| Part1: Effect of Smad4knockdown on the expression ofcardiac core transcription factor and histone acetylation inH9c2cellsObjectiveBuilding the H9c2cardiomyocytes (H9c2cells) model that Smad4knockdown to reaserch the role of Smad4on the expression of cardiac coretranscription factors GATA4, MEF2c, Tbx5, Nkx2.5, and detect he histoneacetylation level both in the total chromatin and the promoter regions ofGATA4, MEF2C and Tbx5, and the expression of histone deacetylase(HAT) subtypes p300and GCN5,and test our hypothesis that Smad4is akey factor in the regulation of histone acetylationMethods(1) Consreucting three lentiviral short hairpin interference RNA vectorstargeted Smad4(Lv-Smad4) in H9c2cells,then transfected H9c2cells to establish stably Smad4knockdown cell lines and detect Smad4proteinexpression by Western-blotting, choose the most efficiency one.(2) Lv-Smad4and negative control (no-load lentivirus) weretransfeted H9c2cells. Real-time quantitive PCR were used to detect theexpression of cardiac core transcription factors GATA4, NKX2.5, MEF2c,Tbx5, and HATs subtype p300and GCN5,Western-blotting were used todetect the totle acetylated histone H3(acH3), and ChIP-Real-Timequantitive PCR were used to detect the histone H3acetylation levels in thepromoter region of GATA4, Nkx2.5, MEF2c and Tbx5.Results(1) The expression of Smad4protein was significantly decreased of86.8%compared with the control groups.(2) The mRNA expression level of cardiac core transcription factorsGATA4, Nkx2.5were decreased in the Smad4knockdown group comparedwith control groups (P <0.05), However,MEF2c Tbx5and HATs subtypesp300and GCN5were not changed.(3) The histone H3acetylation level in the promoter region of GATA4,Nkx2.5,but not MEF2c and Tbx5,were reduced in the Lv-Smad4groupcompared with the control groups,while the whole chromatin histone H3acetylation level was not changed. Conclusions(1) We constructed lentiviral short hairpin interference RNA vectorstargeted Smad4in H9c2cells successfully.(2) The histone H3acetylation level in the promoter region of GATA4and Nkx2.5may lead to reduced mRNA expression, and reduced levels ofSmad4expression may be one of the mechenism of reduced histone H3acetylation level in the promoter region of GATA4, Nkx2.5. Part2: Role of Smad4mediated BMP2signaling pathwayon the cardiac core transcription factors and histone H3acetylation in H9c2cellsObjectiveBlocking Smad4gene expression based on the activation of itsupstream signaling molecules BMP2, to observe the changed expression ofdownstream genes and the histone H3acetylation level,to test ourscientific hypothesis that Smad4mediated the regulation of histone H3acetylation by BMP2in H9c2cells.Methods(1) H9c2cells transfected with adenovirus AdBMP2, total mRNAwere extracted after24h,48h,72h,detect GATA4, Nkx2.5expression toscreen the appropriate time.(2) AdGFP adenovirus AdBMP2were transfected H9c2cells (Blankgroup), negative control group (NC group) and Smad4interferencelentivirus group (Lv-Smad4group), to detect the mRNA expression ofcardiac core transcription factor GATA4, MEF2c, Nkx2.5, Tbx5, and HATssubtypes p300and GCN5by Real time quantitive PCR.(3) Detecting the total histone H3acetylation levels in H9c2cells.(4) To detect the histone H3acetylation levels in the promoter region of GATA4, MEF2C, Nkx2.5and Tbx5.Results(1)AdBMP2transfected H9c2cells at24h,48h and72h, themRNA expression of GATA4and Nkx2.5were significantly higher thanthe control group (P <0.05), and peaked at48h after transfection.(2) The mRNA expression of GATA4, Nkx2.5and HATs subtype p300were decreased (P <0.05) in AdBMP2transfected Smad4knockdnow H9c2cells compared with AdBMP2transfected group,while MEF2c, Tbx5wasnot affected. The GCN5mRNA levels were not changed amongthedifferent groups.(3) Transfeted with AdBMP2,the total histone H3acetylation levelwas incresed compared with the blank control group,however,it decreasedwhen simultaneously treated with Lv-Smad4(P <0.05).