| The nucleolus is the factory for r RNA gene storage, processing r RNA molecules and assembling ribosomal subunits. The nucleolus also participates in nuclear protein degradation, apoptosis, stress, regulation of cell proliferation, tumorigenesis and other important physiological processes. Nucleolar localization protein 12 is a putative RNA binding nucleolar protein associated with r RNA transcription. Previous studies have found that the Drosophila Nol12 homologue viriato is required for the growth and differentiation progression activities of the Dpp pathway during Drosophila eye development and is a d Myc target that regulates nucleolar architecture and is required for d Myc-stimulated cell growth. The study for nucleolar localization mechanism of rats NOL12 have found that nucleolar localization sequence of NOL12 may be located in C-terminus of the protein. Up to now the study about subcellular distribution, localization mechanisms and functions in cell of human NOL12 protein does not delve deeply.In this study, we predicted the possible nucleolar localization signal of NOL12 though bioinformatic analysis. Based on the results, we cut full-length NOL12 into multiple segments to fuse with GFP to construct recombinant plasmids and then transfected to He La cells. By observing the fluorescence of GFP in the cell, we found that there were three relatively independent nucleolar localization signals located in 1-10 aa of the N-terminal region, 166-184 aa and 187-213 aa of the C-terminal region. Meanwhile, we predicted the possible nuclear localization signals of NOL12 by the same way. Interestingly, we found the regions in where possible nuclear localization signals located and the regions of nuclear localization signals appeared to overlap. Since molecular weight of a single GFP is too small, it can freely diffuse into the nucleus. So we fused the predicted segments with the three tandem fluorescent proteins Cherry-Cherry-GFP to construct recombinant plasmids to explore nuclear localization signals. We observed the spatial distribution of fluorescence of m Cherry and found that NOL12 had two relatively independent nuclear localization signals at the least, which are located in the 1-20 aa of the N-terminal and the 166-213 aa of the C-terminus.After we exploring the NOL12 subcellular localization mechanism, we also tested its biological function. We detected the influence of NOL12 on the proliferation of Hela cell by overexpressing NOL12 or transfecting small interfering RNA to interfere the production of NOL12. The results showed that overexpressing NOL12 can promote the proliferation of He La cells and the proportion of cells in S phase increased significantly. On the contrary, silencing NOL12 significantly inhibit the proliferation of He La cells. In order to make NOL12 stably expressing in He La cells to research the biological function of NOL12 better, we established Hela cell lines which express NOL12 and GFP fusion protein stably. In consideration of the impact of GFP to the construction and function of either end of the NOL12, we established a Hela cell line in which GFP expressed at NOL12' N- terminal and another Hela cell line in which GFP expressed at NOL12'C-terminal respectively. We also established a HA-tagged proteins fused NOL12 cell lines. These cell lines can enhance monoclonal colony formation and cell migration under serum-free culture conditions. These phenomena show that NOL12 really participate in the regulation of proliferation, and improving the degree of malignancy of He La cells.Since He La cells derived from cervical cancer, in order to clarify whether NOL12 expression level changes in the cervical carcinoma tissue, we tested expression quantity of NOL12 in several cervical carcinoma pathological sections by immunohistochemistry. The result show that compare to pericarcinomatous tissue, immunoreactivity of NOL12 in cervical cancer nest was significantly increased; and NOL12 distribute in cytoplasm of basal layer cells which has a high proliferative ability and a low differentiation degree; however NOL12 distribute in nucleolus of prickle cell which has a low proliferative ability and is differentiated. For purpose of making it clear that whether spatial translocation and expression quantity increase of NOL12 were related to proliferation ability of Hela cell, we added endothelial growth factor(EGF) or high concentration(30%) fetal bovine serum into culture media.We found that in the cell total expression quantity of NOL12 increased and NOL12 transferred from the nucleus to the cytoplasm. The result suggest that expression level and spatial translocation of NOL12 are significantly associated with the occurrence and development of cervical cancer, and spatial translocation of NOL12 is a relevant factor to malignancy of cervical carcinoma.The present study identified the nuclear/nucleolus localization sequences of NOL12 for the first time. Meanwhile, NOL12 spatial translocation and the high expression were found in cervical cancer; Overexpression of NOL12 could significantly promote the proliferation and degree of malignancy of He La cells. These results suggest that NOL12 may be a new target for cancer therapy. Our results provide new evidences for the research of NOL12 subcellular localization and relations between NOL12 and cervical cancer. |
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