研究三阴性乳腺癌中环状RNA circ_0007823的表达量及其影响三阴性乳腺癌(TNBC)细胞生物学行为的可能机制。

按照纳入及排除标准，纳入2016年1月至2020年12月上海市长宁区妇幼保健院收治的女性TNBC患者120例，按照随机数字表法从中选择30例进行回顾性研究。运用实时定量RT-PCR检测circ_0007823在TNBC组织及癌旁正常组织中的表达量，用t检验比较其差异。按照circ_0007823在TNBC组织中的表达量中位值为截点，将30例患者分为circ_0007823高表达组与circ_0007823低表达组，采用Fisher确切概率法比较circ_0007823高、低表达组间临床病理特征的差异。为了研究circ_0007823过表达在TNBC细胞中的作用，将MDA-MB-231细胞分为2组：circ_0007823过表达质粒转染组和空载体转染组(对照)，分别转染circ_0007823过表达载体质粒及空载体质粒。转染48 h后，使用Transwell实验检测细胞的迁移及侵袭能力，使用CCK-8法检测细胞的增殖活性。为了进一步研究circ_0007823突变在TNBC细胞中的作用，将MDA-MB-231细胞分为3组：circ_NC组、circ_0007823-Mut组、circ_0007823-WT组，分别转染空载体、circ_0007823突变型和circ_0007823野生型质粒。转染48 h后，使用Transwell实验检测细胞的迁移及侵袭能力；转染0、24、48、72、96 h后，使用CCK-8法检测细胞的增殖活性。使用双荧光素酶报告基因实验验证circ_0007823与miR-182-5p的关系。运用RT-PCR及Western blot实验检测过表达circ_0007823后MDA-MB-231细胞中miR-182-5p及靶基因FOXF2的表达。迁移细胞数、侵袭细胞数的2组比较采用t检验，多组比较采用单因素方差分析，两两比较采用Tukey法。细胞吸光度值的2组比较采用t检验，不同时间点的多组比较采用重复测量方差分析，组间两两比较采用Bonferroni法。

TNBC组织中circ_0007823的表达量较癌旁正常组织降低(5.97±3.08比20.28±5.44，t＝16.469，P<0.001)。TNBC组织中circ_0007823的表达量与淋巴结转移相关(P＝0.014)。与空载体组相比，circ_0007823过表达组迁移细胞数降低(120.67±6.81比107.00±3.61，t＝5.857，P＝0.028)，侵袭细胞数也降低(94.00±3.61比58.33±3.06，t＝107.000，P<0.001)，吸光度值下降(0.73±0.02比0.63±0.02，t＝7.187，P＝0.001)。circ_NC组、circ_0007823-Mut组、circ_0007823-WT组迁移细胞数分别为120.67±6.81、109.67±6.51、82.00±17.91，差异有统计学意义(F＝38.550，P<0.001); 3组侵袭细胞数分别为272.33±6.03、261.67±2.52、194.67±9.02，差异有统计学意义(F＝128.648，P<0.001)。circ_0007823-WT组的迁移及侵袭细胞数较对照组、circ_0007823-Mut组均下降(P均<0.001)。circ_NC组、circ_0007823-Mut组、circ_0007823-WT组MDA-MB-231细胞吸光度值比较，差异有统计学意义(组间比较，F＝297.883，P<0.001；不同时间点比较，F＝1 207.174，P<0.001；交互作用，F＝42.433，P<0.001)。双荧光素酶报告基因实验证实circ_0007823与miR-182-5p具有靶向结合位点。与对照组相比，circ_0007823过表达质粒转染MDA-MB-231细胞后，circ_0007823的表达量升高(1.01±0.9比9.30±2.47，t＝-5.794, P＝0.029)，miR-182-5p的表达量降低(1.00±0.11比0.86±0.09，t＝2.838，P＝0.022)，FOXF2的mRNA表达量升高(0.87±0.11比1.06±0.24，t＝2.697，P＝0.027)。

circ_0007823在TNBC中低表达，对TNBC细胞增殖、侵袭及迁移能力有抑制作用，并可与miR-182-5p靶向结合，上调FOXF2表达。

To investigate the expression of circular RNA circ_0007823 in triple negative breast cancer (TNBC) and potential mechanism of circ_0007823 affecting the biological behaviors of TNBC cells.

