H3 Arginine 117 Mono-ADP-ribosylation Regulates Proliferation Through Its Interaction With P300

Objective:To define the roles and the molecular mechanism of H3 Arginine117^th Mono-ADP-ribosylation in the proliferation of human colon carcinoma LOVO cells.Methods:1.Histone H3R117 mono-ADP-ribosylation affects the proliferation of LOVO cells associate with P300.?1?.Histone H3R117 mono-ADP-ribosylation affects the proliferation of LOVO cells.LOVO cells infected with lentivirus containing H3 117^th arginine mutate to alanine?H3R117A?or lysine?H3R117K?were set as research object.The untransfected LOVO cells and the lentivirus blank load transfection LOVO cells were acted as control groups.The CCK8 assay was used for defining the proliferation of LOVO cells in each group.Flow cytometry?FCM?assay was performed to detect the cell cycle of each group.Mutation LOVO cell and control LOVO cell were injected to nude mice for establishing subcutaneous transplanted tumor model,the volume and weight of subcutaneous transplanted tumor were observed.?2?Histone H3R117 mono-ADP-ribosylation affects the proliferation of LOVO cells associate with P300.Mutant groups which were added with N-[4-Chloro-3-?trifluoromethyl?phenyl]-2-ethoxy-6-pentadecylbenzamide?CTPB??the activator of P300?were regarded as treated group.The CCK8 assay was used to define the proliferation of LOVO cells in each group.FCM assay was performed to detect the cell cycle of each group.2.The mechanism about H3R117 mono-ADP-ribosylation affects the proliferation of LOVO cells associate with P300qRT-PCR was performed for detecting the mRNA level of P300;The Histone acetyltransferase?HAT?activity assay kit was performed to define the HAT activity of P300.Western blotting was used to detect the expressions of?-catenin,c-myc and cyclinD1.Immunoprecipitation assay was used to determine the expression of acetylated?-catenin.Meanwhile,theinteractionofP300and?-cateninwasdetectedby co-immunoprecipitation assay.Results:1.Histone H3R117 mono-ADP-ribosylation affects the proliferation of LOVO cells associate with P300.?1?Histone H3R117 mono-ADP-ribosylation affects the proliferation of LOVO cells.?1?The result of CCK8 assay showed that an increase of the inhibitory of LOVO cells proliferation in mutant groups compared to control groups?P<0.05?.However,there was no significant difference between the mutation groups,which was the same between the control groups?P>0.05?.?2?Flow cytometry assay showed that there was an increase ratio of G1 phase and a knockdown proliferation index?PI?in mutant groups,compared to the control groups?P<0.05?.While,there was no obvious difference between the mutation groups,which was the same between the control groups.?3?In the transplanted tumor model of LOVO cells,compared with control groups,the size and weight in the groups which were injected in mutant LOVO cells were decreased?P<0.05?.While,there was no obvious difference between the mutation groups,which was the same between the control groups?P>0.05?.?2?Histone H3R117 mono-ADP-ribosylation affects the proliferation of LOVO cells associate with P300.?1?The CCK8 assay showed that the inhibitory of LOVO cells proliferation reduced in the mutant groups treated with CTPB compared to the mutant groups without CTPB?P<0.05?.However,there was no obviously difference between the mutation groups treated with CTPB?P>0.05?.?2?The results of flow cytometry assay showed,compared to the mutant groups without CTPB,the ratio of G1 phase reduced and the proliferation index?PI?increased in the mutant groups treated with CTPB?P<0.05?.While,there was no significant difference between groups treated with CTPB?P>0.05?.2.The mechanism about histone H3 R117 mono-ADP-ribosylation affects the proliferation of LOVO cells associate with P300.?1?The mRNA level of P300 decreased in the mutation groups compared with the control groups?P<0.05?.Note that there was no significant difference between Non-transfection group and blank load transfection group?P>0.05?,which was the same between H3R117K group and H3R117A group?P>0.05?;?2?HAT activity assay showed that the HAT activity of P300 reduced in mutation groups compared to the control groups?P<0.05?.However,there was no significant difference between the mutation groups?P>0.05?,which was the same between the Non-transfection group and the blank load transfection group?P>0.05?.?3?In vivo and vitro,the protein expression of?-catenin,c-myc and cyclinD1in the mutant groups showed a decrease compared with the control groups?P<0.05?.And the protein expression of?-catenin,c-myc and cyclinD1 in the mutant groups treated with CTPB appeared an increase compared to the mutation groups without CTPB.However,there was no obvious difference between the mutant groups treated with CTPB?P>0.05?.?4?Co-IP assay showed that the mutation of H3 R117 mono-ADPribosylated site could decrease the level of P300 binding with?-catenin in vivo and in vitro?P<0.05?.And,it also could decrease the level of acetylatied?-catenin bingding with?-catenin in vivo and in vitro?P<0.05?.Conclusions:1.Histone H3R117 mono-ADP-ribosylation affects the proliferation of LOVO cells associate with P300.2.Histone H3R117 mono-ADP-ribosylation could regulate the activity of P300 to affect the level of acetylated?-catenin which could mediate the expression of c-myc and cyclinD1 to regulate the proliferation of LOVO cells;and it also could regulate the expression of P300.