| The TGF-βsuperfamily comprises TGF-βs, bone morpho-genetic proteins (BMPs), activins and related proteins with similar structure and cell functions. Transforming growth factor-βsuper family exit in various tissues, and they are involved in cell proliferation and differentiation, embryogenesis, immune response, bone formation, and tumorgenesis and so on. The mechanisms of TGF-βeffective on the target genes are very complicate. The intracellular effectors of TGF-βsignalling, the Smad proteins, are considered as the main regulator of the TGF-βpathway. The E3 ubiquitin ligases, Smurf2 (Smad-ubiquitination-regulatory factor 2), antagonizes TGF-βfamily signalling by interacting with R-Smads and targeting them for degradation. It selectively reduces the Smad2 and Smad3 level which involved in TGF-βpathway, and it also partially reduces the Smadl level which involved in BMP pathway. Yet it also interacts with Smad7 and thereby ubiquitinates the TGF-βreceptor type I in the cytoplasm. In this paper, we found a novel ring finger family protein RLIM (RNF12) which could activate TGF-βand BMP pathway and generate concomitant effects in vivo by interacting with Smurf2.Initially, we showed that RLIM, p53 and Hdm2 could form a ternary or a higher order complex by continuous glycerol gradient experiment. This indicated RLIM should play an important in regulating cell growth since its couterparts, both p53 and Hdm2 gene was critical for the regulation of cell growth. We proofed this idea and found RLIM could inhibit cell growth by using the soft agar assay. As it was frequently reported TGF-βpathway could participate in the process of cell growth inhibition, and there were several papers identified ring finger family proteins like RNF11 and RNF111 could activate TGF-βpathway, we predicated RLIM could also activate TGF-βpathway. After the following bioinformatical analysis, we predicated that the activation of TGF-βpathway might be induced by the interaction between RLIM and Smurf2.To proof this idea, at first, we found RLIM could specifically active TGF-βand BMP pathway by luc-reporter system. The relative reporters'luciferase activities increased with the incremental transfection of RLIM. And then the co-localization of RLIM and Smurf2 could be observed by the Fluorescence co-localization experiment. Through both exogenous and endogenous Immuno-precipitation, it was also proved that there was a pysical interaction between RLIM and Smurf2. After that, we demonstrated Smurf2 could specifically ubiquitinate RLIM in vivo. However, RLIM could not ubiquitinate Smurf2. According the results, we assumed that RLIM competed with R-Smads to bind with Smurf2, which may lead to the promotion of TGF-βsignal transduction pathway. By exogenous gradient transfection experiments, we confirmed RLIM can enhance Smadl, Smad2, Smad3 and Smad4 stability in the U20S cells.Furthermore, we further analyzed RLIM biological function in the following experiments. The metastasis assays through wound healing experiment showed RLIM could empower the TGF-β-dependent U2OS cell migration. ALP activity assay and staining indicated RLIM could promote C3H10 T1/2 cell differentiation into osteoblast; whereas RLIM's fuction in promoting cell differentiation could be reversed by Smurf2. We also got similar result through Alizarin Red S staining.Recently, we also found RLIM could interact with Smad7 and reduced its level in vivo, which indicated RLIM might also lead to the promotion of TGF-βsignal transduction pathway by degrading I-Smad Smad7. In a word, the first part of our work sheds some light in the molecular mechanism and biological function of RLIM.The second part of our work focused on the research about the correlation between TGF-beta gene polymorphism and breast cancer risk. Transcription growth factor beta 1 (TGF-β1) is one of the TGF-βfamily members, having 71% amino acids homology with TGF-β2 and having 77% amino acids homology with TGF-β1. As an important cytokine, TGF-β1 plays important roles in controlling cell proliferation and metastasis involved in breast cancer. The Single Nucleotide Polymorphisms (SNPs) of TGF-β1 were considered as important markers in difference human diseases. It was reported the 29T/C polymorphism in TGF-β1 has been implicated in breast cancer risk. However, studies on the association between this polymorphism and breast cancer remain conflicting.As genetic association study always produce conflicting results for the same variant and disease between different studies, meta-analysis of genetic epidemiology studies has been considered as a good way to resolve the discrepancies between studies. Moreover, meta-analysis can effectively improve the statistical power for detecting the small effect genes and give a more conclusive conclusion.So to derive a more precise estimation of the relationship, a meta-analysis of 10,341 cases and 15,655 controls from fifty published case-control studies was performed. Our analysis suggested that 29T/C has no association with a trend of breast cancer risk when using both dominant [odds ratio (OR)=0.97,95% confidence intervals (CI): 0.87-1.07] and recessive models (0R=l.03,95% CI:0.94-1.12) and other genetic models to analyze the data. In different menopausal status and ethnic subgroups analysis,29T/C also did not appear to be risk factors for breast cancer. As to the meta-analysis of TGF-β1-509C/T polymorphism, seven published case-control studies (5,825 cases and 7,953 controls) were collected. Our analysis suggested that-509C/T has no association with a trend of breast cancer risk when using both dominant (OR=1.00,95% CI:0.86-1.17), recessive models (0R=1.04, 95% CI:0.94-1.16) and other genetic models to analyze the data. In ethnic subgroups analysis,-509C/T also did not appear to be risk factors for breast cancer. However, due to limited publication and due to TGF-βplay different roles in different phase of breast cancer, larger scale primary studies are required to further evaluate the interaction of TGF-β1 29T/C and-509C/T polymorphisms with breast cancer risk in specific populations. |
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