Using random number table, 30 cases were selected from 120 female cases with TNBC who met the inclusion and exclusion criteria in the Changning Maternal and Child Health Hospital, Shanghai from January 2016 to December 2020 for a retrospective study. The expression of circ_0007823 in TNBC tissue and adjacent normal tissue was detected using real-time quantitative RT-PCR and compared by t test. With the median circ_0007823 expression in TNBC tissue as the cutoff value, 30 cases were divided into high expression group and low expression group and the clinicopathological characteristics were compared between two groups using Fisher’s exact test. In order to explore the role of circ_0007823 overexpression on TNBC cells, MDA-MB-231 cells were transfected with circ_0007823 overexpression plasmid and empty plasmid, respectively (overexpression group and control group). At 48 h after transfection, the migration and invasion abilities of cells were detected by Transwell assay, and the proliferation of cells was detected by CCK-8 method. To further investigate the role of circ_0007823 mutation in TNBC cells, MDA-MB-231 cells were transfected with empty, circ_NC mutant and circ_NC wild-type plasmids, respectively (circ_NC group, circ_0007823-Mut group and circ_0007823-WT group). At 48 h after transfection, the migration and invasion abilities of cells were detected by Transwell assay; at 0, 24, 48, 72 and 96 h after transfection, the proliferation of cells were detected by CCK-8 method. The relationship between circ_0007823 and miR-182-5p was verified by the dual-luciferase reporter assay. RT-PCR and Western blot analysis were used to detect the expression of miR-182-5p and target gene FOXF2 in MDA-MB-231 cells with overexpression of circ_0007823. t test was used to compare the number of migrating cells and invasive cells between two groups, one-way analysis of variance with Tukey correction was used for multiple comparison. The optical density of cells was compared between two groups using t test, repeated measures analysis of variance was used for comparison among multiple groups at different time points and Bonferroni correction was used for pairwise comparison.

The expression of circ_0007823 in TNBC tissue was significantly lower than that in adjacent normal tissue (5.97±3.08 vs 20.28±5.44, t=16.469, P<0.001). The expression of circ_0007823 in TNBC tissue was correlated with lymph node metastasis (P=0.014). Compared with control group, a significant decrease was observed in the number of migrating cells, number of invasive cells and optical density of cells in circ_0007823 overexpression group (120.67±6.81 vs 107.00±3.61, t=5.857, P=0.028; 94.00±3.61 vs 58.33±3.06, t=107.000, P<0.001; 0.73 ± 0.02 vs 0.63 ± 0.02, t=7.187, P=0.001). The number of migrating cells in the circ_NC group, circ_0007823-Mut group, and circ_0007823-WT group was 120.67±6.81, 109.67±6.51, 82.00±17.91, respectively, indicating a significant difference (F=38.550, P<0.001); the numbers of invasive cells was 272.33±6.03, 261.67±2.52, 194.67±9.02, respectively, indicating a significant difference (F=128.648, P<0.001). The number of migrating and invasive cells in circ_0007823-WT group was significantly lower than that in control group and circ_0007823-Mut group (all P<0.001). The optical density of MDA-MB-231 cells in the circ_NC group, circ_0007823-Mut group and circ_0007823-WT group indicated a significant difference (comparison between groups, F=297.883, P<0.001; comparison among different time points, F=1 207.174, P<0.001; interaction, F=42.433, P<0.001). The result of dual-luciferase reporter assay confirmed that circ_0007823 had targeted binding sites with miR-182-5p. Compared with control group, after transfection with circ_0007823 overexpression plasmid in MDA-MB-231 cells, the expression of circ_0007823 increased (1.01±0.9 vs 9.30±2.47, t=-5.794, P=0.029), the expression of miR-182-5p decreased (1.00±0.11 vs 0.86±0.09, t=2.838, P=0.022), and the mRNA expression of FOXF2 increased (0.87±0.11 vs 1.06±0.24, t=2.697, P=0.027).

circ_0007823, which is lowly expressed in TNBC tissue, has inhibitory effect on the proliferation, invasion and migration of TNBC cells and its targeted binding with miR-182-5p can up-regulate the expression of FOXF2.