Preliminary Study On The Role Of Sirtuin 3 In Molecular Pathogenesis Of Non-small Cell Lung Cancer

Background: Lung cancer is the leading cause of cancer-related death worldwide,while non-small cell lung cancer(NSCLC)accounts for almost 80%-85% of lung cancer cases,whose effective treatment contributes enormously to enhancing health of human beings.Traditional therapies like surgery,chemotherapy and radiotherapy have shown limited efficacies due to patient adaption,drug-resistance and adverse effects.In recent years,targeted-therapy based on the driving mutations has achieved considerable progress in NSCLC.However,patient insensitivity and resistance to these therapeutics have compromised their clinical application,raising the necessity of exploring the detailed mechanisms underlying NSCLC progression and drug resistance.Protein acetylation and deacetylation play a key role in diverse biological processes.The critical deacetylase sirtuin3(SIRT3)could regulate the function of important proteins via deacetylation,thus determining numerous bio-activities and influencing the outcome of many diseases.Nevertheless,SIRT3 play a contradictory role in NSCLC given the heterogeneity of neoplastic cells,and the differential expression of environmental and other complicated factors.Therefore,exploration on SIRT3-function in NSCLC and the underlying molecular mechanisms will provide novel insights into carcinogenesis and practice treatment.Purposes: to investigate the effect of SIRT3 on malignant progression of NSCLC and the underlying molecular mechanisms.Methods:(1)70 paired NSCLC tumor tissues and corresponding normal tissues with clinciopathological data were collected and subjected to measurement of the different protein and mRNA expression pattern of SIRT3 between NSCLC tumor tissue and normal tissue via immunohistochemistry(IHC),western blot(WB)and real time quantitative PCR(qPCR).(2)Quantitative statistics was used to analyse the relationship between SIRT3 expression and clinicopathological parameters of NSCLC,and the correlation between SIRT3 expression and malignant proliferative biomarker Ki-67,or the activation of the oncoprotein Akt.(3)Small interfering RNAs(siRNAs)were used to build SIRT3 knockdown NSCLC model,while plasmid expression vector was used to establish SIRT3 overexpression NSCLC model(H520: SIRT3-;SW900: SIRT3-;H520: SIRT3+).Then these models were used to estimate the influence of SIRT3 level upon phosphorylation of Akt via WB,and to investigate the interaction of SIRT3 and Akt via laser confocal,co-immunoprecipitation(co-IP)and WB.(4)IHC was used to investigate the status of PTEN,SIRT3 and p53 expression level in NSCLC tumor tissues,and WB was used to explore the expression file of PTEN,SIRT3 and p53 in NSCLC cell lines.According to the results above,SIRT3 knockdown or overexpression under different PTEN level cell models were achieved by siRNAs and plasmid expression vector(H520: PTEN-/SIRT3-,PTEN+/SIRT3-;A549: PTEN-/SIRT3+,PTEN+/SIRT3+).(5)These above models were used to investigate the effect of SIRT3 upon p53 level under the cellular context of different PTEN expression and SIRT3 participation in the ubiquition proteasome pathway dependent degradation of p53 via WB,co-IP and small inhibitors of the ubiquition proteasome pathway.(6)Laser confocal,co-IP and WB were used to investigate the interaction relationship between SIRT3 and p53,and the same technologies were used to explore the deacetylation upon p53 via SIRT3 in the above(4)models.Results:(1)SIRT3 was significantly upregulated in NSCLC tissues compared to normal tissues.(2)SIRT3 expression was correlated to the prognosis of NSCLC,where patients of SIRT3 high expression had a shorter survival time;SIRT3 was differentially expression in NSCLC with different pathological types,in which squamous carcinoma had highest expression;SIRT3 expression had no relationship with other clicopathological parameters.(3)SIRT3 expression was positively related to malignant proliferative biomarker Ki-67 and phosphorylated Akt in NSCLC tissues.SIRT3 could co-localized and co-coprecipitated with Akt in NSCLC cell lines.SIRT3 could enhance the levels of phosphorylated Akt.(4)In NSCLC tissues,PTEN was down-regulated while SIRT3 and p53 were coordinately up-regulated and down-regulated,respectively.In PTEN-deficient but not PTEN-normal NSCLC cell lines,SIRT3 could enhance p53 degradation via ubiquition-dependent proteasome pathway.(5)In PTEN-deficient NSCLC cell lines,SIRT3 could deacetylate p53 at lysine 320/382,which may correlated with p53 stability.Conclusions:(1)STRT3 was up-regulated in NSCLC tissues and was positively correlated with Ki-67,and was related to a poorer prognosis.SIRT3 expression was related to pathological types of NSCLC,but had no evident relation with progression and other malignant hallmarks,which may result from heterogeneity,genetic difference and universality of SIRT3 targets.All these indicated the complicated mechanisms of SIRT3 in NSCLC.(2)Phosphorylation of Akt was the center to promote malignant progression,and deacetylation of Akt could significantly influence its phosphorylation.SIRT3 was the pivotal cellular deacetylase,which suggested that SIRT3 could promote phosphorylation of Akt via deacetylation,leading to malignant progression of NSCLC.In this research,SIRT3 expression was positively related with p-Akt level in NSCLC tissues;SIRT3 could promote phosphorylation activation of Akt in NSCLC cell lines;SIRT3 co-localized and co-coprecipitated with Akt in NSCLC cell lines.All these results demonstrated partly that SIRT3 could regulate NSCLC via mediation of Akt.(3)Deacetylation of p53 could inhibit its tumor suppressive function while PTEN deacetylation promotes its effects as anti-oncogene,which indicates that deacetylation might play different function according to genetic backgrounds.In most NSCLC tissues,PTEN was down-regulated while SIRT3 was up-regulated and p53 was down-regulated,which suggests that SIRT3 might exhibit its oncogenic effect by inhibiting p53 level in PTEN-deficient NSCLC.Meanwhile,we demonstrasted that SIRT3 could enhance p53 degradation via the ubiquition-dependent proteasome pathway in PTEN-deficient NSCLC.Furthermore,we found that SIRT3 could deacetylate p53 at lysine 320/382,which possibly facilitates p53 ubiquition.Therefore,these results indicated that SIRT3 could promote NSCLC pathogenesis via regulation of p53 in the context of PTEN deficiency.