Objectives: This study aims to investigate the role of hypoxia-induced long non-coding small nucleolar RNA host gene 14 (lncRNA SNHG14) in glioma temozolomide (TMZ) resistance and underlying mechanisms.
Methods: According to different treatments, the experiment was divided into a normoxia group and a hypoxia group, a control group and a TMZ group. The lncRNA SNHG14 and O6-methylguanine DNA methyltransferase (MGMT) levels in glioma SNB19 and U251 cell line were detected by real-time PCR and Western blotting, respectively, and the association of lncRNA SNHG14 level with hypoxia and TMZ treatment was analyzed. siRNA was used to knockdown the lncRNA SNHG14 expression in glioma cells, and the transfected glioma cells were divided into a negative control group (si-NC group) and a si-SNHG14 group. The interference efficiency was examined by real-time PCR, the key factor MGMT of lncRNA SNHG14 sensitivity regulation was detected by Western blotting, and the cell apoptosis was detected by cytometry. In addition, MTT method was used to detect the cell viability of gliomas in the different groups under the different TMZ concentrations, and the effect of lncRNA SNHG14 on TMZ sensitivity of gliomas was analyzed. Online tools were used to predict miRNAs that could specifically bind to lncRNAs SNHG14 and MGMT. A si-NC group, a si-SNHG14 group, a normoxia group and a hypoxia group were set up, and the changes of miR-143 abundance in different environments were observed by real-time PCR. miR-143 mimics and inhibitor were used to change the level of miR-143 in glioma cells. A NC inhibitor group, a miR-143 inhibitor group, a NC mimics group and a miR-143 mimics group were set up, the interference efficiency was detected by real-time PCR, the expression level of MGMT was detected by Western blotting, and the effect of miR-143 on the level of MGMT were analyzed. The NC inhibitor group, the miR-143 inhibitor group, the NC mimics group and the miR-143 mimics group were treated with different interventions, and the dual luciferase reporter assay was used to observe the changes of lncRNA SNHG14 and MGMT luciferase activities, and to verify the relationship among lncRNA SNHG14, miR-143 and MGMT. Finally, a NC group and a lncRNA SNHG14 overexpression group were set up, and the changes in the abundance of miR-143 and MGMT in each group were detected by RNA-binding protein immunoprecipitation experiments, and the competitive binding relationship among lncRNA SNHG14, miR-143 and MGMT was analyzed.
Results: Compared with the normoxia group, the hypoxia group could promote the expression of lncRNA SNHG14 in glioma cells. Compared with the control group, the expression of lncRNA SNHG14 could be significantly inhibited in the TMZ group (P<0.05). Compared with the si-NC group, the expression of lncRNA SNHG14 in the si-SNHG14 group could be effectively inhibited, and the expression level of MGMT was significantly decreased, and the apoptosis rate was significantly increased (all P<0.05). With the increase of TMZ concentrations, the glioma cell viability in the si-SNHG14 group was significantly lower than that in the si-NC group, and the cell viability in the hypoxia group was significantly higher than that in the normoxia group (both P<0.05). Online tool prediction found that miR-143 had binding sites with lncRNA SNHG14 and MGMT. The abundance of miR-143 in the hypoxia group was significantly lower than that in the normoxic group, and the abundance of miR-143 in the si-SNHG14 group was significantly higher than that in the si-NC group (both P<0.05). The miR-143 mimics group or the miR-143 inhibitor group could significantly over-express or under-express miR-143 (both P<0.05). But there was no significant difference between the NC mimics group (or the NC inhibitor group) and the control group (both P>0.05). The level of MGMT protein could significantly up-regulate in the miR-143 inhibitor group, and on the contrary which could significantly down-regulate in the miR-143 mimics group (both P<0.01). The dual luciferase reporter assay showed that there was no significant difference between the NC mimics group (or the NC inhibitor group) and the control group (both P>0.05). The wild-type SNHG14 and MGMT luciferase activities were significantly down-regulated in the miR-143 mimics group, which were significantly up-regulated in the miR-143 inhibitor group (P<0.01 and P<0.05, respectively), but there was no significant change in the luciferase activities of mutant SNHG14 and MGMT (both P>0.05). The results of the RNA-binding protein immunoprecipitation experiment showed that: compared with the NC group, more lncRNA SNHG14 was bound to the precipitated argonaute 2 protein in the cells in the lncRNA SNHG14 overexpression group, but the abundance of MGMT mRNA was decreased significantly, and there were significant differences (both P<0.01). There was a targeting regulatory relationship among lncRNA SNHG14, miR-143 and MGMT.
Conclusions: The up-regulated lncRNA SNHG14 can target miR-143, relieve the inhibition of miR-143 on MGMT, and promote the TMZ resistance in the hypoxia-induced glioma cells.
