Tudor-SN Protein Function In The Breast Cancer Progression And Adipocytes Differentiation

Objectives:Tudor staphylococcal nuclease (Tudor-SN) is a highly conserved and ubiquitously expressed multifunctional protein. It has been identified as co-activators of a variety of transcription factors such as STAT6in the nucleus, also participate the pre-mRNA splicing and processing, stress granules’formation. It should be noted that Tudor-SN is one of components of RNAi-induced silencing complex (RISC) and could be involved in the regulation of microRNAs expression. Otherwise, Tudor-SN protein is present in the lipid droplets of secretory cells. Our previous results indicated the effect of Tudor-SN on biological behavior of breast cancer cells, and the main purpose of this study is to find out its potential molecular mechanism. In addition, we try to explore the effect of Tudor-SN protein on adipose differentiation and its possible function mechanism during the process. Methods:Part I:High-throughput microarray was performed to screen the miRNAs regulated by the Tudor-SN. In breast cancer cells, the regulation of Tudor-SN protein on miR-127and BCL6was determind using Stem-Loop PCR method, and western blotting was used to detect the transfection efficiency. In vitro cell wound healing assay and proliferation assay were performed to determine the effect of miR-127on breast cancer cell migration and proliferation. In vitro, parental and stable transfectant MDA-MB-231cells were injected into mammary fat pads of athymic nude mice. The volume of tumor were observed and measured, and miR-127and BCL6expression were detected by Real time quantitative PCR in the tumors.Part II:3T3-L1preadipocytes were transfected with pSG5-Tudor-SN plasmid or Tudor-SN siRNA and induced using MDI, then stained by oil red O to observe the effect of Tudor-SN on adipogenesis. The same experiments were done in Tudor-SN knockout MEF cells. Tudor-SN and PPARy protein or mRNA expression changes were observed by western blot and Real time quantitative PCR during3T3-L1cells differentiation, and the localization of the two proteins were observed by immunofluorescence. A series of key transcription factors and fat factors were detected by quantitative PCR in Tudor-SN knockout and normal MEF cells. Luciferase experiments and ChIP assays were performed to detect the transcription regulation of Tudor-SN promoter region. In vitro GST fusion protein angling and in vivo immune co-precipitation experiments were done to verify and determine the interaction of the two proteins.Results:Part I:miR-127was one of the miRNAs regulated by Tudor-SN protein. Tudor-SN protein negatively regulated miR-127expression and positively regulated the expression of BCL6in breast cancer cells, and in vivo nude mice experiments showed similar results. miR-127overexpression inhibited breast cancer cell MDA-MB-231migration and proliferation.Part Ⅱ:Tudor-SN protein overexpression promoted adipose differentiation, and Tudor-SN knockdown or knockout reduced or blocked differentiation. Tudor-SN and PPARy protein expression and localization were consistent during the adipose differentiation process, but their mRNA expressions were inconsistent. Tudor-SN knockout caused transcription or fat factors C/EBPa, β and8, SREBP1and its target gene FAS, SCD2upregulation, and the target gene expression of PPARy such as aP2, adipsin, LPL, adiponectin and CD36markedly decrease or absence in MEF cells. The transcription of Tudor-SN was regulated by transcription factor C/EBPβ in the3T3-L1differentiation. Tudor-SN interacted with PPARy protein through its SN domain.Conclusion:These findings reveal that the novel mechanism that Tudor-SN promotes the breast cancer progress via reduction of miR-127and consequently increases the expression of tumor promoter BCL6in the breast cancer. Otherwise, Tudor-SN effects the adipose differentiation by interacting with PPARy, Tudor-SN knockout causes PPARy and its upstream transcription or fat factors feedback upregulation, and sequently the expression of PPARy target gene markedly decrease even absence. The transcription of Tudor-SN is regulated by transcription factor C/EBPβ.