Study On The Effect Of Cinobufagin And Bufalin On Cell Cycle And Apoptosis By Deregulating AURKs Expression In Human Hepatoma Cells

ObjectiveTo explore the mechanism of cell cycle arrest and apoptosis induced by Cinobufagin and Bufalin and establish stable cells line with overexpression and knockout of AURKA.MethodsPART ONE:The relationship between the overall survival of patients with hepatocellular carcinoma?HCC?from TCGA and the mRNA expression of aurora kinase was analyzed.Then we cultured HepG2,Huh7 and SMMC7721cells.The viability was detected by MTT assay.The cell cycle and apoptosis were detected by flow cytometry with PI staining and Annexin V/PI staining respectively.The expression levels of cell cycle-related protein cyclin B1 and CDK1,apoptosis-related protein Bcl-2,aurora kinases?AURKA and AURKB?,Xklp2-targeted protein TPX2 and the oncogene protein c-MYC were detected by Western blot.Part TWO:Overexpression and knockout of AURKA of HCC cell lines were built by pLVX-puro and lentiCRISPRv2 plasmids combined with lentivirus packaging technique.Western Blot was used to verify the expression of AURKA in the cell linesResultsThere was a significant negative relationship between overall survival and the mRNA expression of AURKA and AURKB in HCC patients.With the increase of Cinobufagin or Bufalin's concentration,the inhibitory effect on HepG2 Huh7 and SMMC7721 cell growth level was more obvious?R²>0.9?,and both of Cinobufagin and Bufalin could induce G₂/M arrest and apoptosis in HCC cells.And they all modify the expression levels of cell cycle-related protein cyclin B1 and CDK1,apoptosis-related protein Bcl-2,aurora kinases?AURKA and AURKB?,Xklp2-targeted protein TPX2 and the oncogene protein c-MYC for different time-courses in varying degrees?P<0.05?.Plasmids of pLVX-AURKA and CRISPR/CAS9-AURKA was successfully built after sequencing analysis.But only pLVX-AURKA-HepG2 and pLVX-AURKA-SMMC7721 overexpression cell lines and CRISPR/CAS9-AURKA-HepG2 knockout cell line were successfully built.Conclusions1?There was a significant negative relationship between overall survival and the mRNA expression of AURKA and AURKB in HCC patients.2?Both Cinobufagin and Bufalin inhibited the proliferation of HepG2-Huh7 and SMMC7721 cells in a concentration-dependent manner.3?Both Cinobufagin and Bufalin could induce G2/M arrest and apoptosis in the 3 types of HCC cell by affecting AURKs,then down-regulated of the translation level of TPX2,c-MYC,CDK1,Cyclin B1 and Bcl-2.