LncRNA LY6E-DT在人脑胶质瘤细胞替莫唑胺耐药中的作用及机制研究

Preliminary exploration of relevant mechanisms and effects of LncRNA LY6E-DT on temozolomide resistance in high grade glioma cell U251

目的探究长链非编码RNA(LncRNA)LY6E-DT对高级别人脑胶质瘤(high grade glioma,HGG)细胞替莫唑胺(temozolomide,TMZ)抵抗的影响及相关机制。方法生物信息学筛选与HGG组织中O6-甲基鸟嘌呤-DNA-甲基转移酶(O-6-methylguanine DNA methyltransferase,MGMT)表达密切的LncRNA,并挖掘潜在相关的微小RNA(miRNA)。培养HGG细胞U251并随机分为对照组、抵抗组及干扰组,抵抗组及干扰组采用0.2~24.0μg/ml TMZ梯度递增诱导,等渗PBS则处理对照组细胞;对照组、抵抗组采用慢病毒转染随机对照载体,干扰组转染靶向LY6E-DT的shRNA干扰载体。CCK-8实验检测各组细胞TMZ半抑制浓度(IC50),实时荧光定量PCR(RT-qPCR)技术检测各组细胞LY6E-DT、miR-125a-5p及MGMT mRNA的表达水平,免疫印迹实验(Western bolt)检测MGMT蛋白表达水平,荧光素酶报告基因法检测miR-125a-5p对MGMT的调控作用及LY6E-DT的影响。结果LY6E-DT表达与MGMT显著正相关(Pearson相关系数=0.32,P<0.001),且发现LY6E-DT在HGG组织中显著低表达,但不利于HGG患者预后;对照组、抵抗组及干扰组LY6E-DT的表达分别为(1.000±0.047)、(3.704±0.402)及(0.743±0.064);miR-125a-5p表达分别为(1.000±0.049)、(0.351±0.031)及(0.934±0.050);MGMT mRNA的表达分别为(1.000±0.017)、(5.205±0.462)及(3.183±0.667);MGMT蛋白表达分别为(0.108±0.012)、(0.891±0.063)及(0.375±0.038);TMZ IC50分别为(6.79±0.71)、(30.52±3.69)及(15.64±2.25)μg/ml。相比对照组,抵抗组LY6E-DT、MGMT及TMZ IC50显著增高,而干扰组LY6E-DT表达显著降低,MGMT及TMZ IC50显著增高(P均<0.05);相比抵抗组,干扰组LY6E-DT、MGMT mRNA及蛋白的表达、TMZ IC50均显著降低(P均<0.05),miR-125a-5p可显著抑制MGMT 3’UTR的Luciferase表达(P<0.01),而LY6E-DT可显著恢复该表达水平(P<0.01)。结论LY6E-DT可促进HGG细胞MGMT表达及TMZ抵抗,其机制与抑制miR-125a-5p的表达有关。

Objective To investigate the effect of long non-coding RNA(LncRNA)LY6E-DT on temozolomide(TMZ)resistance in high grade glioma cells(HGG)cells and its mechanism.Methods Bioinformatics screened the LncRNA correlated to the expression of O6-methylguanine-DNA methyltransferase(MGMT)in HGG tissue,and excavated potentially related microRNA(miRNA).HGG cell U251 was cultured and randomly divided into control group,resistance group and interference group.Resistance group and interference group were treated with 0.2-16.0μg·ml-1 TMZ gradient incremental induction,and isosmotic PBS was used to treat control group cells.Control group and resistance group were transfected with control vector,while the interference group was transfected with shRNA interference vector targeting LY6E-DT by lentivirus.The semi-inhibitory concentration(IC50)of TMZ in each group was detected by CCK-8 experiment,the expression levels of LY6E-DT,miR-125a-5p and MGMT mRNA in each group were detected by real-time fluorescent quantitative PCR(RT-qPCR),and the expression levels of MGMT protein were detected by Western blot.The effect of miR-125a-5p on MGMT and LY6E-DT was detected by luciferase reporter gene method.Results LY6E-DT expression was positively correlated with MGMT(Pearson correlation coefficient=0.32,P<0.001),and it was found that LY6E-DT expression was significantly lower in HGG tissues,but it was not conducive to the prognosis of HGG patients;In the control group,resistance group and interference group,the expressions of LY6E-DT were 1.000±0.047,3.704±0.402 and 0.743±0.064;the expressions of miR-125a-5p were 1.000±0.049,0.351±0.031 and 0.934±0.050;the expressions of MGMT mRNA were 1.000±0.017,5.205±0.462 and 3.183±0.667;the expressions of MGMT protein are 0.108±0.012,0.891±0.063 and 0.375±0.038;TMZ IC50 are 6.79±0.71,30.52±3.69 and 15.64±2.25μg·ml-1.Compared with the control group,LY6E-DT,MGMT and TMZ IC50 in the resistance group were significantly higher,while LY6E-DT expression in the interference group was significantly lower,MGMT and TMZ IC50 were significantly higher(P<0.05);Compared with the resistance group,the expression of LY6E-DT,MGMT mRNA and protein and TMZ IC50 decreased significantly in the interference group(P<0.05).miR-125a-5p significantly inhibited luciferase expression of MGMT 3'UTR(P<0.01),while LY6E-DT significantly restored the expression level(P<0.01).Conclusion LY6E-DT can promote MGMT expression and TMZ resistance in HGG cells,which is related to the inhibition of miR-125a-5p expression.