SIRT1 Protein Expression And Gene Status In DLBCL And Its Effects On Biologic Behaviour Of DLBCL Cell Lines

SIRT1 protein expression and gene status in DLBCL and its effects on biologic behaviour of DLBCL cell linesBackground:Diffuse large B-cell lymphoma (DLBCL) is an aggressive malignancy of mature B lymphocytes accounted for 30%-40% ofNon-Hodgkin lymphoma. DLBCL is a heterogeneous tumor, clinically, morphologically and genetically. The molecular mechanisms are largely unknown and need to be further investigated. SIRT1 gene, the closest homologue of the yeast silent information regulator-2(Sir2), is 33kb and located on human chromosome 10q21.3. It encoded a nicotinamide adenine dinucleotid(NAD+)-dependent deacetylase which is a cellular growth-regulated protein composed of 747 amino acids. It had been shown to deacetylate a wide range of histone substrates and non-histone substrates including P53, FOXO family proteins, DNA repair protein Ku70, nuclear factorκB. SIRT1 was called longevity gene as it had broad biological functions in growth regulation, stress response and maintenance of genomic instability. However, the antiapoptotic effects of SIRT1 may paradoxically increase the risk of cancer. Transcription factor FOXO3a was an important target protein of SIRT1 which have effects on cellular proliferation and apoptosis. All of the functions may contribute to the development of cancer. SIRT1 was shown to highly express in a number of tumour types such as prostate cancer, skin cancer, breast cancer and so on. And targeted inhibition of SIRT1 in vitro and in vivo can effectively limit cancer progression and induce apoptosis. However, in DLBCL there was still rare both at home and abroad.Objective:To observe SIRT1 expression and gene status in DLBCL and to investigate the relationship with their clinicopathologic significance. To study the effects of lentivirus-mediated stable RNAi targeting SIRT1 gene on cellular biologic behavior in DLBCLcell lines.Methods:1. A306 dot tissue microarray consisting of 138 cases of DLBCL and 20 cases of RLH was made. All the paraffin-embedded tissues were retrieved from the files of the Department of Pathololy, Cancer Hospital, Fudan University between1995 and 2004 and all the histo logical slides were reviewed for confirmation of pathologic diagnosis by two professional pathologists. The diagnostic criteria refers to"WHO Classification:Tumors of Haematopoietic and Lymphoid Tissues(2008)". The immunophenotypic and clinical features were collected and analyzed.67 patients were followed up for 2-120 months until December,2009.2. Immunohistochemical analyses of SIRT1 and FOXO3a were performed in DLBCL compared withRLH. The relationship between SIRT1 and FOXO3a expression and their clinicopathologic significance were analyzed respectively.3. The locations of BAC (RP11-44L18) included whole SIRT1 gene region were compiled from information archived by the UCSC and the NCBI. Probes were labeled by nick translation with Red-dUTP. The copy number of SIRT1 and CEP-10 were evaluated by fluorescence in situ hybridization (FISH) in the tissue array and lyl, ly8 DLCBCL cells. The correlation between SIRT1 gene copy number and protein expression was analyzed.4. Two DLBCLcell lines, lyl and ly8 were maintained in10%FBS culture medium. Cell Counting Kit-8(CCK-8) was used to detect the cell proliferation status. The expression of SIRT1 and FOXO3a were detected by Westernblotting. The relation between proteins expression and cell proliferation was analyzed respectively. Plasmids pMagic4.0-SIRT1 was constructed, which was combined with pRsv-REV, pMDlg-pRRE and pMD2G to constitute vector system of four plasmids. The lentiviral vector system was transfected into 293T cell to produce EGFP-SIRT1 lentivirus. EGFP-NClentivirus was also produced as a negative control. Lyl and ly8 cells were infected with both EGFP-NC and EGFP-SIRT1 lentivirus. Flow cytometry(FCM) were used to pick clones stably expressing EGFP-NC and EGFP-SIRT1 fragment. SIRT1 protein level was detected by western blotting. CCK-8 was used to