The Role Of XIAP 3’-Untranslated Regions On Biological Behavior Of Human Breast Cancer Cells

Objective: To construct a vector carrying human XIAP gene 3’ untranslated region(3’UTR) and its effect on biological behavior of human breast cancer cells.Methods: Human breast cancer cells MCF-7 and MDA-MB-231 were cultured for the following experiments:1. Serum starvation assay was used to observe the detect XIAP m RNA and protein at different time points in two cell lines.2. Construction of human XIAP 3’UTR expression vector by cloning technology and the method of direct selection after plasmid transfection was used to construct XIAP3’UTR overexpression MCF-7 and MDA-MB-231 stably cell lines.3. MTT, colony formation, apoptosis, Transwell and wound healing assays were used to detect the functions of XIAP 3’UTR on proliferation, invasion, migrasion and apoptosis of two breast cancer cells in vitro.4. MCF-7 cells in nude mice were used to observed the effect of xenograft formation in vivo.5. Immunohistochemical Ki-67 and TUNEL assays were used to detect proliferation and apoptosis of orthotopic tumorgrafts in nude mice.6. qRT-PCR and Western-blot assays were used to detect XIAP m RNA and protein levels in XIAP 3’UTR stably breast cancer cell lines.Results:1. Under serum starvation conditions, MCF-7 and MDA-MB-231 cells showed increased XIAP m RNA levels and decreased protein levels.2. The XIAP 3’UTR expression vector was successfully constructed, which was confirmed by the assessment of sequencing.3. The result of q RT-PCR and western blot assays showed that the XIAP m RNA and protein levels were elevated stably in XIAP 3’UTR overexpression stably transfected cell lines.4. The results of MTT, colony formation, apoptosis, Transwell and wound healing assays showed that XIAP 3’UTR has the potential to promote proliferation, invasion,migration and inhibit apoptosis of breast cancer cells in vitro(P<0.05).5. Nude mice assay showed that stable expression of XIAP 3’UTR MCF-7 breast cancer cells has the potential to promote the tumor growth and metastasis in vivo(P<0.05).6. Immunohistochemical Ki-67 and TUNEL assays showed that stable expression of XIAP 3’UTR orthotopic tumorgrafts in nude mice has a higher proliferation and lower apoptosis rate compared to control group(P<0.05).7. The expression levels of XIAP 3’UTR m RNA and protein were significantly increased in XIAP 3’UTR MCF-7 and MDA-MB-231 stably breast cancer cells(P<0.05).Conclusion: Overexpressin of XIAP 3’UTR may significantly promote the proliferation,invasion, migration, metastasis and inhibit apoptosis of breast cancer cells, suggesting that XIAP 3’UTR acts as an oncogene, may be a new molecular mechanisms for cancer develoment.