Preliminary Studies On The Effects Of SKA2 And FAM3B Genes On Osteosarcoma Metastasis And Apoptosis

Objective:To investigate the effects of SKA2 and FAM3B genes on cell epithelial mesenchymal transition(EMT),proliferation,apoptosis,invasion and migration after differential expression in human osteosarcoma cells,and to explore the possible molecular mechanisms.Methods:(1)Human osteosarcoma saos-2 and 143B cell lines were cultured,siRNA designed to synthesize SKA2 gene was transfected into saos-2 and 143B cells to form cells with low expression of SKA2.The mRNA levels of SKA2 and EMT-related genes E-cadherin,Vimentin and Snail2 were detected by qRT-PCR assay after siRNA transfection.The protein levels of SKA2,E-cadherin,Vimentin and Snai12 were detected by western blot assay after siRNA transfection.transwell assay was used to detect changes in cell invasion ability after differential expression of SKA2 gene,and changes in migration ability were detected by scratch assay.The proliferation ability was detected by CCK-8 assay,and the apoptosis and cell cycle changes were detected by flow cytometry.(2)Construction of osteosarcoma saos-2 cell lines over-expressing and knock-down expressing FAM3B gene by electrotransfection and siRNA transfection.The electroporation efficiency was detected by flow cytometry,and the mRNA level of FAM3B in cells after electroporation and siRNA transfection was detected by qRT-PCR.The changes of FAM3B protein after electroporation and siRNA transfection were detected by western blot assay.Transwell assay detects changes in cell invasion ability,scratch assay detects changes in migration ability,and CCK-8 assay detects changes in proliferation ability.Results:(1)The expression of SKA2 gene in the transfected cells was detected by qRT-PCR and western blot,and it was confirmed that saos-2 and 143B cells with low SKA2 expression were established(P<0.05).The expression of EMT-related genes Vimentin and Snai12 decreased(P<0.05),and the expression of E-cadherin increased(P<0.05)were detected by qRT-PCR and western blot,indicating that the process of EMT was reversed.The results of CCK-8 assay,transwell chamber and scratch assay showed that the proliferation,invasion and migration of osteosarcoma cells were inhibited after siRNA transfection(P<0.05).Flow cytometry results showed the ability to increase cell early apoptosis Stayed in G0/G1 stage(P<0.05).(2)The qRT-PCR method and western blot detected that the electrotransfection plasmid method successfully established the over-expression of FAM3B gene in osteosarcoma saos-2 cells.siRNA transfection established cells with knock-down.FAM3Bexpression.The proliferation,invasion,and migration of saos-2 cells were enhanced after overexpression,while all were inhibited after siRNA transfection.Conclusion:(1)The SKA2 gene regulates human osteosarcoma cell invasion and metastasis through the EMT process,and can affect proliferation and apoptosis.(2)The expression level of FAM3B gene is positively correlated with the proliferation,invasion and migration ability of human osteosarcoma cells.(3)SKA2 and FAM3B are expected to be potential molecular markers for the treatment and diagnosis of osteosarcoma.