目的: 探讨低氧诱导的长链非编码核内小RNA宿主基因14(long non‑coding small nucleolar RNA host gene 14，lncRNA SNHG14)在胶质瘤替莫唑胺(temozolomide，TMZ)耐药中的作用和潜在机制。方法: 根据不同处理将实验分为常氧组、低氧组、对照组(NC组)和TMZ组，利用real-time PCR和蛋白质印迹法分别检测胶质瘤细胞SNB19和U251中lncRNA SNHG14和O6-甲基鸟嘌呤DNA甲基转移酶(O6-methylguanine DNA methyltransferase，MGMT)的表达水平，分析lncRNA SNHG14表达水平与低氧和TMZ处理的关系。利用siRNA干扰胶质瘤细胞中lncRNA SNHG14表达，将转染后的胶质瘤细胞分为si-对照组(si-NC组)和si-SNHG14组，利用real-time PCR检测干扰效率，并采用蛋白质印迹法检测TMZ敏感性调控关键因子MGMT的表达变化，采用流式细胞术检测细胞凋亡；此外，增设常氧组和低氧组，应用MTT法检测不同TMZ浓度梯度下各组胶质瘤的细胞活性，分析lncRNA SNHG14对胶质瘤TMZ敏感性的影响。利用在线工具针对性地预测与lncRNA SNHG14和MGMT结合的miRNAs。应用real-time PCR观察不同环境下si-NC组、si-SNHG14组、常氧组和低氧组miR-143的丰度变化。利用miR-143拟似剂(mimics)和抑制剂(inhibitor)改变胶质瘤细胞中miR-143水平，将实验设置为NC inhibitor组、miR-143 inhibitor组、NC mimics组和miR-143 mimics组，采用real-time PCR检测干扰效率，应用蛋白质印迹法检测MGMT的表达水平，分析miR-143对MGMT水平的影响。对NC inhibitor组、miR-143 inhibitor组、NC mimics组和miR-143 mimics组进行不同干预，采用双荧光素酶报告实验观察lncRNA SNHG14和MGMT荧光素酶的活性变化，验证lncRNA SNHG14、miR-143和MGMT之间的靶向调控关系。最后，设置NC组和lncRNA SNHG14过表达组，通过RNA结合蛋白免疫沉淀实验检测各组中miR-143和MGMT的丰度变化，分析lncRNA SNHG14、miR-143和MGMT间的竞争结合关系。结果: 与常氧组相比，低氧组可以促进胶质瘤细胞中lncRNA SNHG14表达；与NC组相比，TMZ组能显著抑制lncRNA SNHG14的表达(均P<0.05)。与si-NC组相比，si-SNHG14组可有效抑制lncRNA SNHG14表达，且MGMT表达水平呈现显著下降，细胞凋亡率显著升高(均P<0.05)。随着TMZ浓度的升高，si-SNHG14组胶质瘤细胞活力显著低于si-NC组，低氧组的细胞活力显著高于常氧组(均P<0.05)。在线工具预测发现：miR-143与lncRNA SNHG14和MGMT存在结合位点，低氧组miR-143丰度显著低于常氧组，si-SNHG14组miR-143丰度显著高于si-NC组(均P<0.05)。miR-143 mimics组或miR-143 inhibitor组可显著过表达或低表达miR-143(均P<0.05)，但NC mimics组或NC inhibitor组与NC组相比，差异均无统计学意义(均P>0.05)；miR-143 inhibitor组MGMT蛋白水平显著上调(P<0.01)，miR-143 mimics组则反之(P<0.01)。双荧光素酶报告实验显示：NC mimics组或NC inhibitor组与各自的对照组相比，差异均无统计学意义(均P>0.05)；miR-143 mimics组野生型SNHG14和MGMT的荧光素酶活性显著下调，而miR-143 inhibitor组野生型SNHG14和MGMT的荧光素酶活性显著上调(分别P<0.01和P<0.05)；但突变型SNHG14和MGMT的荧光素酶活性均无明显变化(均P>0.05)。RNA结合蛋白免疫沉淀实验结果显示：与NC组相比，过表达lncRNA SNHG14组细胞中沉淀的argonaute 2蛋白上结合有更多的lncRNA SNHG14，但MGMT mRNA丰度却出现显著下降，差异均有统计学意义(均P<0.01)，lncRNA SNHG14、miR-143和MGMT之间存在靶向调控关系。结论: 低氧下胶质瘤细胞中表达上调的lncRNA SNHG14靶向miR-143，解除miR-143对MGMT的抑制，从而促进胶质瘤细胞产生TMZ耐药。.
Keywords: drug resistance; glioma; hypoxia; long non-coding RNA; temozolomide.