Eastablishment Of Notch Signaling Pathway Reporter System In Prostate Cancer Cell Lines And Study Of TRIM59 Biological Function In Breast Cancer

We set up a Notch signaling reporter system by first constructing a lentivirus plasmid(pLVX-8XCSL-acGFP).Then the 293 T cells were transfected with pLVX-8XCSL-acGFP plasmids for 6h or with pLVX-acGFP plasmids as a control group,respectively.Furthermore,293 T cells in each of the above group were re-transfected with pETP-NICD or pETP control plasmids.The green fluoroscence was observed with fluorescence-microscope at 48 h,and the fluoroscenece indensity was quantified via flow cytometry analysis.Next,pLVX-8XCSL-acGFP plasmids were packed to produce pLVX-8XCSL-acGFP lentivirus to transduce prostate cancer cell line LnCaP.The green fluoroscence was viewed under fluorescence-microscope at 72 h.Furthermore,the cells were sorted into two groups with flow cytometry according to the the fluorencence intensity: the 5% highest intensity group and the 5% lowest group.Then the expressions of Notch1,Notch2 and Hey1 were monitored by Q-PCR in both groups.Green fluoroscenece indensity was significantly higher in NICD-overexpressing pLVX-8XCSL-acGFP 293 T cells than in the control group.While overexpression of NICD in the 293 T cells transfected with pLVX-acGFP plasmid did not lead the fluoroscenece indensity change.Quantitative analysis revealed that the green fluoroscenece indensity was increased about 10 times in NICD-overexpressing pLVX-8XCSL-acGFP 293 T cells compared to the control.Q-PCR test displayed that the expression of Notch1,Notch2 and Hey1 in the 5% highest fluorencence intensity cells were significant higher than in the 5% lowest intensity group.We researches testify pLVX-8XCSL-acGFP can be used as an effective tool to report Notch signal level and provide a foundation for further research of the effect of Notch signal pathway in prostate cancer.Growth rate of Breast cancer incidence in China is twice the global average,especially in city since 1990 s.It is the highest incidence of femal cancer,and is the sixth cause of death in China.The purpose of our present study is to investigate the biological function of TRIM59 in breast cancer for providing a new target for breast cancer therapy.We firstly analyzed microarray data sets from the Oncomine data-base and found that TRIM59 is highly overexpressed in breast cancer.Furthermore,we constructed two MDA-MB-231 cell lines respectively,which TRIM59 is knocked down or overexpressed.In addition,TRIM59 biological role in breast cancer were determined by cell proliferation assay,clone-forming assay,transwell assay in vitro and growth of xenograft tumors in nude mice assay in vivo.Our results revealed that TRIM59-knockdown resulted in a decrease in cell proliferation,clone formation,and migration capacity of breast cancer cells,as well as growth of xenograft tumors in nude mice;overexpression of TRIM59 had the opposite effects.