目的

研究LINC01232 在三阴性乳腺癌（TNBC）中的作用机制，探索与miR-516a-5p/BCL9 轴的调控关系。

方法

基因表达综合数据库（GEO）筛选与TNBC 恶性进展相关的差异表达基因。实时定量RT-PCR 检测LINC01232 在TNBC 中的表达水平，细胞功能实验探索LINC01232 对TNBC 进展的潜在影响。 生物信息学预测LINC01232 与miR-516a-5p 和B 细胞淋巴因子9（BCL9）之间的结合位点，并利用双荧光素酶报告基因和RNA 免疫沉淀实验验证它们之间的结合活性。

结果

与邻近正常组织相比，TNBC 组织中LINC01232 表达异常升高（0.797±0.449 比2.524±1.099，t=11.273，P＜0.001）。 对LINC01232 进行敲低，各组增殖细胞数量比较差异有统计学意义（F=884.428， P＜0.001），分组与时间点之间存在交互作用（F=54.276， P＜0.001），与对照组相比，72、96 h 敲低组细胞增殖能力明显降低（t=8.933， P＜0.001； t=8.378， P＜0.001）。 克隆形成实验结果显示，与对照组相比，敲低组克隆数量明显减少（t=35.308， P＜0.001）。 Transwell 实验结果显示，与对照组相比，敲低组中迁移和侵袭细胞数量明显减少（t=40.455， P＜0.001； t=48.039， P＜0.001）。 LINC01232 能够与miR-516a-5p 竞争性结合，抑制miR-516a-5p 表达，从而上调BCL9 表达。 MTS 实验结果显示，sh-NC，sh-LINC01232 和sh-LINC01232 及miR-516a-5p 抑制剂共转染组，3 组之间差异有统计学意义（F=4412.680， P＜0.001），分组与时间点之间存在交互作用（F=64.100， P＜0.001）。 与对照组相比，72、96 h 敲低LINC01232 可以显著抑制乳腺癌细胞的增殖（t=4.011， P=0.007； t=13.993， P＜0.001），共转染一定程度恢复乳腺癌细胞的增殖能力（t=-1.734， P=0.134； t=-1.091， P=0.317）。 克隆形成实验中，3 组克隆形成数差异有统计学意义（F=891.520， P＜0.001），组间内两两比较，敲低LINC01232 可以显著抑制乳腺癌细胞的增殖（t=44.111， P＜0.001），共转染一定程度恢复乳腺癌细胞的增殖能力（t=-0.220， P=0.810）。 Transwell 实验结果显示，3 组迁移及侵袭细胞数差异有统计学意义（F=2114.691， P＜0.001； F=810.413， P＜0.001），组间内进一步两两比较，敲低LINC01232 后，乳腺癌细胞的迁移和侵袭明显减少（t=55.103， P＜0.001， t=51.879，P＜0.001），共转染后乳腺癌细胞迁移和侵袭能力与对照组相比有所恢复（t=-0.223， P=0.822， t=-9.470，P＜0.001）。

结论

在三阴性乳腺癌细胞中，LINC01232 通过miR-516a-5p/BCL9 轴促进TNBC 肿瘤细胞体外迁移和侵袭能力。

Objective

To investigate the role of LINC01232 in triple negative breast cancer （TNBC）and explore the regulatory relationship with miR-516a-5p/BCL9 axis.

Methods

The Gene Expression Omnibus （GEO） database was screened for differentially expressed genes associated with TNBC malignant progression. Real-time quantitative RT-PCR was performed to detect the expression of LINC01232 in TNBC，and cell function experiments were performed to explore the potential effect of LINC01232 on TNBC progression.Bioinformatics analysis was performed to analyze the association between LINC01232 and miR-516a-5p and B-cell lymphokine 9 （BCL9）， and their binding activities were verified using dual-luciferase reporter gene and RNA immunoprecipitation assays.

Results

Compared with adjacent normal tissue， the expression of LINC01232 was abnormally elevated in TNBC tissue （0.797±0.449 vs 2.524±1.099， t=11.273， P＜0.001）.When LINC01232 was targeted for knockdown， breast cancer cell proliferation among different groups showed a statistically significant difference （F=884.428， P＜0.001）， with an interaction between subgroups and time points （F=54.276， P＜0.001）. Compared with the control group， cell proliferation ability was reduced in the LINC01232 knockdown group at 72， 96 h（t=8.933， P＜0.001； t=8.378， P＜0.001）. Clone formation assay showed that the number of clones in the LINC01232 knockdown group was reduced compared with the control group （t=40.455，48.039， both P＜0.001）. Transwell assay showed that the number of migrated and invaded cells in the LINC01232 knockdown group was significantly reduced compared with the control group （539.3±0.1 vs 344.3±0.6， t=35.308， P＜0.001）. LINC01232 was able to competitively bind to miR-516a-5p and inhibit the expression of miR-516a-5p， thus upregulating the expression of BCL9. The results of MTS experiments showed that there was a statistical difference between sh-NC， sh-LINC01232 and sh-LINC01232 and miR-516a-5p inhibitor co-transfection groups （F = 4412.680， P ＜0.001） and an interaction between subgroups and time points （F= 64.100， P ＜0.001）. Knockdown of LINC01232 at 72， 96 h significantly inhibited the proliferation of breast cancer cells （t = 4.011， P = 0.007； t = 13.993， P ＜0.001）， and cotransfection somewhat restored the proliferation ability of breast cancer cells （t=-1.734， P=0.134； t=-1.091， P=0.317）. In the clone formation experiment， Statistical differences existed between groups （F=891.520， P＜0.001）， and pairwise comparison within groups showed the knockdown of LINC01232 inhibited the proliferation of breast cancer cells （t=44.111， P＜0.001）， and co-transfection restored the proliferative ability of breast cancer cells to a certain extent （t=-0.220， P=0.810）.The results of Transwell assay showed that statistical differences were observed in the migrated cells and invasive cells among three groups （F=2114.691，P＜0.001； F=810.413， P＜0.001）， and pairwise comparisons within the groups showed that knockdown of LINC01232 reduced the migration and invasion of breast cancer cells （t=55.103， P＜0.001， t=51.879， P＜0.001）， and the migration and invasion ability of breast cancer cells was reduced after cotransfection （t=-0.223， P=0.822， t=-9.470， P＜0.001）.

Conclusions

In triple negative breast cancer cells， LINC01232 promotes the migration and invasion capacity of TNBC cells in vitro via the miR-516a-5p/BCL9 axis.