| Human medical ultimate goal is to overcome the aging. It not only leads to the structure changes and function decline of organs and tissues, but also gives rise to multiple senescent-related diseases, like diabetes,Parkinsonism, Alzheimer’s disease and some tumors. Now the researches on the senescence pathways were far to be clear. SWI/SNF complex is a protein family that remodels the contracture of chromosomes in an ATP-dependent manner. The SWI/SNF complex is composed of multiple subunits, transcriptional activates or inhibits the expressions of targets downstream. Human SWI/SNF complex has a function in cell cycle process,like DNA replication,extension,splicing, and DNA damage repair. Recently there are some studies reveled that the catalytic subunit of SWI/SNF complex, BRG1 was also involved in regulation of cell senescence, while few studies on the others subunits in this part. In our research, hydrogen peroxide(H2O2, 150150μM, 1 h) was used to induce the senescent models of Ha Cat and GLL19 cells, and then screen the subunits of SWI/SNF that differentially expressed in the senescent cells. The recombinant expression vectors and siRNAs were used to overexpression or knockdown the three subunits,and then using SA-β-gal, MTT assay, Flow cytometry and Western blot to study the regulation of SWI/SNF subunits on senescence and the possible mechanism. The mainly results were as follows,①H2O2(150μM) was used to treat the cells for 1 h, and cultured for 4 days. Cell morphological observation,SA-β-gal staining, MTT assay, and flow cytometry were used to confirm the senescence state. The results showed that, after 4 days treatment, the cells were bigger and plate, fuzzy boundary,most of the cells were staining positively, the cell proliferation were suppressed, and the cell cycles were arrested in G2 phase.② Semi RT-PCR was used to screen the differential expression subunits of SWI/SNF complex, and then Western Blot was used to confirm the up-regulation of these subunits. The results showed that, after H2O2 treatment, BAF57, BAF60 a, SNF5 and BRG1 were up-regulated in the senescent cells, since there already have been the studied on the BRG1 involved in senescence, BAF57, BAF60 a and SNF5 were selected as candidates which would been studied in the further experiments.③ Recombinant vectors pc-BAF57, pc-BAF60 a and pc-SNF5 were constructed successfully and transfected into the cells. Semi RT-PCR and Western Blot were used to confirm the over-expression of the three subunits. Then SA-β-gal staining, MTT assay, and flow cytometry were used to detect the responses of the cells after transfection. The results showed that, the recombinant vectors were expressed in the cells. There were no obvious positive staining cells of SA-β-gal, while the cell proliferation were significantly suppressed, and the cell cycles were arrested in S phase.④ SiRNAs were used to knockdown the expression of BAF57, BAF60 a, SNF5, and then the cells were treated by H2O2, SA-β-gal and flow cytometry were used to detect the responses of the cells that were pretreated by siRNAs. The results showed that, pretreatment by siRNAs, the SA-β-gal staining positive cells were decreased compared to the cells only treated by H2O2, the cell cycle arrest was reduced. Which meant knockdown of the three subunits led to some cells bypass the senescence pathway.⑤ Semi RT-PCR was used to detect the expression of senescent-related factors after transfection of pc-BAF57, pc-BAF60 a and pc-SNF5, and then Western Blot was used to detect the expression of senescent-related factors after knockdown the subunits and H2O2 treatment. The results showed that, after transfection of pc-BAF57,pc-BAF60 a and pc-SNF5, p53,p21,p16 and pRB were up regulated. Western Blot results showed that, compared to the H2O2 treatment group, p53,p21,p16 and pRB were obviously down regulated in the pretreatment groups. The results reveled that BAF57, BAF60 a, SNF5 promote the cell senescence involved in p53/p21 and p16/pRB pathways. |
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