长链非编码RNA HOTAIR 对胃肠间质瘤 细胞化疗敏感性的影响

Effect of long chain noncoding RNA HOTAIR on chemo-sensitivity of gastrointestinal stromal tumor cells

摘要:

目的 研究长链非编码RNA（lncRNA）HOTAIR 对胃肠间质瘤细胞伊马替尼化疗敏感性的影响， 并探讨其作用机制。方法 首先在胃肠间质瘤组织中检测HOTAIR 的表达水平。选择胃肠间质瘤细胞GIST-T1 为研究对象，si 干扰HOTAIR 后，采用dUTP 缺口末端标记测定法（TUNEL）和半抑制浓度（IC50）法检 测GIST-T1 对伊马替尼化疗敏感性的变化。通过实时荧光定量聚合酶链反应（qRT-PCR）法、RNAhybird 软件分析和荧光素酶报告法，筛选并验证与HOTAIR 存在内源性竞争关系的miRNAs。最后将miRNA 与 HOTAIR 共转染，观察HOTAIR 与miRNA 的竞争性结合能否改变GIST-T1 细胞对伊马替尼的化疗敏感性。 结果 HOTAIR 在胃肠间质瘤组织中表达含量高于正常组织（P <0.05）。伊马替尼药物作用下，si-HOTAIR 组细胞IC50 低于si-NC 组（P <0.05）；TUNEL 阳性细胞比例高于si-NC 组（P <0.05）；差异有统计学意 义。qRT-PCR 结果显示，si-HOTAIR 组细胞miRNA-21 表达水平高于si-NC 组（P <0.05）；RNA hybird 分析及荧光素酶验证结果显示HOTAIR 与miR-21 核心序列区存在碱基互补。将miRNA-21 与HOTAIR 共转染GIST-T1 细胞后，与miRNA-21+HOTAIR- 突变型组比较，miRNA-21+HOTAIR- 野生型组细 胞IC50 增高（P <0.05）；TUNEL 阳性细胞比例降低，差异有统计学意义（P <0.05）。结论 长链非编码RNA HOTAIR 可通过内源性竞争结合miRNA-21，降低胃肠间质瘤细胞对伊马替尼的化疗敏感性。

Abstract:

Objective To investigate the effect of long chain noncoding RNA (lncRNA) HOTAIR on chemosensitivity of gastrointestinal stromal tumor (GIST) cells to Imatinib. Methods Expression level of HOTAIR in GIST tissues and GIST-T1 cell line was measured. HOTAIR was knocked down by siRNA. Chemo-sensitivity of GIST-T1 cell line against was identified by TUNEL and IC50. Endogenously competitive binding miRNAs was screened with RNA-hybird software analysis, qRT-PCR and luciferase reporter HOTAIR. Chemo-sensitivity of the cell line against Imatinib was further validated through co-transfection of miRNA and HOTAIR. Results Expressed of HOTAIR was enhanced in gastrointestinal stromal tumor tissue compared with normal tissue (P < 0.05). The IC50 of Imatinib in GIST-T1 cells transfected with si-HOTAIR was decreased when compared with that in cells transfected with si-NC (P < 0.05). TUNEL analysis indicated that positive staining cells was increased in GIST-T1 cells transfected with si-HOTAIR compared with cells transfected with si-NC under imatinib stimulation (P < 0.05). QRTPCR showed that expression of miRNA-21 was upregulated in GIST-T1 cells transfected with si-HOTAIR compared miRNAwith GIST-T1 cells transfected with si-NC (P < 0.05). RNA-hybird analysis and luciferase validation confirmed that HOTAIR obtained complementary base pair to "seed" zone of miR-21. Moreover, co-transfection of GIST-T1 cells and miRNA-21 induced increased concentration of IC50 and decreased levels of positive staining of cells in TUNEL analysiswhen compared with those in genetically mutant cell line (P < 0.05). Conclusions Long chain noncoding RNA HOTAIR reduces the sensitivity of gastrointestinal stromal tumor cells against Imatinib through endogenously competitive miRNA-21.