assess the cell proliferation status. FCM was used to analyze cell cycle and apoptosis. Western blotting was used to detect FOXO3a, caspase-3, caspase-8 and caspase-9 which might relevant to cell apoptosis.Results:1. Clinical and Morphological features of DLBCL:there are 71 males and 63 females(M:F=1.1:1). The age ranged from9 to 85 years with median age of55 years.80 cases were younger and 56 cases were older than 59 years.86 cases were had at least on lymph node involved, and 51 were extranodal DLBCL. Of them,30 cases were in the gastroenteric tract de nevo. Patients were divided into 1(30),11(42),Ⅲ(27) and IV(6) according to their clinical stages.74 cases were 0-2 scores of the international prognostic index (IPI) and 19 cases were IPI scores of 3-4. There are 47 patients with normal serum LDH and 35 with high LDH. With the TMA algorithm described by Hans et al, 51 (37%) patients had GCB type and 87 patients(63%) had non-GCB types.of DLBCL.2. The results of immunohistochemistry:(1) The positive rate of SIRT1 expression in DLBCL(67.2%) was significantly higher than that in RLH(33.1%) (P<0.01). (2) In RLH, FOXO3a was charactistically expressed in the quiescent cells of the follicular mantle zone and in interfollicular lymphocytes, and only rare cells in the germinal center(GC) were positive. The possitive rate was 90%. But in DLBCL, FOXO3a was uniformly expressed with possitive rate of 81.7%. (3) No difference ofFOXO3a expression was found between RLH and DLBCL, but the strong positive expression rates of FOXO3a in RLH and DLBCL were significantly different(P<0.01). (4) No significant correlation was shown between SIRT1 and FOXO3a expression in RLH. However in DLBCL, SIRT1 and FOXO3a were significantly positively correlated (x 2=7.316,γ=0.237, P0.05). (7) No relationship was found between SIRT1 and prognosis of DLBCL(P>0.05).3. The results of FISH detection:(1) 3 of the 127 DLBCL cases(2.4%) showed SIRT1 gene amplification. And 33 tetrasomy or trisomy(26.0%) and 6 polysomy cases(4.7%) were found. Overall,42 out of 127 DLBCL cases demonstrated SIRT1 gene alteration, either gene copy number increase or amplification, accounts for 33.1%. (2) SIRT1 gene alteration was significantly correlated with SIRT1 protein expression(γ=0.281, P=0.001). (3) No SIRT1 gene alteration was found in 15 RLH. (4) Less than 5% cells showed SIRT1 gene copy number increase in DLBCL cell lines lyl and ly8.4. The results of In Vitro study:(1) SIRT1 expression was much higher at the early phase after transfer of culture of lyl and ly8 cells and decreased at the later phase. (2) Lyl and ly8 cells stably expressing EGFP and EGFP-SIRT1 fragment were successfully picked. SIRT1 contents were obviously decreased by 78%in ly1 cell and 75% in ly8 cell(P<0.05). (3) The results of growth ability by CCK-8 method and cell cycle by flow cytometry showed no significant difference in all these cells. (4) Apoptosis by flow cytometry show significant difference by the effect of SIRT1 gene stably-silencing both in lyl and ly8 cells(P<0.05). (5) SIRT1 silencing up-regulated caspase-3,8 and 9 level in ly8 cell, but had no effect on FOXO3a level in all of the cells. Conclusions:1. SIRT1 expression can be observed more often in DLBCLthan in RLH. SIRT1 may play an important role in the tumorigenesis of DLBCL.2. FOXO3a expression was stronger in RLH than in DLBCLwhich suggest that FOXO3a gene is a putative tumor suppressor gene3. SIRT1 expression are positively correlated with nuclear expression of FOXO3a in overall DLBCL. SIRT1 may be involved in the development of DLBCL though the regulation of FOXO3a expression and its functioa4. SIRT1 may play an important role in de nevo gasterointestinal DLBCL.5. SIRT1 amplification was rare in DLBCL while gene copy number increase was very common, accounts for 1/3 cases. SIRT1 gene alteration significantly contributes to protein expression of SIRT1 in DLBCL.6. SIRT1 silencing induces apoptosis in DLBCL cells, and both extrinsic and intrinsic pathway maybe required which suggests that SIRT1 had anti-apoptosis effects in DLBCL.