(4) The histone H3acetylation level in the promoter regions ofGATA4, Nkx2.5,but not MEF2c,Tbx5， were decreased in AdBMP2transfected Smad4knockdnow H9c2cells compared with AdBMP2transfected group.Conclusions(1) BMP2can promote the expression of cardiac transcription factorGATA4, MEF2c, Nkx2.5and Tbx5.(2) BMP2can improve the overall level of histone H3acetylation in H9c2cells, while the knockdown of Smad4inhibited the ability of BMP2upregulating the histone H3acetylation levels, suggesting that Smad4iscritical for histone modifications induced in BMP2.(3) BMP2can increase the mRNA expression of GATA4, MEF2c,Nkx2.5, Tbx5by increasing the histone H3acetylation level in thepromoter regions,and also can upregulate HATs subtype p300.However,the mRNA expression of GATA4, Nkx2.5and HATs subtype p300weredecreased (P <0.05) in AdBMP2transfected Smad4knockdnow H9c2cellscompared with AdBMP2transfected group, while the histone H3acetylation in the promoter regions of MEF2c and Tbx5were notchanged,indicating that HATs subtype p300was involved in theregulation of transcription factor GATA4, Nkx2.5and histone H3acetylation by BMP2, and there maybe exist other molecules mediated thisprocess. Part3: Role of Smad4mediated TGF-β2signalingpathway on the cardiac core transcription factors and histoneH3acetylation in H9c2cellsObjectiveBlocking Smad4gene expression based on the activation of itsupstream signaling molecules TGF-β2, to observe the changed expressionof downstream genes and the histone H3acetylation level,to test ourscientific hypothesis that Smad4mediated the regulation of histone H3acetylation by TGF-β2in H9c2cells.Methods(1) H9C2cells treated with different concentrations (5μg/L,10μg/L,20μg/L,40μg/L) of human recombinant transforming growth factor(rhTGF-β2)，detected the mRNA expression of TGF-β2downstream genesGATA4,MEF2c after treated24h,48h,72h, screening the appropriateconcentration and time point.(2) Treated with rhTGF-β2in H9C2blank group, negative controlgroup and Lv-Smad4group respectively, detected the mRNA expression ofGATA4, MEF2c, Nkx2.5, Tbx5and HATs p300, GCN5by Real timequantitive PCR.(3) Detecting total histone H3acetylation levels in H9c2cells by Western blot.(4) Detecting the histone H3acetylation levels in the promoterregions of GATA4, MEF2c, Nkx2.5, Tbx5by ChIP.Results(1) Compared with the control group, the TGF-β2treatement group,the mRNA expression of GATA4and MEF2c significantly increased (P<0.05), and peaked at48hours,20μg/L.(2)The mRNA expression of GATA4, Nkx2.5but not MEF2cdecreased (P <0.05) in Lv-Smad4transfected and TGF-β2treated cellscompared with TGF-β2treatment group. While the mRNA expression ofTbx5, HATs subtype p300and GCN5showed no significant changesamong the different treatment groups.(3) Treated with TGF-β2,the total histone H3acetylation level wasincresed compared with the blank control group,however, it decreasedwhen simultaneously treated with Lv-Smad4(P <0.05).(4) The histone H3acetylation level in the promoter regions ofGATA4, Nkx2.5but not MEF2c were decreased in Lv-Smad4transfectedand TGF-β2treated cells compared with TGF-β2treatment group, andTbx5was not changed in all of groups.Conclusions(1) TGF-β2may promote the expression of cardiac core transcription factor GATA4, MEF2c, and Nkx2.5,and maybe has no effect on thewxpression of Tbx5,HATs subtype p300,and GCN5.(2) TGF-β2can improve the overall histone H3acetylation levels,andit can be inhibited by the Lv-Smad4transfected,suggesting that Smad4playa key role in the histone modifications by TGF-β2.(3) The histone H3acetylation level in the promoter regions ofGATA4,Nkx2.5but not MEF2c,were upregulated by TGF-β2, which can beinhibited when simultaneously transfected with Lv-Smad4,indicating thatSmad4was essential for the upregulation of histone acetylation level byTGF-β2induced, in addition,there maybe still exist other moleculemediated TGF-β2to regulate the histone modification. |
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