Effect And Mechanism Of LncRNA-LINC00470 In Serum Exosomes On Proliferation And Autophagy Of Glioma Cells

Objective:To explore the correlation between the expression of lnc RNA LINC00470 in serum exosomes of patients with glioma and their prognosis,and to explore the effect and molecular mechanism of lnc RNA LINC00470 in regulating autophagy and proliferation of glioma cells through experiments.Methods:1.Serum exosomes(GC-exo)were extracted from 45 patients with glioma and 10 healthy subjects(HC-exo):(1)Exosomes were identified by transmission electron microscopy,particle size analysis,Western Blot and flow cytometry.(2)The expression of LINC00470 was detected by q RT-PCR.(3)Combined with the clinicopathological features of patients with glioma,the relationship between the expression of LINC00470 in serum exosomes and the overall survival of patients was analyzed.2.When studying the effects of LINC00470 in serum exosomes of patients with high-grade glioma on the autophagy and proliferation of glioma cells:(1)The glioma model of nude mice was established,and the tumor changes were observed by fluorescence imaging in vivo.HE staining and immunohistochemistry were used to detect the tumor proliferation.Western blot was used to detect the expression of autophagy related proteins in tumor tissues.(2)In vitro,exosome incubation and LINC00470 transfection were performed,and autophagy levels were detected by transmission electron microscopy,MDC staining,LC3 immunofluorescence staining and Western blot.Cell proliferation was detected by CCK8 assay,clone formation assay and flow cytometry.3.When studying the regulation mechanism of LINC00470 in serum exosomes of patients with high-grade glioma on autophagy and proliferation of glioma cells:(1)In vitro,through exosome incubation,LINC00470 transfection and rapamycin intervention were performed,and autophagy levels were detected by transmission electron microscopy,MDC staining,LC3 immunofluorescence staining and Western blot.Cell proliferation was detected by CCK8 assay,clone formation assay and flow cytometry.(2)In vitro,the binding of LINC00470 and m RNA-WEE1 to miR-580-3p was detected by dual luciferase reporter gene assay and RNA immunoprecipitation assay.(3)In vitro,the expressions of LINC00470,m RNA-WEE1 and miR-580-3p were up-regulated or down-regulated by cell transfection technology,and proteins related to PI3K/AKT/m TOR pathway were detected by Western blot.Results:1.Compared with HC-exo,LINC00470 was highly expressed in GC-exo(P < 0.05),and the expression level of exosome lnc RNA LINC00470 was negatively correlated with postoperative survival(P<0.001).2.Compared with HC-exo,high-grade GC-exo enhanced the tumorigenesis of U251 cells in primary nude mouse glioma model(P<0.01).Down-regulating the expression of LINC00470 by transfection with sh-LINC00470 could inhibit the promoting effect of high-level GC-exo on the growth of xenograft tumors(P<0.05).In vitro,GC-exo inhibited autophagy and promoted cell proliferation of U251 and SWO-38 cells,while down-regulating expression of LINC00470 can reverse the effect of GC-exo(P < 0.05).At the same time,high-grade GC-exo has stronger inhibition of autophagy and promotion of cell proliferation than low-grade GC-exo.3.LINC00470 can inhibit autophagy of U251 and SWO-38 cells and promote cell proliferation(P<0.05).Autophagy induced by rapamycin can reverse the effect of LINC00470(P < 0.05).LINC00470 and m RNA-WEE1 can competitively bind with miR-580-3p.Incubation with high-level GC-exo,overexpression of LINC00470 and knockdown of miR-580-3p can activate PI3K/AKT/m TOR pathway(P < 0.05).Overexpression of miR-580-3p can inhibit PI3K/AKT/m TOR pathway,however up-regulating expression of m RNA-WEE1 can reverse the inhibition effect of miR-580-3p on PI3K/AKT/m TOR pathway(P <0.05).Conclusions:1.The expression level of LINC00470 in serum exosomes of patients with glioma is up-regulated,and the expression level of lnc RNA LINC00470 in serum exosomes is negatively correlated with postoperative survival of glioma patients.2.The serum exosome LINC00470 of high-grade glioma can regulate the PI3K/AKT/m TOR pathway through the LINC00470/miR-580-3p/WEE1 axis,thereby inhibiting the glioma cells autophagy and promoting the glioma cells proliferation.