The Role Of SETD4 In Inflammation And Proliferation And Apoptosis

14 years ago,Stephen and others first discovered SET(suppressor of variegation,enhancer of zeste and trithora)domain family proteins.One of the common features of the members of the family is a highly conservative SET structure domain.The evolutionarily conserved SET domain(roughly 130 amino acids)was first recognized in Drosophila proteins:Suppressor of variegation(Su(var)3-9),Enhancer of zeste(E(z)),and Trithorax.There is homology between the SET domain and a plant enzyme,Rubisco large subunit lysine methyltransferase(LSMTs).SET-N and SET-C,which contains a lot of activity related amino acid residues,are highly conservative.Between the fields in SET domain,SETD-I can specificity recognition methyltransferase substrates and cofactors.A growing body of evidence shows that the SET-domain containing proteins have histone and nonhistone methyltransferase activity.These proteins also can regulate gene expression,participate in gene transcription inhibition and heterochromosome silencing,and affect the individual development and cell growth cycle.Once SET-domain containing proteins expression dysregulation,will lead to abnormal transformation and proliferation,and even lead to the occurrence of tumor.We can see SET-domain containing proteins play an extremely important role in the process of inflammation and the occurrence of tumor development.And thus became a research hot spot in recent years.SETD4,which also contains highly conservative SET domain structure,is one of the members of the SET family.In eukaryotes,SET domain has catalytic activity,and involved in the regulation of diverse cellular processes.It was presumed that SETD4 have histone and nonhistones methyltransferase activity.Although it has been found in hundreds of proteins in eukaryotic cells,just one tiny part of its function has been Confirmed by experimental study.At present,one of the biggest challenges is how to reveal the biological function of these proteins.So far,there is less research reports about SETD4 protein.As a member of the SET family,SETD4 protein probably has histone or nonhistone methyltransferase activity.The malfunction of SETD4 protein may lead to the dysfunction of body or the development of some diseases.At present about SETD4 protein function is less research reports,the recent article reported that down-regulated the expression of SETD4 can inhibit the proliferation of human breast cancer cell lines.Cell apoptosis or cell proliferation is one of the important characteristics of life.Abnormal cell apoptosis or cell proliferation will cause many diseases and even cancer.Therefore,exploring the role of methylation transferase in cell proliferation and apoptosis plays an important role in elucidating the mechanism of the occurrence of diseases.In addition,in this laboratory prophase research work shows that SETD4 proteins involved in the gene regulation of cytokine induced by bacteria lipopolysaccharide(LPS).However,the molecular mechanism remains to be seen.TLR4 recognized LPS mediated inflammation.We do not know whether SETD4 involved in the inflammation induced by other microbial products.The work will lay the foundation of further analysis of the mechanism of its action.According to these scientific problems,the experiment is divided into the following parts:The first part:Explore the role of SETD4 protein in the inflammatory response.At first,we studied the changes of SETD4 mRNA in RAW264.7 cells with bacterial lipoprotein(BLP)stimulate or polyinosinic acid-polycytidylic acid(Poly(I:C))stimulate;Second,we studied the role of SETD4 protein in inflammatory cytokines released downstream.We used synthetic effective specificity RNA interference fragment down-regulated the expression of SETD4 in RAW264.7 cells,then detected the release of cytokines in cell conditioned medium of RAW264.7 cells with BLP stimulate or Poly(I:C)stimulate by liquidchip.The second part:To investigate the influence on cell proliferation and apoptosis of SETD4 protein in different expression level.Based on the known sequences of setd4 gene in mice,we constructed a eukaryotic expression plasmid,and synthesized an effective specificity RNA interference fragment,so as to get cells overexpression of SETD4 or low expression of SETD4.Then determined the cell proliferation by MTT,and determined the cell apoptosis by flow cytometry,to analyse the influence on cell survival of the level of expression SETD4 protein.To sum up,this thesis has made the experimental results as follows:At first,SETD4 mRNA level increased obviously in RAW264.7 cells after BLP stimulation for 6 h;SETD4 mRNA level fall in RAW264.7 cells after Poly(I:C)stimulation for 1 h,then back to normal after Poly(I:C)stimulation for 2 h.After Poly(I:C)stimulation for 6 h,SETD4 mRNA level increased obviously in RAW264.7 cells,too.Second,inflammatory stimulation can induce the release of cytokines in RAW264.7 cells.Down-reguited expression of SETD4 protein in RAW264.7 cells by siRNA interference,We can see the release of tumor necrosis factor ?(TNF-?)and interleukin-6(IL-6)in cell conditioned medium of RAW264.7 cells with BLP stimulate can obviously decrease;While down-reguited expression of SETD4 protein in RAW264.7 cells by siRNA interference,We can see the release of TNF-a in cell conditioned medium of RAW264.7 cells with Poly(I:C)stimulate can obviously increase;At last,overexpressed SETD4 protein in 3T3 cell by eukaryotic plasmid transfection,we can see the cell proliferation and vitality among differences groups had no statistical significance by MTT test(P>0.05).The cell apoptosis among differences groups had no statistically significant difference by flow cytometry(P>0.05);Down-regulated expression of SETD4 protein in 3T3 cells by siRNA interference,the results of MTT showed that the cell proliferation and vitality among differences groups had no statistical significance(P>0.05),and results of flow cytometry results showed that the cell apoptosis among differences groups had no statistically significant difference(P>0.05);Down-regulated expression of SETD4 protein in RAW264.7 cells by siRNA interference,the results of MTT showed that the cell proliferation and vitality among differences groups had no statistical significance(P>0.05).According to the experimental results,we can get following preliminary conclusions:With different TLR agonist stimulation,the level of SETD4 mRNA will changes.It illustrate that SETD4 protein is likely to be involved in the development process of inflammation reaction.Down-reguited expression of SETD4 protein will affect the release of cytokines.There is different impact of SETD4 protein on the release of cytokines in different types of inflammatory response.Maybe SETD4 protein can selective effect on inflammatory reaction.MTT experiment reported that neither overexpression of SETD4 protein nor down-regulated expression of SETD4 protein does affect cell proliferation;Flow cytometry indicate that neither overexpression of SETD4 protein nor down-regulated expression of SETD4 protein does affect cell apoptosis.It turned out that the level of SETD4 protein expression does not directly affect proliferation and apoptosis in 3T3.SETD4 may not on the apoptosis pathway.In a word,we know next to nothing of SETD4 protein at the present stage.In this study,we discussed the role of SETD4 protein in inflammatory response and the function of SETD4 protein in cell proliferation and apoptosis.But the detailed molecular mechanism remains to be determined.