Construction And Functional Analysis Of Breast Cancer Cell Lines With Irx5 Gene Overexpression

Irx5 gene belongs to the Iroquois homeobox gene family, which has six members from Irx1 to Irx6. Studies have reported that some members of Irx family were correlated with multiple cancers, of which Irx4 and Irx1 were proved to be suppressor genes in gastric cancer and prostate cancer, respectively. However, Irx5 gene was down-regulated in high metastatic human and mouse breast cancer cell lines, and its expression level was negatively correlated with the metastatic potential of breast cancer cells, which implied that Irx5 may be a suppressor gene in breast cancer. In order to further study the functions of Irx5 gene in the occurrence, development and migration of breast cancer cells. We constructed MDA-MB-231 breast cancer cell lines with Irx5 overexpression, and used proliferation, invasion and migration assays to confirm the inhibited effect of Irx5. The results are shown as follows:(1) According to the sequence of human Irx5 gene in Gen Bank, we designed a pair of primers and the full length sequence of Irx5 gene was amplified from 293 T cells by RT-PCR. The purified products were digested by Xba I and Bam HI enzyme and connected to the linearized lentiviral vector with ligase to obtain the recombinant lentiviral plasmid(PEB-3xflag-GP-Irx5). By digestion, electrophoresis and sequencing technology, we found that the inserted sequence was consistent with the Irx5 sequence reported in Gen Bank, which suggested that the recombinant vector was constructed successfully.(2) The correct recombinant lentiviral plasmid and packaging plasmid were co-transfected into 293 T cells to produce recombinant virus. We used gradient dilution method to measure the titer of recombinant virus, the result showed that the titer of virus was 3~8×108 TU/m L. MDA-MB-231 cells were transfected with recombinant lentivirus. After cells were cultured for 48 h, we screened the stable cells by puromycin with a final concentration 2.5 μg/m L. After 2~3 weeks, we detected the expression of the exogenous Irx5 gene by Western blotting. The results showed that the exogenous Irx5 gene overexpression existed in breast cancer cells infected with recombinant lentivirus, but breast cancer cells infected with empty vector were no exogenous Irx5 gene expression. The above results indicated that breast cancer cell lines with the exogenous Irx5 overexpression were constructed successfully, and Irx5 was stable expression in breast cancer cells.(3) MTT, colony formation, cell wound-healing and Transwell assays were performed to detect breast cancer cells proliferation, migration and invasion. MTT and colony formation assays showed that breast cancer cells proliferation with Irx5 overexpression were reduced compared to the control. Wound-healing and Transwell migration assays showed that breast cancer cells migration with Irx5 overexpression were reduced compared to the control. Transwell invasion assays showed that breast cancer cells invasion with Irx5 overexpression were reduced compared to the control. These results indicated that Irx5 gene overexpression inhibited breast cancer migration and invasion.The inhibitory effect of Irx5 gene in breast cancer cells was confirmed, which not only provided an experimental foundation for further elucidating the molecular mechanism and the transcriptional regulatory networks of Irx5, but also provided a new idea for molecular diagnosis, individualized treatment and targeted drugs therapy to prevent the metastatic breast cancers.