| Objective: glioma is the most common primary malignant tumor of the central nervous system,more than half of which are malignant glioma with poor prognosis.The clinical efficacy of glioma is still unsatisfactory with the combination of surgical resection,radiotherapy and chemotherapy.Glioma has caused a huge burden to the society and the family,so it is urgent to improve its treatment status.In recent years,immunocheckpoint molecules have been found to have good prospects as therapeutic targets for glioma,and are expected to improve the prognosis of glioma patients.B7-H3(CD276)is one of the key molecules of tumor immune checkpoint,which plays a key role in the proliferation,invasion and tumorigenesis of various human malignant tumors and is associated with poor prognosis of tumor patients.Some studies have reported that B7-H3 is also closely related to the progression of malignant glioma,but its specific role and mechanism are still unclear.Therefore,this study hopes to clarify the relationship between the two,so as to provide a basis for finding new therapeutic targets for glioma.Method:1.Immunohistochemistry was used to detect the expression of B7-H3 protein in tumor tissue specimens of high-grade glioma and low-grade glioma,and the relationship between the expression of B7-H3 in tumor tissue specimens of 79 patients with glioblastoma and their survival time was analyzed retrospectively.2.B7-H3 lentivirus was transfected into glioma cells LN229 to construct B7-H3 stable overexpression glioma cells(LN229-B7-H3 OE)and B7-H3 silencing glioma cells(LN229-B7-H3 KO),and empty vector control cells(LN229-B7-H3 Cas-9)were set.Conduct cell culture for subsequent experiments.3.The proliferation and colony formation ability of glioma cells after B7-H3 overexpression/silencing were detected by CCK-8 analysis and cloning formation experiments,respectively.4.Transwell assay was used to detect the changes in the invasion ability of glioma cells after B7-H3 overexpression/silencing.5.Subcutaneous tumor model in nude mice were conducted to observe the changes in the tumorigenesis ability of glioma cells after B7-H3 overexpression/silencing.6.The activation of JAK2/STAT3 signaling pathway and the expression of related proteins in glioma cells after B7-H3 overexpression were detected by Western blot,and the activation of JAK2/STAT3 signaling pathway in tumor tissues was detected by immunofluorescence.7.FLLL32 was used to inhibit the JAK2/STAT3 signaling pathway in B7-H3 overexpression glioma cells to further elucidate the role of the JAK2/STAT3 signaling pathway in B7-H3-induced glioma progression.Results: 1.The expression of B7-H3 in the tumor tissues of high-grade glioma was significantly higher than that of low-grade glioma(P<0.05).In the analysis of survival of 79 patients with glioblastoma,it was found that the relapse-free survival and overall survival time of patients with B7-H3 overexpression were significantly shortened(all P<0.05).2.The proliferation capacity of B7-H3 overexpressed LN229-B7-H3 OE cells was significantly increased compared with that of LN229 cells(P<0.01),and that of B7-H3 silenced LN229-B7-H3 KO cells was significantly decreased compared with that of empty carrier LN229-B7-H3 Cas-9 cells(P<0.05).3.The clone formation experiment showed that the overexpression of B7-H3 significantly improved the clone formation ability of LN229 cells(P<0.01),and the clone formation ability decreased after B7-H3 knockout(P<0.05).4.In the subcutaneous tumorigenesis model of nude mice,the in vivo tumorigenesis ability of B7-H3 overexpressed tumor cells was significantly enhanced(P<0.05),and was significantly decreased after the silencing of B7-H3.5.Transwell assay showed that overexpression of B7-H3 enhanced the invasion of tumor cells(P < 0.01),and silence of B7-H3 inhibited the invasion of tumor cells(P<0.05).6.Western blotting was used to detect the activation of related signal pathway after overexpression of B7-H3 in glioma cells.Enhanced expression of p-JAK2 and p-STAT3 was observed in B7-H3 overexpressed glioma cells,and enhanced phosphorylation of Src was detected,while the negative regulator SOCS3/ SHP-1 and its phosphorylation form were reduced.In addition,enhanced expression of p-JAK2 and p-STAT3 was also observed in tumor tissues of patients with high-grade glioma by immunofluorescence.The results showed that the overexpression of B7-H3 in glioma induced the activation of Src and the down-regulation of the negative regulator SOCS3/ SHP-1 activated the JAK2/STAT3 signaling pathway.At the same time,the increase of N-cadherin expression and the decrease of E-cadherin expression were also detected in B7-H3 overexpressed glioma cells,suggesting the occurrence of EMT.7.After treatment of LN229-B7-H3 OE cells with FLLL32,a JAK2 / STAT3 inhibitor,it was observed that the ability of cell proliferation and clone formation was decreased(all P < 0.01),the ability of tumorigenesis and cell invasion was inhibited(all P < 0.05),and the expression of E-cadherin and N-cadherin was reversed.It is suggested that JAK2/STAT3 signaling pathway is involved in the proliferation,invasion and tumorigenesis of glioma cells,and can promote the EMT process.Conclusion: 1.The expression of B7-H3 is increased in glioma tissues,and overexpression of B7-H3 can significantly promote the proliferation and invasion of glioma cells in vitro and in vivo,leading to poor clinical prognosis.2.B7-H3 promotes the growth,cell invasion and EMT of glioma by activating the JAK2/STAT3 signaling pathway.3.B7-H3 is a potential therapeutic target for glioma and has the value of further research. |
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