MiR-210 regulates cell cycle in nasopharyngealcarcinoma cell line (CNE-1) under hypoxic condition by reducing the expression of cyclin D1

Objective： The aim of our study was to determine the underlying mechanism of miR-210 on regulation of the cell cycle in nasopharyngeal carcinoma cell line CNE-1, particularly through regulation of cyclin D1, under hypoxic conditions. Methods： The CNE-1 cell line was induced with hypoxia, and the expression levels of endogenic miR-210 and cyclin D1 were detected by real-time PCR and Western blotting. Next, the luciferase assay was used to confirm that cyclin D1 is a target gene for miR-210. Cell cycle and cell proliferation were detected in CNE-1 cells that were cultured under hypoxic conditions with either overexpression or knockout of miR-210 using flow cytometry and MTT assay, respectively. Results： Hypoxia induced the expression of miR-210, resulting in reduced mRNA and protein levels of cyclin D1 and repression of cyclin D1 in CNE-1 cells. Further analysis indicated that miR-210 directly binded to the 3＇UTR of the cyclin D1 gene, thus regulated the expression of cyclin DI. The flow cytometry assay showed that, under hypoxic conditions, miR-210 blocked CNE-1 cells in the G1 phase, and miR-210 also inhibited the proliferation of CNE-1 cells. Conclusion： Under hypoxic conditions, miR-210 directly reduced the expression of cyclin D1, leading to CNE-1 cells blocked in G1 phase.

p16和cyclin D_1蛋白在鼻咽癌组织中的表达及其意义

目的 探讨鼻咽癌组织中p16和cyclinD1蛋白的表达及其意义。方法 应用免疫组化S P法检测p16和cyclinD1蛋白在 85例鼻咽癌和 18例鼻咽部炎性黏膜组织中的表达。结果 85例鼻咽癌中p16的表达率为 41 2 %(35 / 85 ) ;cyclinD1的过表达率为 6 3 5 % (5 4/ 85 )。 18例鼻咽部炎性黏膜中 ,p16的表达率为 10 0 % ;cyclinD1的表达全部为阴性。高分化鳞癌p16的表达率为 75 0 % (6 / 8) ,低分化鳞癌为 38 2 % (2 6 / 6 8) ,两者差异显著 (P <0 0 5 )。有淋巴结转移的p16表达率为 2 8 6 % (14/ 49) ,无淋巴结转移的为 5 8 3% (2 1/ 36 ) ,两者差异非常显著 (P <0 0 1)。 35例p16 (+)的癌组织中有 2 8例cyclinD1呈过表达 ;5 0例p16 (- )的癌组织中有 2 6例cyclinD1呈过表达。结论 p16的缺失表达和cyclinD1的过表达两者可能单独或共同参与了鼻咽癌的发生发展过程。p16的缺失可能还与鼻咽癌细胞的分化。

EFFECT OF TEA POLYPHENOLS AND EGCG ON NASOPHARYN-GEAL CARCINOMA CELL PROLIFERATION AND THE MECHANISMS INVOLVED

Objective: Tea polyphenols present in green tea show cancer chemopreventive effects in many tumor models. Epidemiological studies have also suggested that green tea consumption might be effective in the prevention of certain human cancers. In the present study, we investigated the molecular mechanisms of the inhibition of cell proliferation by tea polyphenols in nasopharyngeal carcinoma (NPC) cell line CNE1-LMP1. Methods: CNE1-LMP1 cells were treated with tea polyphenols at various doses (0, 25, 50, 100, 200 μg/ml) for 24 hours, the morphology of cells was observed by light microscopy, and cell survival rate was determined by MTT assay. At the same time, cell cycle of CNE1-LMP1 was analyzed by flow cytometry. Cyclin D1 transcription was analyzed by cyclin D1 promoterluciferase reporter system and expression of cyclin D1 protein by Western blot analysis. Transactivities of NF-κB and AP-1 was analyzed by Dual-fluorescence reporter gene system. Results: After treatment of CNE1-LMP1 cells with tea polyphenols, the number of proliferating cells was obviously decreased as determined by light microscopy and MTT assay (from 100% to 89.4%, 83.3%, 74.8% and 38.1%). With the increase of tea polyphenols concentrations, the number of cells in S-phase was obviously decreased, and the number of cells in G1-phase from 22.20% to 13.16%, and the number of cells in G0/G1 phase was elevated from 68.5% to 74.08%. It suggests that tea polyphenols could arrest the cell cycle at both of the two checkpoints. Furthermore, transcription and were obviously declined 7–8 folds (100–200 μg/ml tea polyphenols or EGCG group) and expression of cyclin D1 protein also decreased in a dose-dependent manner. Transactivities of NF-κB and AP-1 were obviously down-regulated in CNE1-LMP1 cells. Conclusion: Green tea polyphenols could inhibit cell proliferation, by suppressing the activity of NF-κB and AP-1, and by down-regulation of the transcription of cyclin D1.