miR-20a/106b调控NKG2D途径介导儿童型脑干胶质瘤免疫逃逸的实验研究

目的探讨miR-20a/106b在儿童脑干胶质瘤(BSG)恶性进展中的作用及其机制。方法回顾性纳入2012年1月至2017年12月天津市环湖医院(天津市脑系科中心医院)神经外科接受手术切除的13例儿童BSG患者和5例成人BSG患者。采用免疫组织化学染色法检测肿瘤组织标本中自然杀伤细胞2家族成员D(NKG2D)配体MICA的表达情况,采用生物信息学方法筛选出调控MICA的miRNA。应用人脑胶质瘤LN229和U251细胞株通过荧光素酶报告基因方法验证筛选出的miRNA与MICA的靶向调控关系,通过蛋白质免疫印迹(WB)实验检测miR-20a/106b转染LN229和U251细胞株后MICA的表达情况,通过乳酸脱氢酶(LDH)释放法检测免疫细胞对LN229细胞株的毒性能力。通过向去胸腺幼年(3周龄)和成年(10周龄)雄性SD大鼠脑桥区立体定向注射LN229细胞株的方法制备BSG幼鼠(J-rat)和成年鼠(A-rat)模型,并根据注射细胞株不同各分为3组,依次为J-rat组、J-rat-scr组、J-rat-miR-20a/106b-d组和A-rat组、A-rat-scr组、A-rat-miR-20a/106b-u组(每组各3只),通过免疫组织化学染色方法检测MICA的表达情况,采用MCID软件检测肿瘤的侵袭性,采用TUNEL法检测细胞凋亡。结果成人患者的MICA总阳性表达率较儿童患者高(分别为5/5、10/13,P<0.05)。检索结果显示,有23个可能调控MICA的microRNA,其中包括miR-20a和miR-106b。在LN229细胞株中,荧光素酶报告基因结果显示,转染pGL3-MICA-wt(野生型)质粒的荧光强度较对照组和转染pGL3-MICA-mut(突变型)质粒的强(均P<0.05);WB结果显示,与空白对照组和无义序列组比较,AS-miR-20a组、AS-miR-106b组及AS-miR-20a/106b组MICA的表达均增加(均P<0.05)。上述实验在U251细胞株得到相似的结果。不同效靶比(20∶1,10∶1,5∶1)情况下,AS-miR-20a/106b组LDH的活性高于对照组和AS-miR-20a/106b+MICA mAb组(均P<0.05),但后两组的差异无统计学意义(均P>0.05)。术后头颅MRI结果显示,3组幼鼠和3组成年鼠均成功制备脑干胶质瘤模型。MCID软件检测结果显示,J-rat-miR-20a/106b-d组I值较J-rat组、J-rat-scr组均降低(均P<0.05);A-rat-miR-20a/106b-u组I值较A-rat组、A-rat-scr组均增加(均P<0.05)。免疫组织化学染色结果显示,J-rat-miR-20a/106b-d组的MICA染色强度均较J-rat组和J-rat-scr组增加(均P<0.05),A-rat-miR-20a/106b-u组MICA的染色强度均较A-rat组和A-rat-scr组降低(均P<0.05)。体内凋亡实验显示,J-rat-miR-20a/106b-d组的细胞凋亡指数均较J-rat组和J-rat-scr组增加(均P<0.05),A-rat-miR-20a/106b-u组的细胞凋亡指数较A-rat组和A-rat-scr组均降低(均P<0.05)。结论miR-20a/106b通过NKG2D途经达到的免疫逃逸是导致儿童型BSG恶性度高的重要原因。

Experimental research on the immune evasion of pediatric brainstem gliomas mediated by the regulation of NKG2D by miR-20a/106b

Objective To explore the role and mechanism miR-20a/106b in the malignant progression of pediatric brainstem gliomas(BSG).Methods A total of 13 patients with BSG(13 pediatric and 5 adult patients)who underwent surgical treatment at Department of Neurosurgery,Tianjin Huanhu Hospital(Tianjin Central Hospital for Neurosurgery and Neurology)from January 2012 to December 2017 were retrospectively enrolled into this study.The level of MICA was detected in pediatric and adult BSG tissues by immunostaining.The miRNAs regulating the expression of MICA was screened out by bioinformatics.The human glioma cell lines LN229 and U251 were used to verify the relationship between the selected miRNA and MICA by the luciferase reporter assays,and the expression of MICA following transfection of antisense miR-20a/106b into LN229 and U251 were detected by Western blotting(WB).The lactate dehydrogenase(LDH)release assay was used to detect the cytotoxic activity of immune cells against the LN229 cell line.BSG juvenile(J-rat)and adult(A-rat)models were prepared for regain of function experiments by stereotactic injection of the LN229 cell line into the pontine area of athymic juvenile(3 weeks old)and adult(10 weeks old)male Sprague Dawley rats.Each of those two groups was further divided into 3 subgroups according to the different cell lines injected,which were J-rat,J-rat-scr,J-rat-miR-20a/106b-d and A-rat,A-rat-scr,A-rat-miR-20a/106b-u subgroups with each subgroup containing 3 rats.The expression of MICA in each subgroup was detected by immunohistochemical staining,the invasion of tumors in each subgroup was detected by MCID,and the apoptosis of cells in each subgroup was detected by TUNEL method.Results The total positive expression rate of MICA in adult patients was higher than that in children(5/5 vs.10/13,P<0.05).The screening results showed that there were 23 miRNAs that might regulate MICA,including miR-20a and miR-106b.In the LN229 cell line,the luciferase reporter assay showed that the fluorescence activity of those transfected with wild-type pGL3-MICA plasmid was stronger than those of controls and cells transfected with mutant pGL3-MICA plasmid(both P<0.05).WB results showed that,compared with the sham control group and the nonsense sequence group,the expression of MICA in the antisense miR-20a group,antisense miR-106b group and antisense miR-20a/106b group all increased(all P<0.05).In the above experiments,similar results were obtained in U251 cell line.Under different effector to target ratios(20∶1,10∶1,5∶1),the LDH activity of the antisense miR-20a/106b group was higher than that of the control group and the group with combined blocking of MICA(all P<0.05),while the latter two groups had no significant difference(all P>0.05).The postoperative head MRI results showed that the 3 groups of juvenile rats and 3 groups of adults rats were all successfully modeled.The results of the MCID method showed that the I value of the J-rat-miR-20a/106b knockdown group of the juvenile rat model was lower than that of the J-rat group and the J-rat nonsense control group(both P<0.05).The I value of A-rat-miR-20a/106b overexpression group was higher than that of A-rat group and A-rat nonsense control group(both P<0.05).The results of immunohistochemical staining showed that the intensity of MICA staining in the J-rat-miR-20a/106b knockdown group of the juvenile rat model was higher than that of the J-rat group and the J-rat nonsense control group(both P<0.05).The staining intensity of MICA of the model A-rat-miR-20a/106b overexpression group was lower than that of the A-rat group and the A-rat nonsense control group(both P<0.05).In vivo apoptosis experiments showed that the apoptosis index of the J-rat-miR-20a/106b knockdown group in the juvenile rat model was higher than that of the J-rat group and the J-rat nonsense control group(both P<0.05).The apoptosis index of the A-rat-miR-20a/106b overexpression group in the adult rat model was lower than that of the A-rat group and A-rat nonsense control group(both P<0.05).Conclusion The immune evasion mediated by miR-20a/106b through NKG2D is an important factor for the high malignancy of pediatric BSG.