Biological Effect And Mechanism Of SIRT1 In Lung Adenocarcinoma

Lung cancer, as one of the tumors with the highest incidence and fatality rate, poses a severe threat to human health. So far, surgery, chemotherapy and radiotherapy are still the mostly applied counter measures for the clinical management of lung cancer. With the development of surgery-based multi-discipline comprehensive treatment, substantial progress has been making about the treatment for lung cancer in the past decade. However, patients with lung cancer are faced with poor prognosis with a 15% five-year survival rate. Precision medicine is a prevailing trend leading current tumor treatment, the key to which is to identify effective biomarkers and distinguish target groups so that anti-tumor treatment can be customized for specific groups and gain higher efficiency. Therefore, it is essential to identify new effective molecular markers and potential treatable targets.Brand new understanding was obtained after the completion of Human Genome Project. It is safe to conclude that the lung adenocarcinoma and lung squamous carcinoma are two different diseases in the molecular and gene level. Growing evidences suggested that it may be more likely to find biomarkers in lung adenocarcinoma, and may be benefit from it.Silent information regulator 1(SIRT1) is a silent information regulator 2 related enzyme in mammals, holding similar functions with NAD-dependent histone deacetylase. SIRT1 could regulate not only histones, but many other genes and proteins related to cell aging, apoptosis, differentiation and oncogenesis.Previous studies show that SIRT1 expression level is closely related with tumors and is implicated as a potential target for anti-tumor treatment. Moreover, SIRT1 is thought to have entirely different functions in different tumors. On the one hand, SIRT1 may act as a tumor promoter by downregulating p53 and other tumor inhibitors. On the other hand, SIRT1 also has anti-tumor function as it suppresses inflammation and oncogene transcription factors, and helps maintain gene stability. Recently, a small number of studies concerning SIRT1 function in lung cancer reached different conclusions, leaving this topic in controversy. Thus it is necessary to investigate the effect of SIRT1 on biological behavior and its possible molecular mechanisms in the lung adenocarcinoma, in hope of providing theoretical and experimental bases for further illustration of SIRT1 function in tumorigenesis and tumor development.Objection:1.To observe SIRT1 expression in lung adenocarcinoma, analyze its correlation with clinical characteristics and clinical values2. To study the function of SIRT1 on lung adenocarcinoma biological effect3.To discuss the possible molecular mechanism of SIRT1 influence on lung adenocarcinoma biological behaviorMethods:1. SIRT1, Survivin and VEGF expression in lung adenocarcinoma and corresponding noncancerous tissues(NCTs) was detected by immunohistochemistry using a tissue microarray. ALK and EGFR expression was detected by fluorescent in situ hybridization. We statistically analyzed the correlation between SIRT1 and lung adenocarcinoma clinical pathological characteristics in order to reveal the potential clinical values of SIRT1.2. The effect of SIRT1 inhibitors(nicotinamide and toxoflavin) on lung adenocarcinoma A549 cell proliferation was evaluated by CCK-8, cell apoptosis by APC-Annexin V and flow cytometry, and cell migration by wound healing assay.3. The m RNA profile of lung adenocarcinoma A549 cells and those co-cultured with SIRT1 inhibitors(nicotinamide and toxoflavin) was detected using whole human oligonucleotide microarray.4. Bioinformatics technique was applied to conduct pathways analysis and functional annotation on differentially expressed genes, among which those with significantly elevated expression levels in nicotinamide and toxoflavin groups were chosen as SIRT1 downstream target genes. Correlation between the chosen target genes and SIRT1 was confirmed by TCGA database.5. Target genes expression in lung adenocarcinoma A549 cells co-cultured with nicotinamide was detected by RT-PCR and western blotting in m RNA and protein level to form a preliminary assumption concerning molecular mechanism of how SIRT1 affects the malignant biological behaviors of lung adenocarcinoma.Results:1. Correlation between SIRT1 expression in lung adenocarcinoma and clinical pathological characteristicsIn the present study, we found the SIRT1 expression was significantly higher in lung adenocarcinoma than NCTs. SIRT1 expression was significantly associated with high pathological stage, while no difference was found in sex, age, TNM stage, ALK and EGFR expression. SIRT1 expression was related to overall survival(OS) and associated with poor prognosis. Study showed that SIRT1 was significantly related to VEGF expression but unrelated to Survivin expression.2. The influence of SIRT1 on lung adenocarcinoma biological effectThe present study revealed as the concentration and time of toxoflavin use climbed up, its inhibition efficiency on lung adenocarcinoma A549 cell proliferation increased, forming a dose and time dependent relationship. Annexin V-FTTC/PI cell apoptosis testing demonstrated a similar dose and time dependent relationship between toxoflavin and cell apoptosis Cell migration in toxoflavin group was significantly weaker than the control group.Similar but slightly different results were obtained in the nicotinamide groups. As the time of nicotinamide use climbed up, its inhibition efficiency on lung adenocarcinoma A549 cell proliferation increased. Within 24 hours a dose dependent relationship was also observed. Annexin V-FTTC/PI cell apoptosis testing demonstrated that as the concentration and time of nicotinamide use increased, lung adenocarcinoma A549 cell apoptosis rate was elevated, presenting a statistically significant time and dosage dependent relationship3. The possible molecular mechanism of SIRT1 influence on lung adenocarcinoma biological behaviorm RNA microarrays profile revealed that, compared to lung adenocarcinoma A549 cell, toxoflavin group contains certain genes whose expression is increased(745 genes) or decreased(803 genes) more than two times. m RNA microarrays profile revealed that, compared to lung adenocarcinoma A549 cell, nicotinamide group contains certain genes whose expression is increased(2505 genes) or decreased(745 genes) more than two times. Analysis concerning the shared differentially expressed genes between nicotinamide and toxoflavin groups revealed increased expression of 159 genes and decreased expression of 65 genes in both groups. Providing that SIRT1 was correlated with lung adenocarcinoma malignant biological behavior, further analysis discovered that Bcl-2 associated transcription factor 1(BCLAF1) may be the downstream target gene of SIRT1.Analysis based on the TCGA database proved simultaneous expression of SIRT1 and BCLAF1 in lung adenocarcinoma tissue, whose m RNA expression are correlated with each other.Using RT-PCR technique, we discovered that the m RNA expression of BCLAF1 gene in lung adenocarcinoma A549 cells co-cultured with nicotinamide was significant higher as compared with the control group. Besides, western blotting illustrated high expression of BCLAF1 protein.Conclusion:The present study proves that SIRT1 expression in lung adenocarcinoma is significantly higher than that in the NCTs. SIRT1 expression is also associated with OS and works as a significant prognostic indicator for lung adenocarcinoma patients. SIRT1 inhibitors can suppress lung adenocarcinoma A549 cell proliferation and migration, and promote cell apoptosis. Using human whole genome oligonucleotide array, BCLAF1, a specific gene differentially expressed in A549 cell is selected by bioinformatics technique. RT-PCR and western blotting show significantly higher BCLAF1 expression in the m RNA and protein level in lung adenocarcinoma A549 cells co-cultured with SIRT1 inhibitors. Taken together, our findings suggest that SIRT1 may be a tumor promoter of lung adenocarcinoma, probably through the target regulation of BCLAF1. These findings provide theoretical and experimental bases for further study concerning the molecular mechanism of SIRT1.