circRNA_0128846靶向miR-1183介导人非小细胞肺癌细胞放射抵抗的研究

目的 探讨环状RNA（circRNA）_0128846对人非小细胞肺癌（NSCLC）细胞放射抵抗的影响及机制。 方法 应用GEO数据库筛选、采用荧光实时定量聚合酶链反应（qRT-PCR）检测在人NSCLC细胞A549及其放射抵抗细胞（A549R）中差异表达最显著的circRNA作为目标circRNA，qRT-PCR检测其在人支气管上皮细胞Beas-2B及人NSCLC细胞A549、H460、H1299、H1975中的表达水平。在A549细胞中转染目标circRNA过表达载体，在A549R细胞中转染目标circRNA沉默载体，并检测以上细胞经8 Gy X射线照射后的克隆形成能力。应用人类环状RNA数据库和circinteractome数据库预测目标circRNA下游的miRNA，qRT-PCR检测A549R细胞中目标circRNA沉默后表达水平上升最显著的miRNA作为目标miRNA。qRT-PCR检测A549细胞中过表达目标circRNA后目标miRNA的表达情况；通过双荧光素酶报告基因分析人胚肾细胞293T中过表达目标miRNA后目标circRNA的表达情况；qRT-PCR检测目标miRNA在A549、A549R细胞中的表达水平。在A549细胞中转染目标miRNA抑制剂以沉默目标miRNA，在A549R细胞中转染目标miRNA模拟物以过表达目标miRNA，并检测以上细胞经8 Gy X射线照射后的克隆形成能力；将过表达目标circRNA的A549细胞进行8 Gy X射线照射并在该细胞中过表达目标miRNA，检测细胞的克隆形成能力。组间数据的比较采用独立样本t检验。 结果 qRT-PCR检测结果显示，circRNA_0128846为目标circRNA，且其在人NSCLC细胞A549、H460、H1299、H1975中的相对表达水平均高于人支气管上皮细胞Beas-2B（t=6.200、7.903、6.010、6.132，均P<0.01）。过表达circRNA_0128846的A549细胞经照射后的克隆形成能力增强（0.22%对0.45%，t=4.427，P<0.05）；沉默circRNA_0128846后A549R细胞经照射后的克隆形成能力降低（0.23%对0.10%，t=3.780，P<0.05）。qRT-PCR检测结果显示，miR-1183为目标miRNA，在A549细胞中过表达circRNA_0128846显著下调miR-1183的表达（t=6.002，P<0.01）；双荧光素酶报告基因分析证实过表达miR-1183可显著下调circRNA_0128846的表达（t=4.562，P<0.05）；miR-1183在A549R细胞中的相对表达水平显著低于亲本A549细胞（t=6.025，P<0.01）。过表达miR-1183显著降低A549R细胞经照射后的克隆形成能力，沉默miR-1183显著增强A549细胞经照射后的克隆形成能力（0.26%对0.15%、0.21%对0.31%，t=3.671、3.293，均P<0.05），且过表达miR-1183显著降低了过表达circRNA_0128846对A549细胞克隆形成能力的促进作用（1.90%对1.20%，t=6.325，P<0.01）。 结论 circRNA_0128846作为miR-1183的吸附海绵，可抑制miR-1183的表达进而抑制其放疗增敏作用，从而促进人NSCLC细胞的放射抵抗。

The study of circRNA_0128846 targeting miR-1183 to mediate radioresistance in human non-small cell lung cancer cells

Objective To investigate the effect and mechanism of circular RNA (circRNA)_0128846 on the radioresistance of human non-small cell lung cancer (NSCLC) cells. Methods The circRNA with the most significant differential expression in the human NSCLC cell line A549 and its radioresistant cell line (A549R) was considered as the target circRNA. It was screened by using the GEO database and detected through fluorescence real-time quantitative polymerase chain reaction (qRT-PCR). qRT-PCR was utilized to detect the expression of the target circRNA in the human bronchial epithelial cell strain Beas-2B and NSCLC cells A549, H460, H1299, and H1975. A549 cells were transfected with a silencing vector, and A549R cells were transfected with an overexpression vector. Clone formation assay was used to detected the clone formation ability of the cells after 8 Gy X-ray irradiation. The downstream miRNA of the target circRNA was predicted with the human circular RNA database and circinteractome database. qRT-PCR was applied to detect the miRNA with the expression level that had increased most significantly after the target circRNA was silenced in the A549R cells, and regarded it as the target miRNA. The expression of the target miRNA after the overexpression of the target circRNA in A549 cells was detected with qRT-PCR. A dual-luciferase reporter gene system was employed to analyze the expression of the target circRNA after the target miRNA was overexpressed in human embryonic kidney cells 293T. The expression level of the target miRNA in A549 and A549R cells was detected through qRT-PCR. The silencing and overexpression vectors of the target miRNA were transfected into A549 and A549R cells, respectively. Then, clone formation ability was detected after 8 Gy X-ray irradiation. The A549 cells overexpressing the target circRNA were irradiated with 8 Gy X-ray, and the target miRNA was overexpressed in the cells to detect their clone formation ability. Data were compared between groups by using independent sample t test. Results qRT-PCR results revealed that circRNA_0128846 was the target circRNA, and its relative expression level in human NSCLC cells A549, H460, H1299, and H1975 was higher than that in the human bronchial epithelial cell line Beas-2B (t=6.200, 7.903, 6.010, 6.132; all P<0.01). After circRNA_0128846 was overexpressed, the clone formation ability of irradiated A549 cells was enhanced (0.22% vs. 0.45%, t=4.427, P<0.05). After circRNA_0128846 was silenced, the clone formation ability of irradiated A549R cells decreased (0.23% vs. 0.10%, t=3.780, P<0.05). qRT-PCR results showed that miR-1183 was the target miRNA, and the overexpression of circRNA_0128846 in A549 cells significantly down-regulated the expression of miR-1183 (t=6.002, P<0.01). Dual-luciferase reporter gene analysis confirmed that the overexpression of miR-1183 could significantly reduce the expression of circRNA_0128846 (t=4.562, P<0.05). The relative expression level of miR-1183 in A549R cells was significantly lower than that in parental A549 cells (t=6.025, P<0.01). The overexpression of miR-1183 significantly reduced the clone formation ability of A549R cells after irradiation; the silencing of miR-1183 significantly enhanced the clone formation ability of A549 cells after irradiation (0.26% vs. 0.15%, 0.21% vs. 0.31%; t=3.671, 3.293; both P<0.05); and the overexpression of miR-1183 significantly reduced the promoting effect of the overexpression of circRNA_0128846 on the clone formation ability in A549 cells (1.90% vs. 1.20%, t=6.325, P<0.01). Conclusion In human NSCLC cells, circRNA_0128846 acts as a miR-1183 adsorption sponge that can inhibit the radiosensitization effect of miR-1183 by reducing miR-1183 expression, thereby promoting NSCLC radioresistance.