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

Irx5 gene is one of the members in Iroquois homeobox gene family.The proteins which are encoded by the Iroquois homeobox gene family have a highly conservative homology domain structure,containing 63 amino acids,and it has the activity of transcription factors that can be combined with specific DNA.Some reports indicate that many members of the Irx gene family were correlated with the tumor occurrence.Irx1 expression can inhibit gastric cancer cell growth,invasion and tumor formation,and it is a potential tumor marker for gastric carcinoma.Irx2 can promote cell proliferation and invasion in osteosarcoma cells.Irx3 is downregulated in LNCaP prostate cancer cells.Irx4 can suppress prostate cancer growth through the interaction with vitamin D receptor.When Irx5 is knockout,it can reduce the number of prostate cancer cells and increase the cell apoptosis.In addition,Irx5 gene was down-regulated in high metastatic breast cancer cell lines,and was up-regulated in low metastatic breast cancer cell lines.Finally,the expression levels of Irx5 were significantly negative correlation with the cancer metastasis ability of the patients.We have been constructed MDA-MB-231 breast cancer lines with Irx5 overexpression and confirmed that Irx5 gene can inhibit MDA-MB-231 breast cancer cell proliferation,migration and invasion.In order to further study the effect of Irx5 gene in breast cancer cells,we constructed three MCF7 breast cancer cell lines with Irx5 gene silencing through RNAi technology.MCF7-shRNA2 with the best effect of Irx5 gene silencing and MCF7-control were used to detect the cell proliferation,migration and invasion.The results are shown as follows:1.According to the sequence of human Irx5 gene（NM₀05853）,we designed and synthesized three pair of shRNA sequence.The lentiviral vectors were digested by BamH（40）and EcoR（40）enzyme,and the shRNA sequences were connected to the linearized lentiviral vector by T4 ligase.We obtained three recombinant lentiviral plasmids with shRNA sequence through transformation: miRZip-shRNA1,miRZip-shRNA2 and miRZip-shRNA3.The shRNA interference plasmid and packaging plasmid MIX were co-transfected into HEK293 T cells.We obtain the final shRNA interferencing lentivirus packaged in HEK293 T cells.2.After transfecting and screening of puromycin,we obtained three MCF7 breast cancer cell lines with Irx5 gene silencing through transfected with different shRNA interference lentiviral respectively.The results of Western blotting and qPCR indicated that Irx5 gene silencing were detected in MCF7-shRNA1,MCF7-shRNA2 and MCF7-shRNA3,of which the best effect of Irx5 gene silencing is MCF7-shRNA2 whose expression is reducesed by 49%.3.MCF7-shRNA2 and MCF7-control were used for functional analysis: The results of MTT assays showed that there are no obvious difference between MCF7-shRNA2 and MCF7-control,which suggested that Irx5 gene silencing have a little or no effect on cell proliferation in MCF7 cells;The results of wounding-healing and Transwell migration assays showed that the metastatic capability of MCF7-shRNA2 is stronger than MCF7-control,which indicated that knockdown of Irx5 gene can promote the metastasis of MCF7 cells.;The results of transwell invasion assays showed that the capability of invasion of MCF7-shRNA2 is stronger than MCF7-control which suggested that knockdown of Irx5 gene can promote the invasion of MCF7 cellsThis study further confirmed the inhibitory effect of Irx5 gene in breast cancer cells,which not only provided certain theoretical basis for understanding the development and the mechanism of metastasis and invasion of breast cancer,but also provided a solid theoretical basis for the molecular diagnosis and targeting therapy of metastatic breast cancer.