Fig 1: NLGN3 promoted the proliferation and metastasis of glioma cells. (A) NLGN3-specific siRNA and an overexpression plasmid was transferred into glioma cells to generate NLGN3 knockdown and overexpression cell lines, respectively. Western blot was used to detect NLGN3 expression. CCK-8 assay was performed to determine the proliferation of U87 (B) and U251 (C) cells. (D) Colony formation assay was performed to further confirm the proliferation of U87 (E) and U251 (F) cells. (G) The migration of U87 (H) and U251 (I) cells was detected using wound healing assay. (J,K) The invasion of glioma cells was determined using transwell assay. NC, negative control group; KD, NLGN3 knockdown group; OE, NLGN3 overexpression group; *p < 0.05; **p < 0.01; ***p < 0.001.
Fig 2: ADAM10 promoted the migration and invasion of glioma cells. (A) Correlation analysis between expression of ADAM10 and NLGN3 in glioma and normal brain tissue was performed using GEPIA. (B) Correlation analysis between expression of ADAM10 and LYN in glioma and normal brain tissue was performed using GEPIA. (C) The expression of ADAM10 in LGG and GBM was analyzed on GEPIA. (D) U87 cells were treated with ADAM10 inhibiter, GI254023X (10 μM), and CCK-8 assay was performed to detect the proliferation of each group of cells. (E,F) ADAM10 inhibiter, GI254023X, suppressed the migration of U87 cells. (G,H) GI254023X inhibited the invasion of U87 and U251 cells. (I) The relationship between ADAM10 and prognosis of patients with LGG and GBM was analyzed on GEPIA. *p < 0.05.
Fig 3: NLGN3 was overexpressed in human glioma. (A) The expression level of NLGN3 in human glioma was detected by IHC, and normal brain tissue was used as the control. The magnification of the image was 100x. (B) Statistical analysis of NLGN3 expression in glioma and normal tissues. (C) The percentage of NLGN3 positive specimens in high-grade glioma was significantly higher in comparison to low-grade glioma. (D) NLGN3 expression in five human glioma cell lines, U87, U251, HS683, A172, and U373, was detected using Western blot analysis. U251 and U87 cells showed higher levels of NLGN3 and were used for subsequent experiments. *p < 0.05.
Fig 4: NLGN3 activated the LYN pathway to upregulate ADAM10, which in turn promoted cleavage of NLGN3. (A) The NLGN3 plasmid carrying the Flag fragment was transfected into U251 cells with an empty vector as the control. Western blot was performed to detect the Flag label. SS = signal sequence; M = maker. (B) The level of phosphorylation of AKT and LYN was detected after treatment with a NLGN3 plasmid, with an empty vector as the control. WT = wild type. (C) U251 cells were transfected with LYN or treated with bafetinib along with NLGN3 plasmid transfection, with an empty vector or DMSO as the control. Western blot was performed to detect the Flag label. WCL = whole cell lysates. (D) qPCR was performed to detect the expression of ADAM10 in U251 cells. (E) Western blot was performed to detect the expression of ADAM10 in U251 cells. ADAM10 expression was upregulated by LYN and inhibited by bafetinib. (F) Compared with the control cells, LYN overexpression increased the level of cleaved NLGN3, whereas decreased cleaved NLGN3 was observed after treatment with the ADAM10 inhibitor compared with LYN overexpression cells. (G) Overexpression of ADAM10 increased cleaved NLGN3 levels, but there was no significant change of cleaved NLGN3 levels after treatment with LYN inhibitors compared with ADAM10 overexpression group. *p < 0.05; **p < 0.01.
Fig 5: Schematic illustration of the positive feedback circle, s-NLGN3/LYN/ADAM10. Secreted NLGN3 derived from neurons and glioma cells activates LYN and then up-regulates ADAM10 expression, which can cleave NLGN3 to promote its secretion.
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