosis-related extracellular matrix proteins at both 2 and 12 weeks post-spinal cord injury. More Biotinylated dextran amine (BDA)-traced corticospinal tract axons crossed the scar area and extended into the distal region after IM7 administration. A recovery of motor and sensory function was observed based on Basso Mouse Scale (BMS) scores and tail-flick test. In vitro experiments revealed that inhibiting CD44 and JAK2/STAT3 signaling pathway decreased the proliferation, differentiation, and migration of fibroblasts induced by the inflammatory supernatant. Collectively, these findings highlight the critical role of CD44 and its downstream JAK2/STAT3 signaling pathway in fibrotic scar formation, suggesting a potential therapeutic target for SCI

Immunohistochemical analysis of neogenin in primary gliomas.

<p>(A–D) Representative immunohistochemical pictures of grade I-IV glioma respectively, (A’–D’) Magnification of the red squares in (A–D), (A–D) 200× magnification; (A’–D’) 400× magnification. (E) Scatter diagram of neogenin expression in 69 primary gliomas, blue bars are the means of four grades, n = 69, One-Way ANOVA was used in statistical analysis. (F) Histogram of mean neogenin expression in low-grade gliomas and high-grade gliomas, statistical analysis was performed with independent <i>t</i>-test. *<i>p</i><0.05, **<i>p</i><0.01, error bars indicate standard error means.</p

Additional file 1 of Plasma exosomes improve peripheral neuropathy via miR-20b-3p/Stat3 in type I diabetic rats

Additional file 1: Figure S1. The diabetic model was successfully constructed after STZ injection. Figure S2. Internalization of plasma exosomes of sciatic nerve in NC rats. Figure S3. Biological distribution of plasma exosomes in vivo. Figure S4. Effects of plasma exosomes on RSC96 and DRG cells. Figure S5. Effects of plasma exosomes on RSC96 and DRG cells. Figure S6. Statistical analysis of pstat3/stat3. Figure S7. Characterization of ageing plasma exosomes. Figure S8. Ageing-exos did not improve nerve damage caused by high glucose. Figure S9. Ageing-exos augments the motor and sensory innervation of the targets. Table S1. Random blood glucose and total cholesterol levels after exosome treatment. Table S2. Random blood glucose and total cholesterol levels after miR-20b-3p agomir treatment. Table S3. Sequence information used in the article. Table S4. Reagent information used in the article

Clinicopathologic features and relative neogenin level profile in 13 paired surrounding and glioma tissues (January – November, 2011).

<p>SD: standard deviation;</p>*<p>: paired <i>t</i>-test.</p

Primary and recurrent clinicopathologic features of 16 patients (January, 2001 – January, 2011).

<p>IOD: integral optical density;</p><p>IOD values were supplied as Mean±Standard Deviation.</p>*<p>: paired <i>t</i>-test;</p>†<p>: undefined grade gliomas.</p

Kaplan-Meier survival curves for overall progression to high-grade glioma.

<p>Failure event for computation of this curve was diagnosed as the high-grade glioma. Higher neogenin patients are marked in green color, lower neogenin patients are marked in blue color.</p

Overexpression of neogenin in SHG-44 cell line.

<p>(A) Western blot results showed neogenin expression in the blank control which was untreated (Con), the negative control which was transfected by empty vector (Vec) and the over-expressive group which was transfected by neogenin expression plasmid (Neo) in SHG-44 cell line at 48 hours after transfection; (B-D) Status of cells in the blank, negative and over-expressive group of neogenin respectively at 48 h after transfection. (E) Apoptotic distribution maps of cells in the blank, negative and over-expressive group were drew by flow cytometry assay respectively. (F) Average apoptotic rate of cells in the blanck, negative and over-expressive group in the flow cytometry assay, One-Way ANOVA, n = 3, **<i>p</i><0.01, error bars indicate standard error means.</p

Expression of neogenin in 16 paired primary and recurrent glioma sections.

<p>(A-B) Representative immunohistochemical pictures (200× magnification): (A) primary glioma, (B) matching recurrent glioma. (C) statistical graph of mean neogenin expression in primary and recurrent gliomas. Paired t-test, n = 16, * <i>p</i><0.05, error bars indicate standard error means.</p