The Effect Of SIRT1-△AExon8 And SIRT1-FL On 293T Cells Injury Model Induced By H₂O₂

Oxidative stress is fundamental to the occurrence and development of many diseases. The cell injurand carcinogenic effects induced by oxidative stress have a close relationship with many organ damage and tumor disease. Oxidative stress can produce reactive oxygen species(ROS). ROS includes all kinds of oxygen free radicals, the main member is H2O2. H2O2 leads to the destruction of proteins and lipids, especially DNA in the mitochondrial and genome, thereby induces apoptosis or necrosis. H2O2 could effectively improve the levels of intracellular oxidative stress, So it is widely used to establish cellular oxidative stress damage model.SIRT1(silent information regulator 1) is the first member of Sirtuin protein family discovered in mammals, belongs to histone deacetylases(HDACs) Ⅲ. It affects different downstream molecules, such as p53, PGC-1α, PCAF, FOXO, P300 through deacetylation, thus regulates different process of gene transcription, cell cycle regulation, energy metabolism and cell apoptosis and so on.Numerous studies show that SIRT1 may affect cell survival due to the role of regulating oxidative stress. It is known that SIRT1 protect against oxidative stress in a variety of oxidative stress related diseases. But there are also studies points out that activating SIRT1 may aggravate the injury of oxidative stress and plays an negative role on cell survival. Therefore, most research has confirmed that SIRT1 involves in regulation of cell damage induced by oxidative stress, but there is no consistent theory about the role of SIRT1 during oxidative damage.Lynch’s study reported that human and mice SIRT1 existed a novel alternative splicing isoform(SIRT1-ΔExon8). The SIRT1-ΔExon8 was the first reported SIRT1 splice variant, in which the 558 bp region of exon 8 was precisely excluded. Comparing with SIRT1-Full-Length(SIRT1-FL), the activity of the deacetylases of SIRT1-ΔExon8 decreased. And SIRT1-ΔExon8 is more sensitive to extracellular stress than SIRT1-FL. For example, SIRT1-ΔExon8 expression increased in dose-dependent after UV irradiation, while the change of SIRT1-FL is not obvious. These results have shown that SIRT1-ΔExon8 has distinct characteristics and functions with the SIRT1-FL.Therefore, in this study, we used 293 T cells induced by H2O2 to establish oxidative stress damage model, to observe the role of the two different forms of SIRT1. This study provides a new perspective for different functions of SIRT1, and may provide a reasonable explanation for the contradictory conclusions about SIRT1 under the same stress injury. Objectives:1. To explore the role of SIRT1-ΔExon8 and SIRT1-FL in cell damage model induced by H2O2 by detecting the expression level of SIRT1-ΔExon8 mRNA and SIRT1-FL mRNA.2. To clarify the different roles of SIRT1-FL and SIRT1-ΔExon8 by observing the effect of overexpression SIRT1-FL and interference SIRT1-ΔExon8 on cell damage induced by H2O2. Methods:In the first part, there were two groups: normal control group(H2O2: 0 μM) and experimental groups(H2O2: 50 μM, 100 μM, 200 μM, 400 μM, 800 μM). All groups were treated with different concentrations of H2O2 for 3 hours respectively. Morphologic characteristic of the cells were observed by optical microscope. Cytotoxicity index was detected by CCK-8 assay and LDH release assay. Apoptosis rate and intracellular reactive oxygen species(ROS) level were determined by flow cytometry. The expression of SIRT1-FL and SIRT1-ΔExon 8 mRNA were detected by RT-PCR.In the second part, there were four groups: normal control group, blank plasmid group, SIRT1-ΔExon8 interference group and SIRT1-FL overexpression group. Recombinant vectors were constructed by phMGFP. Cells were transfected with blank plasmid, over-expression plasmid and shRNA plasmid. All groups were induced with H2O2 for 3 hours. Then detected the toxicity effects, apoptosis and intracellular ROS level. Results:1. Different concentrations of H2O2(50 μM, 100 μM, 200 μM, 400 μM, 800 μM) caused decreasing of cell viability, increasing of LDH release, increasing of cell apoptosis rate and intracellular ROS in dose-dependent. With the increasing of H2O2 concentration, SIRT1-FL mRNA expression decreased in dose-dependent, while SIRT1-ΔExon8 mRNA expression increased in dose-dependent.2. Overexpression of SIRT1-FL and interference SIRT1-ΔExon 8 can antagonize the toxicities induced by H2O2(400 μM), including increased cell viability, decreased LDH leakage, decreased cell apoptosis rate and intracellular ROS level. Conclusions:1. The expression of SIRT1-FL mRNA decreased gradually, while SIRT1-ΔExon8 mRNA increased with increasing H2O2 concentration. As a result, cell viability decreased gradually and the number of apoptotic cell increased. It indirectly suggests that SIRT1-ΔExon8 may promote cell damage induced by H2O2, and SIRT1-FL may inhibit the cellular oxidative stress injury.2. Overexpression of SIRT1-FL and interference of SIRT1-ΔExon8 can antagonize cytotoxicity and apoptosis induced by H2O2. It further proves SIRT1-ΔExon8 may promote cell oxidative damage while SIRT1-FL may play the opposite role.3. Overexpression of SIRT1-FL and interference of SIRT1-ΔExon8 can reduce the level of intracellular ROS, suggested that SIRT1-ΔExon8 may promote cell oxidative damage by increasing intracellular ROS, while SIRT1-FL may play the role of anti-oxidative damage by decreasing intracellular ROS.