摘要
Background Periostin originally designated osteoblast-specific factor 2 （OSF-2） is frequently found to be highly expressed in various types of human cancer cell lines in vitro and human cancer tissues in vivo. We proposed that periostin was a key factor during the process of proliferation and invasion in cancer cells. We investigated the effect of periostin on the function of human osteosarcoma cell line （U2OS）, such as proliferation, apoptosis, invasion and the associated signal pathway.Methods A human PGCsi/U6 promoter-driven DNA template was adopted to induce short hairpin RNA （shRNA）-triggered RNA interference （RNAi） to block periostin gene expression in the cell line U2OS. U2OS cells were divided into three groups： cells transfected with phosphate buffered saline as control group （the U2OS group）, cells transfected with pGCsi as negative control group （the NC group） and cells transfected with periostin/pGCsi as experimental group （the pGCsi-periostin group）. Then, transfection efficiency of cell was observed under fluorescent microscope. The expressions of periostin and the related genes in cells were detected by reverse transcription polymerase chain reaction and Western Blotting. Cell viability was determined using the methyl-thiazolyl tetrazolium bromide （MTT） quantitative colorimetric assay. The invasion and migration capability of cells were tested by transwell plates with or without extracellular matrix gel. Furthermore, the changes of cell cycle and apoptosis were analyzed by flow cytometry.Results The transfection efficiency of periostin/pGCsi to U2OS cells was about 70%-80%. When compared with the NC group, the levels of mRNA and protein of periostin in the pGCsi-periostin group decreased by 82% （F=564.71, P＜0.001） and 58% （F=341.51, P ＜0.001 ）, respectively. Meantime, the earlier apoptosis value increased by 417 （F=28.69,P ＜0.001）. The percentage of S phase pGCsi-periostin cells decreased by 21% （F=47.00, P ＜0.001）, however, that of G0-G1 phase cells increased by 12% （F=14.50, P ＜0.001）. The capability of migration and invasion reduced by 41%（F=17.79, P ＜0.001） and 72% （F=197.08, P ＜0.001）, respectively. The cell proliferation in the pGCsi-periostin group decreased by 59% and 72% at 48 and 120 hours after transfection, respectively. The mRNA expressions of transforming growth factor-β and vascular endothelial growth factor decreased by 17% （F=73.99, P ＜0.001） and 47% （F=30.25, P ＜0.001）, respectively. A tendency of lower focal adhesion kinase （FAK） was shown in pGCsi-periostin cells but without any statistically significant difference. Otherwise the expression of p-FAK in those cells had markedly decreased by 21%（F=16.81, P＜0.001）.Conclusions RNAi against periostin can effectively down-regulate periostin gene expression. Periostin increases the hyperplasia and invasion of cancer cells. Periostin might be involved in and served as a tumor promoter gene in the pathogenesis of osteosarcoma.
Background Periostin originally designated osteoblast-specific factor 2 （OSF-2） is frequently found to be highly expressed in various types of human cancer cell lines in vitro and human cancer tissues in vivo. We proposed that periostin was a key factor during the process of proliferation and invasion in cancer cells. We investigated the effect of periostin on the function of human osteosarcoma cell line （U2OS）, such as proliferation, apoptosis, invasion and the associated signal pathway.Methods A human PGCsi/U6 promoter-driven DNA template was adopted to induce short hairpin RNA （shRNA）-triggered RNA interference （RNAi） to block periostin gene expression in the cell line U2OS. U2OS cells were divided into three groups： cells transfected with phosphate buffered saline as control group （the U2OS group）, cells transfected with pGCsi as negative control group （the NC group） and cells transfected with periostin/pGCsi as experimental group （the pGCsi-periostin group）. Then, transfection efficiency of cell was observed under fluorescent microscope. The expressions of periostin and the related genes in cells were detected by reverse transcription polymerase chain reaction and Western Blotting. Cell viability was determined using the methyl-thiazolyl tetrazolium bromide （MTT） quantitative colorimetric assay. The invasion and migration capability of cells were tested by transwell plates with or without extracellular matrix gel. Furthermore, the changes of cell cycle and apoptosis were analyzed by flow cytometry.Results The transfection efficiency of periostin/pGCsi to U2OS cells was about 70%-80%. When compared with the NC group, the levels of mRNA and protein of periostin in the pGCsi-periostin group decreased by 82% （F=564.71, P＜0.001） and 58% （F=341.51, P ＜0.001 ）, respectively. Meantime, the earlier apoptosis value increased by 417 （F=28.69,P ＜0.001）. The percentage of S phase pGCsi-periostin cells decreased by 21% （F=47.00, P ＜0.001）, however, that of G0-G1 phase cells increased by 12% （F=14.50, P ＜0.001）. The capability of migration and invasion reduced by 41%（F=17.79, P ＜0.001） and 72% （F=197.08, P ＜0.001）, respectively. The cell proliferation in the pGCsi-periostin group decreased by 59% and 72% at 48 and 120 hours after transfection, respectively. The mRNA expressions of transforming growth factor-β and vascular endothelial growth factor decreased by 17% （F=73.99, P ＜0.001） and 47% （F=30.25, P ＜0.001）, respectively. A tendency of lower focal adhesion kinase （FAK） was shown in pGCsi-periostin cells but without any statistically significant difference. Otherwise the expression of p-FAK in those cells had markedly decreased by 21%（F=16.81, P＜0.001）.Conclusions RNAi against periostin can effectively down-regulate periostin gene expression. Periostin increases the hyperplasia and invasion of cancer cells. Periostin might be involved in and served as a tumor promoter gene in the pathogenesis of osteosarcoma.
基金
This study was supported by a grant from the National Natural Science Foundation of China （No. 0973126）.
