溶质运载蛋白16A家族成员10(SLC16A10)对胶质瘤干细胞增殖及自我更新能力的影响

Effect of solute carrier family 16 member 10(SLC16A10) on proliferation and self-renewal of glioma stem cells

目的探索溶质运载蛋白16A家族成员10(SLC16A10)在胶质瘤干细胞(GSC)中的功能,为针对GSC的脑胶质瘤治疗提供新的潜在靶点。方法387GSC培养于含10%胎牛血清的DMEM培养基中7 d,分化为387非干性肿瘤细胞(387NSTC),RT-qPCR检测387GSC和387NSTC中SLC16A10、性别决定区Y框蛋白2(SOX2)、少突细胞谱系转录因子2(Olig2)和神经胶质纤维酸性蛋白(GFAP)mRNA水平。构建靶向SLC16A10基因的干涉质粒,并进行慢病毒包装。慢病毒感染387GSC 48 h,嘌呤霉素(2 mg·L^(-1))筛选2 d,Western印迹法检测SLC16A10蛋白表达水平,验证敲低效果。CellTiter-Glo试剂盒检测敲低SLC16A10后387GSC相对细胞活力和肿瘤细胞球形成数目。免疫荧光法检测387GSC中SLC16A10蛋白的细胞定位。结果与387GSC相比,387NSTC中SLC16A10 mRNA水平显著降低(P<0.01),同时肿瘤干细胞标志物SOX2和Olig2 mRNA水平表达显著降低(P<0.01),非干细胞标志物GFAP表达显著升高(P<0.01),表明387GSC已分化为387NSTC。敲低SLC16A10后,387GSC相对细胞活力显著下降(P<0.01),肿瘤细胞球数目明显减少(P<0.01)。免疫荧光法结果表明,387GSC中SLC16A10未定位于细胞核。结论SLC16A10在387GSC中高表达,其敲低后387GSC增殖及肿瘤自我更新能力均被显著抑制,提示SLC16A10在脑胶质瘤发生发展中发挥重要作用。

OBJECTIVE To explore the function of solute carrier family 16 member 10 (SLC16A10)in glioma stem cells (GSCs),and to provide a new potential target for GSC therapy.METHODS387GSCs were cultured in DMEM medium containing 10%fetal bovine serum for 7 d and induced into387 non-stem-tumor cel s (387NSTCs).RT-q PCR was used to detect the m RNA expressions of SLC16A10,sex determining region Y-box 2 (SOX2),oligodendrocyte lineage transcription factor 2 (Olig2) and Glia fibrillary acidic protein (GFAP) in 387GSCs and 387NSTCs.The interference plasmid targeting SLC16A10 gene was constructed,and the virus was packaged.After 48 h of lentivirus infection,purinomycin (2 mg·L^(-1)) was screened for 2 d.The SLC16A10 protein level was detected by Western blotting to verify the knockdown efficiency.The cell viability of 387GSCs after SLC16A10 knockdown was detected by Celltiter-Glo kit,the formation of tumor cell spheres was observed,and the number of tumor cel spheres was counted.The cell localization of SLC16A10 protein in 387GSCs was detected by immunofluorescence assay.RESULTS Compared with 387GSCs,the SLC16A10 mRNA level in 387NSTCs was significantly decreased (P<0.01).At the same time,the mRNA expressions of tumor stem cel markers SOX2 and Olig2 were decreased (P<0.01),and the mRNA expression of non-stem-cell marker GFAP was increased (P<0.01).These results indicated that 387GSCs had differentiated into 387NSTCs.After SLC16A10 was knocked down by two interference sequences in 387GSCs,the cell viability of387GSCs was significantly decreased (P<0.01),and the number of tumor cell spheres of 387GSCs was significantly inhibited (P<0.01).Immunofluorescence results suggested that SLC16A10 was localized outside the nuclei of 387GSCs.CONCLUSION SLC16A10 is highly expressed in 387GSCs,and387GSC viability and sphere formation ability are significantly inhibited when SLC16A10 is knocked down,which suggests that SLC16A10 may play an important role in the occurrence and development of glioma.