| The dissertation consists of two parts:the first part describes the role of human growth differentiation factor 3 in breast cancer; the second part describes cloning and characterization of a novel human CDK5 splicing varian.In the first part, transforming growth factor-β(TGF-β) are a superfamily of secreting polypeptides, which play a pivotal role in the regulation of a wide variety of physiological processes. At present time, the characterization of GDF3 mainly focuses on three aspects: 1) as an adipogenic cytokine; 2) as a marker of stem cells; 3) functioning in the early embryonic differentiation. However, less is known about the role of GDF3 in the progression of human breast cancer. First of all, we found that GDF3 was specifically down-regulated in tumor at protein levels when compared with neighboring pathological normal breast tissues. The recombinant human GDF3 protein could inhibit the growth of breast cancer cells. We generated lentivirus containing sh-RNA targeting GDF3 to knockdown its expression. Compared with control virus, the sh-RNA containing lentivirus have no effect on the cell cycle, cell adhesion and cell migration. However, we found that down-regulation of GDF3 resulted in the promotion of colony formation, and enhanced the ability of anchorage-independent cell growth in soft agar. In addition, overexpression of GDF3 could promote the apoptosis induced by Taxol or Sorafenib. The study of the biological functions of GDF3 will be of great help not only for further elucidating the mechanism of breast cancer genesis but also for the diagnosis and chemotherapy of breast cancer.In the second part, The cyclin-dependent kinases (CDKs) are a family of serine/threonine kinases, playing an essential role in regulating cell-cycle progression. In our present work, human CDK5 and a novel CDK5 splicing variant, named as CDK5-SV, were cloned from the cDNA library of human testis. CDK5-SV lacking the exon 7 of CDK5 encodes a protein of 260 amino acids. Through RT-PCR analysis in different human tissues, CDK5-SV was found to be expressed in testis, skeletal muscle, colon, bone marrow and ovary, while CDK5 was ubiquitously expressed. Immunofluorescence experiment in HeLa cells showed that the subcellular localizations of CDK5-SV and CDK5 were totally different. CDK5 mainly located in the cytoplasm, while CDK5-SV accumulated in nucleus. Reporter gene assay showed that when co-transfected withβ-catenin, CDK5 and CDK5-SV could both strongly inhibit the Wnt/β-catenin signaling pathway. Consistently, CDK5-SV could also interact withβ-catenin as CDK5 does. Taken together, our findings suggest that CDK5-SV might also be a negative regulator of Wnt/β-catenin signaling pathway.
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