| Objective:To investigate the role of nicotinamide adenine dinucleotide oxidized form(NAD+)-dependent histone deacetylase sirtuin 7(SIRT7)and its molecular mechanism in non-small cell lung cancer(NSCLC).Methods:(1)The human lung cancer tumor and adjacent non-tumor tissue specimen were obtained from randomly selected patients who have undergone surgery for lung cancer at the Department of Cardio-Thoracic Surgery,the First Affiliated Hospital of Soochow University.The clinicopathological characteristics of these patients were collected based upon medical history.A total of 102 pairs of paraffin-embedded lung cancer tumor and adjacent non-tumor tissue specimen were selected for preparation of human lung cancer tissue microarray(TMA)with a sample diameter of 1.5 mm(102 cases,204 dots).The lung cancer TMA was then cut into 3 ?m-thick sections for immunohistochemistry analysis of SIRT7 expression.The expression level of SIRT7 inlung cancer tumor tissues and its correlation with clinicopathological parameters of lung cancer patientswas analyzed.(2)The total cellular RNA and protein derived from a panel of human NSCLC cell lines such as A549,H292,H1299 and H1975 as well as a human normal bronchial epithelial cell line HBE(used as a control)was extracted,respectively.The expression of SIRT7 in human NSCLC cells was detected by qRT-PCR(SIRT7-F1:5'-ACT TGG TCG TCT ACA CAG GC-3',SIRT7-R1:5'-GGT GAT GCT CAT GTG GGT GA-3';PCR product size:158bp)and Western blot analysis.(3)The human SIRT7 coding sequence(CDS)fragment(1203 bp)was amplified by PCR using pGEM/SIRT7 cloning plasmid carrying human SIRT7 CDS as a template and using a human full-length SIRT7-specfic primer pair(SIRT7-F2:5'-CTA GCT AGC GCC ACC ATG GCA GCC GGG GGT CTG AG-3',SIRT7-R2:5'-TTG GCG CGC CTT ACG TCA CTT TCT TCC TTT TTG TGC G-3')and subsequently subcloned into a pLenti6.3/IRES/GFP lentiviral plasmid between NheI and SgsI restriction enzyme sites to construct a recombinant lentiviral plasmid pLenti6.3/SIRT7/IRES/GFP,which was then identified by PCR,double enzyme digestion and DNA sequencing.(4)The pLenti6.3/SIRT7/IRES/GFP expressing human SIRT71entiviral plasmid or pLenti6.3/IRES/GFPwas cotransfected into 293T human embryonic kidney cells(a packaging cell line for lentivirus generation)with helper packaging plasmids including pLP1,pLP2 and VSVG by Lipofectamine 2000.The lentivirus expressing SIRT7(LV-SIRT7)and the blank lentivirus(LV)were consequently generated and the titre was then determined,respectively.(5)The A549 human NSCLC cells that expressed the lowest level of SIRT7 was infected with LV-SIRT7 or LV(control)at a multiplicity of infection(MOI)of 50 and selected with drug Blasticidin S(BSD),leading to the generation of A549-SIRT7 and A549-mock(control)transgenic cells.The H292 human NSCLC cell line that expressed the highest level of SIRT7 was infected with LV-shSIRT7 or LV-shcontrol(control)and selected with drug Puromycin(PURO),resulting in formation of H292-shSIRT7 and H292-shcontrol(control)transgenic cells.The transgene efficiency in A549 NSCLC cells was analyzed according to GFP reporter by fluorescence microscopy and flow cytometry.The overexpression or knockdown efficiency of SIRT7 in A549 or H292 NSCLC cells was determined by qRT-PCR and Western blot analysis.(6)The effect of SIRT7 overexpression(A549-SIRT7 vs A549-mock)or knockdown(H292-shSIRT7 vs H292-shcontrol)on in vitro proliferation,cell cycle,migration and invasion of NSCLC cells was evaluated by Cell Counting Kit-8(CCK-8)assay,colony formation assay,propidium iodide(PI)cell cycle assay,wound healing assay and Transwell migration and invasion assays.