Effect Of RNAi Silencing S100A9 Gene On Proliferation And Self-renewal Of Glioma Stem Cells U87 And Its Mechanisms

ObjectiveMalignant glioma is the most common primary tumor of the central nervous system.It has the characteristics of short course,rapid progress and poor prognosis.Current treatment methods have no obvious effect,seriously threatening human life and health.The 2-year survival rate is less than 30%.Recent discoveries of cancer stem cells(CSCs)provide new strategies and directions for the diagnosis and treatment of glioma.It has been found that there are a group of glioma stem cells(GSCs)in glioma,which have the ability of infinite proliferation,self-renewal,multi-directional differentiation and reconstruction,although their content is very small.They are related to the occurrence,invasion and metastasis of tumors,resistance to radiotherapy and chemotherapy,and recurrence of tumors.Therefore,killing glioma stem cells may become a feasible means to cure glioma.In our previous proteomic studies,we found that calcium binding protein S100A9 was significantly up-regulated in glioma stem cells,and the proliferation of glioma decreased after knocking down S100A9.S100A9 is a member of the calcium binding protein S100 family.It is widely involved in cell skeleton construction,calcium homeostasis regulation,differentiation and inflammatory response,and is closely related to tumors.In this study,human U87 glioma stem cells were infected with shRNA-S100A9 lentivirus to observe the proliferation and self-renewal ability of U87 glioma stem cells,and to reveal the potential mechanism of its role.MethodTo construct a plasmid silencing S100A9 gene(including 3 silencing sequences and 1 negative control sequence of S100A9 gene),and to infect human U87 glioma stem cells with lentiviruses silencing S100A9 gene in a three-plasmid system.The expression of green fluorescence was observed by fluorescence microscopy.Puromycin screened U87 glioma stem cells after infection,and purinomycin-resistant cell clones were obtained for culture.The expression of S100A9 was detected by real-time quantitative PCR(RT-qPCR),and the expression of S100A9 protein was detected by Western blot.The experimental groups were as follows: blank group,control group and experimental group.Blank group: U87 glioma stem cells without any treatment,control group: U87 glioma stem cells transfected with empty vector,experimental group:shRNA-S100A9-1,shRNA-S100A9-2,shRNA-S100A9-3.MTT assay was used to detect cell proliferation in vitro,stem cell globulation assay was used to detect cellself-renewal ability,RT-qPCR and Western blot were used to detect the expression of β-catenin gene and protein respectively.ResultsThe expression of S100A9 in U87 glioma stem cells was significantly inhibited after lentivirus infection.Compared with the control group,the proliferation of cells in the experimental group was slower(P < 0.05),the ability of stem cells to globulize was significantly inhibited(P < 0.05),and the expression of β-catenin mRNA and protein was significantly decreased(P < 0.05).ConclusionTargeted interference with S100A9 can significantly inhibit the proliferation and self-renewal of U87 glioma stem cells in vitro.Its mechanism may be related to the inhibition of wnt/β-catenin signaling pathway,suggesting that S100A9 may be a potential target for glioma therapy.