| Tumor cells exhibit morphological plasticity in response to environmental cues.Special geometric features allow tumor cells migration,proliferation or metastasis with higher efficiency.In addition,tumor cells can sense and transduce mechanical forces into biochemical signals to elicit a cellular response such as gene activation in the nucleus.Therefore,mechanical cues induced diversity of tumor cells with geometric morphologies may have their unique biological functions.Poor prognosis and recurrence are the main reasons that cancer is difficult to cure,which is related to the self-renewal ability of tumor cells.However,we don’t know much about whether the geometric morphologies of tumor cells are related to their self-renewal ability and the regulation mechanism.The purpose of this study is to explore the regulation of single-cell morphology control on tumor cell self-renewal ability and its molecular mechanism.In this study,osteosarcoma U-2 OS cells were confined to micro-patterned arrays substrates with square,round and rectangular with an area of 1000(?)m~2.The aspect ratio of rectangular was 4:1.After the cells were cultured for 24 hours,immunofluorescence staining was used to detect the expression of the self-renewal markers Nanog,Oct4,and Sox2 of tumor cells with differential geometric morphologies.The results showed that U-2 OS cells grown on circular micro-patterns with curved borders and rectangular micro-patterns with an aspect ratio of 4:1 had higher expression of self-renewal markers than cells grown on square micro-patterns.Furthermore,we stained the cytoskeleton protein F-actin and detected the expression of p-MLC,MLCK and the protein expression of WDR5 and H3K4me3 in the nucleus by immunofluorescence staining.The experimental results showed that the expression of F-actin,p-MLC and MLCK in U-2 OS cells grown on circular and rectangular micro-patterns were increased,while the expression of WDR5 and H3K4me3 in the nucleus increased significantly.Subsequently,after the round and rectangular cells were treated with the MLCK inhibitor ML-7,the expression of p-MLC was significantly reduced.The Myosin II inhibitor Blebbistatin and ML-7 were used to treat round and rectangular cells.And immunofluorescence staining was used to detect the protein expression of the self-renewal markers Nanog,Oct4 and Sox2 in the cells after the inhibitor treatment.We found that the expression of the self-renewal markers was significantly decreased.Furthermore,after the round and rectangular cells were treated with ML-7,the protein expression of WDR5 in the nucleus was analyzed by immunofluorescence staining,and the co-localization of WDR5 and H3K4me3 nucleus was observed by confocal laser scanning microscope.The experimental results showed that after inhibitor treatment,the expression of WDR5 in the nucleus was significantly reduced,and the co-localization of WDR5 and H3K4me3 was reduced.After transfected with sh WDR5,the expression of H3K4me3 in the nucleus and the expression of self-renewal markers of round and rectangular cells were detected by immunofluorescence staining.The results showed that after the knockout of WDR5,the level of H3K4me3 in the nucleus of round and rectangular cells and the self-renewal markers were significantly decreased.In summary,we have found that when U-2 OS cells grown in circular micro-patterns with curved borders and rectangular micro-patterns with an aspect ratio of 4:1,the expression of MLCK was increased,and the cytoskeleton contractility was increased.Elevated cytoskeletal contractility promoted WDR5 protein expression in the nucleus.And overexpression of WDR5 increased H3K4me3 and up regulated the expression of self-renewal markers Nanog,Oct4 and Sox2.Ultimately,the self-renewal ability of osteosarcoma cells was improved.This study links the single cell morphological characteristics of tumor cells with biological functions,which can provide a new perspective for tumor diagnosis and prognosis. |
