Abstract:

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.

Key words: Triple negative breast cancer, Long non-coding RNA, microRNA, LINC01232