(7)Theeffect of SIRT7 on NSCLC cell growth in vivo was monitored by measurement of tumor volume and tumor weight using a NSCLC subcutaneously(s.c.)transplanted tumor model in athymic BALB/c nude mouse.Theeffect of SIRT7 on NSCLC cell metastasis to the lung of athymic BALB/c nude mouse in vivo was detected by intravenous(i.v.)injection of NSCLC cells through murine tail vein.(8)The effect of SIRT7 overexpression(A549-SIRT7 vs A549-mock)or knockdown(H292-shSIRT7 vs H292-shcontrol)on expression of p-AKT(T308),p-AKT(S473),AKT,p-ERK1/2,ERK1/2,p21,p27,Cyclin D1,Cyclin E,CDK4,CDK2,E-cadherin,N-cadherin,Vimentin,Snail(Snail1)and Slug(Snail2)in NSCLC cells was analyzed by Western blot.Moreover,the regulatory effect of SIRT7 on in vivo expression level of p-AKT(T308),p-AKT(S473),AKT,p-ERK1/2,ERK1/2,p21,p27,Cyclin D1,Cyclin E,CDK4,CDK2,E-cadherin,N-cadherin,Vimentin,Snail(Snail 1)and Slug(Snail2)in NSCLC(A549-SIRT7 vs A549-mock;H292-shSIRT7 vs H292-shcontrol)s.c.transplanted tumor tissues in athymic BALB/c nude mouse was detected by immunohistochemistry analysis.Results:(1)The expression of SIRT7 in human NSCLC tumor tissues was significantly higher than that in adjacent non-tumor tissues,which was positively associated with the tumor size,the presence of lymph node metastasis and the pathologic stage(p<0.05).Compared with a normal bronchial epithelial cell line,the both mRNA and protein levels of SIRT7 are also upregulated in human NSCLC cells(p<0.05).(2)The stable SIRT7-overexpressed A549-SIRT7 vs A549-mock and the SIRT7-knocked down H292-shSIRT7 vs H292-shcontrol NSCLC transgenic cell lines were successfully established.The lentivirus-mediated SIRT7 overexpression remarkably promoted in vitro proliferation,G1/S phase cell cycle transition,migration and invasion in A549 NSCLC cells,whereas knockdown of SIRT7 suppressed proliferation,migration and invasion as well as induced G1 phase cell cycle arrest in H292 NSCLC cells(p<0.05).Overexpression of SIRT7 also obviously facilitated vivo growth and metastasis to lung in A549 NSCLC cells transplanted in athymic BALB/c nude mouse,whereas knockdown of SIRT7 in H292 NSCLC cells repressed these processes(p<0.05).(3)Overexpression of SIRT7 significantly upregulated level of p-AKT and p-ERK1/2 in A549 NSCLC cells in vitro and in vivo in athymic BALB/c nude mouse,leading to enhanced activation of AKT and ERK signaling(p<0.05).SIRT7 also upregulated cyclins including Cyclin D1 and Cyclin E and cyclin-dependent kinases including CDK4 and CDK2 as well as downregulated CDK inhibitors including p21 and p27 in A549 NSCLC cells in vitro and in vivo(p<0.05).Moreover,SIRT7 upregulated mesenchymal markers such as N-cadherin,Vimentin,Snail and Slug while downregulated epithelial marker E-cadherin in A549 NSCLC cells in vitro and in vivo(p<0.05).However,knockdown of SIRT7 exerted an opposite effect in H292 NSCLC cells in vitro and in vivo in athymic BALB/c nude mouse(p<0.05).Conclusions:SIRT7 is elevated in human lung cancer.SIRT7 promotes growth and metastasis possibly by activation of AKT and ERK signaling as well as by promotion of cell cycle G1 to S1 transition via modulating G1 phase checkpoint molecules and induction of epithelial-mesenchymal transition(EMT)in human lung cancer(NSCLC).SIRT7 exhibits an oncogenic role in human lung cancer,which may be a novel therapeutic target for human lung cancer. |
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