Abstract:
Background Cyclin dependent kinase 4 (CDK4) belongs to the serine/threonine kinase family, which is a potential target for many cancers. Its expression level and function in osteosarcoma remain unclear and need to be further studied.
Objective To investigate the expression level and function of CDK4 gene in osteosarcoma cells and verify its relationship with the proliferation and migration of osteosarcoma cells.
Methods The microarray data of gene expression profiles related to osteosarcoma were searched in GEO database. Osteosarcoma MG63 cell line and normal osteoblast HOB cell line in the dataset were compared to analyze the differences of CDK4 gene expression between the two groups. Using the human mammary gland library as a template, polymerase chain reaction (PCR) was carried out to amplify the CDS region coding sequence of human CDK4 gene to obtain the amplification product. Subsequently, the amplification product was inserted into the pEGFP-C1 vector to obtain the recombinant plasmid, which was then transfected into the human osteosarcoma MG63 cell line. The expression of the plasmid was detected by Western blot, and the location of the fluorescent labeled CDK4 protein within cells was observed by fluorescence microscope. The proliferation and migration of MG63 cells transfected with CDK4 recombinant plasmid were studied by CCK-8 assay, clone formation assay and scratch test.
Results The gene expression profile microarray dataset analysis showed that the expression of CDK4 in human osteosarcoma MG63 cell line was significantly higher than that in normal osteoblast HOB cell line. The obtained plasmids were digested and sequenced. The results showed that the eukaryotic expression vector pEGFP-CDK4 was successfully constructed. Western blot proved that the target protein was successfully expressed. Fluorescence microscopy showed that CDK4 protein was located in the nucleus of osteosarcoma cells. CCK-8 assay, colony formation assay and scratch test showed that overexpression of pEGFP-CDK4 promoted the proliferation and migration of MG63 cells.
Conclusion The expression of CDK4 in human osteosarcoma MG63 cells is significantly higher than that in normal osteoblast HOB cells. CDK4 protein is located in the nucleus of MG63 cells and can probably promote the proliferation and migration of osteosarcoma cells.
下载: