Effects Of Histone H3 Arginine-specific Mono-ADP-ribosylation On Proliferation And Apoptosis Of Human Colon Cancer Cell And Its Possible Mechanism

Objective:To explore the effects of histone H3 arginine-specific mono-ADP-ribosylation on proliferation and apoptosis of human colon cancer and its possible mechanismMethods:Lentivirus vectors named pLenti-H3 mut1-IRES-eGFP and pLenti-H3 mut2-IRES-eGFP of histone H3 arginine-specific mono-ADP-ribosylation site-directed mutation were constructed to infect LOVO cells.Stable transfected mutants were selected by BSD.At the same time,all of Cells can be divided into four groups:Untransfected group of LOVO cells,empty-vector group of LOVO cells,H3-mut1(H3A117)group of LOVO cells,H3-mut2(H3K117)group of LOVO cells.The effect of LOVO cells proliferation in each group was measured by CCK8 assay.The flow cytometry was performed to analysis cell apoptosis,cell circle distribution,respectively.The xenografts tumor model in nude mice of human colon cancer were constructed to observe their growth status.The mRNA expression of RASSF1 A was tested by qRT-PCR.The expression of H3K9 dimethylation and RASSF1 A,Bcl-2,Bax,and Cyclin D1 proteins of LOVO cells were detected by Western Blot,separately.Results:1.Lentivirus of LV-control,H3-mut1(H3A117)and H3-mut2(H3K117)were infected into LOVO cells successfully,and the LOVO cell line with stable mutation of H3-arginine-specific mono-ADP-ribosylation were successfully screened.2.The results of CCK8 showed that the inhibitory rate of LOVO cells proliferation in the H3-mut1(H3A117)and H3-mut2(H3K117)groups were significantly higher than that in the untransfected and LV-control groups(P?0.05).However,there was no obviously difference between the untransfected and LV-control groups(P?0.05),and it is the same between the H3-mut1(H3A117)and H3-mut2(H3K117)groups(P?0.05).3.In the cell cycle analysis,when compered with the untransfected and LV-control groups,the percentage of the LOVO cells in G1 phase in the H3-mut1(H3A117)and H3-mut2(H3K117)groups were markedly increased(P ? 0.05),and the proliferative indices were significantly decreased(P?0.05),but there was no obviously difference in cell circle distribution and proliferative indice between the untransfected and LV-control groups(P ? 0.05),and it is the same between the H3-mut1(H3A117)and H3-mut2(H3K117)groups(P?0.05).4.The result of the flow cytometry suggested that the apoptosis rate of the LOVO cells in H3-mut1(H3A117)and H3-mut2(H3K117)groups were higher than that in untransfected and LV-control groups(P?0.05),but there was no obviously difference about the apoptosis rate of the LOVO cells between the untransfected and LV-control groups(P?0.05),and it is the same between the H3-mut1(H3A117)and H3-mut2(H3K117)groups(P?0.05).5.In the xenografts tumor model of nude mice,the weight and volume of the tumor reduced in H3-mut1(H3A117)and H3-mut2(H3K117)groups compered with the untransfected and LV-control groups(P?0.05).Nonetheless,there was no statistically difference between the untransfected and LV-control groups(P ? 0.05),and it is the same between the H3-mut1(H3A117)and H3-mut2(H3K117)groups(P?0.05).6.Compered with the untransfected and LV-control groups,the mRNA expression of RASSF1 A was obviously increased in the LOVO cells in H3-mut1(H3A117)and H3-mut2(H3K117)groups(P?0.05),but there was no statistically difference between the untransfected and LV-control groups(P ? 0.05),and it is the same between the H3-mut1(H3A117)and H3-mut2(H3K117)groups(P?0.05)..7.The expression level of RASSF1 A,Bax proteins of LOVO cells in H3-mut1(H3A117)and H3-mut2(H3K117)groups were higher than that in untransfected and LV-control groups(P?0.05).However,The expression level of H3K9 dimethylation as well as Bcl-2,Cyclin D1 proteins of cells and tumor xenografts in H3-mut1(H3A117)and H3-mut2(H3K117)groups were lower than that in untransfected and LV-control groups(P?0.05).there was no obviously difference in the above index between the untransfected and LV-control groups(P?0.05),and it is the same between the H3-mut1(H3A117)and H3-mut2(H3K117)groups(P?0.05).Conclunsion:Histone H3 arginine-specific mono-ADP-ribosylation site-directed mutantion can inhibit the proliferation of human colon cancer LOVO cells,and promote apoptosis.The results indicate that histone H3 arginine-specific mono-ADP-ribosylation plays an important role in the process of proliferation and apoptosis of human colon cancer LOVO cells.The mechanism of this effect probably associates with histone poly-ADP-ribosylation,H3K9 di-methylation,DNA methylation,followed by the activity of RASSF1 A,and finally influence the expression of the downstream factors like Bcl-2,Bax and Cyclin D1.