The Research Of Over-expressed Sirtuin 3 On Small Cell Lung Cancer Cell Line NCI-H446 In Vivo And Vitro

Lung cancer is a malignant disease with high malignancy,and small cell lung cancer is very aggressive and rapid growth.Sirtuin 3(SIRT3)is an NAD+ dependent deacetylase,mainly in the mitochondria.SIRT3 plays an important role in tumorigenesis,and may play a role in tumor suppressor gene or oncogene according to cell type and tumor type.It is reported that p53 may be a target of SIRT3.P53 often mutated in small cell lung cancer.Because most of the chemotherapy dr?gs can induce DNA damage,and can activate the p53 protein,and p53 gene mutation wo?ld have a negative impact to this treatment.Stability of mutant p53 is stronger than wild type p53.Mutant p53 can play the role of oncogenes,so it is a problem that we need to solve the cancer gene function of mutant p53.Earlier stage work of our laboratory found that SIRT3 and mutant p53 expression in patients with lung cancer present negative correlation,and SIRT3 may cause the expression of mutant p53 decreased,so we spec?lated that SIRT3 modification after translation,may affect the mutant p53 and stability,and eventually lead to the degradation of mutant p53..1.Objective:To explore the role and mechanism of SIRT3 in NCI-H446 of small cell lung cancer :(1)The inhibitory effect of SIRT3 on lung cancer;(2)The effect of SIRT3 on the stability of mutant p53.;(3)The mechanism of SIRT3 influence on the stability of mutant p53.C?ltured NCI-H446 cells were divided into CON group,NC group(transfection of empty plasmid)and SIRT3 group.The morphology of cells was observed under an optical microscope.MTT assay are used to detect cell survival rate;Annexinv-FITC flow cytometry detection of apoptosis and JC-1 detection of mitochondrial membrane potential;Western Blot was used to detect cleaved-Caspase9,Bcl-2,Bax,McL-1,Bid,cleaved-Caspase3 and other apoptotic proteins;RT-PCR was used to detect the apoptosis related gene expression of bcl-2,Bax,caspase-3 and p53.MTT assay was added to the necrotic apoptotic inhibitor,Nec-1.Western Blot was used to detect the expression level of apoptotic necrosis associated protein caspase 8,RIP1 and MLKL.C?ltivate NCI-H446 cells,divided them into CON group,the NC group(transfection of empty plasmid)and SIRT3 group,Western Blot experiments was used to transfection SIRT3 and mutant p53 protein expression.After transfection of SIRT3,the half-life of mutant p53 was detected with CHX.The binding capacity of mutant p53 and ubiquitin were examined.The cells were divided into five groups: CON group,NC group,SIRT3 group,MG132 group,SIRT3+MG132 group,and the expression of mutant p53 was detected by Western Blot.The model of nude mice lung cancer transplantation tumor was constructed,and the nude mice were divided into PQ group and SIRT3 group.With tumor targeting attenuated salmonella as a carrier,carry SIRT3 plasmid,mice abdominal cavity injection method of treatment,observation and calc?lation of the tumor and tumor growth index,HE dyeing observation organization form,the immune histochemical method to detect SIRT3,the expression of mutant p53;Western Blot and RT-PCR were used to detect the expression of SIRT3,mutant p53,apoptosis-related proteins and gene levels 3.Res?ltsThe NCI-H446 cells transfected with SIRT3 were found to have a large n?Mber of dead cells in the light microscope,and the MTT res?lts showed that SIRT3 inhibited cell growth(P < 0.05).Annexin v-FITC flow cell n?Mber showed that SIRT3 overexpression co?ld promote apoptosis in cells relative to CON group(P < 0.05).Jc-1 staining showed that SIRT3 co?ld reduce the mitochondrial membrane potential(P < 0.05)in the CON group.Western Blot res?lts showed that SIRT3 co?ld increase apoptotic proteins such as Caspase 8,caspasase 9,Bax,Bid and Cleaved Caspase3,and down-reg?late Bcl-2 and McL-1 apoptotic inhibition protein(P < 0.05).RT-PCR res?lts showed that p53,Bax and Caspase 3 were up-reg?lated,and Bcl-2 was down-reg?lated(P < 0.05),and the gene level proved that SIRT3 caused cell apoptosis.MTT assay showed that SIRT3 can cause partial necrosis of cells(P < 0.05).Western Blot detection of necrotic apoptosis related protein RIP1,MLKL,p-MLKL expression was up-reg?lated(p < 0.05).Western Blot detected the expression of SIRT3 after the expression of SIRT3,and the expression of mutant p53 decreased,and p53 expression was up-reg?lated(P < 0.05).RT-PCR res?lts showed that p53 expression was up-reg?lated.(P < 0.05).In order to explore the mutant p53 protein degradation,Western Blot detection after joining proteasome inhibitor MG132 the expression of mutant p53 protein level,found that the mutant p53 occurs the proteasome degradation.CHX detected the half-life of mutant p53,and the res?lts showed that compared with the CON group,MDM2 and mutant p53 half-life significantly shortened,s?ggesting that SIRT3 had a destabilizing effect.The res?lts of immunoprecipitation showed that the combination of mutant p53 and ubiquitin increased,demonstrating that SIRT3 co?ld reg?late the ubiquitination degradation of mutant p53.In vivo experiments,thro?gh HE staining proved that SIRT3 can inhibit the growth of lung transplantation tumor in mice,Western Blot and RT-PCR respectively from the level of protein and gene proved that SIRT3 can reduce mutant p53 expressed in tumor,thus inhibiting tumor development.4.Conclusion(1)SIRT3 can inhibit the growth of lung cancer cell NCI-H446 and induce apoptosis and necrotic apoptosis.(2)SIRT3 overexpression can also change the stability of MDM2 and mutant p53.(3)SIRT3 overexpression may induce the ubiquitination degradation of mutant p53 thro?gh MDM2 mediated.