The Study On The Role Of Lentivirus-mediated TIPE2 Expression In Non-small Cell Lung Cancer And Its Underlying Mechanism

Objective: To examine the role of lentivirus-mediated expression of tumor necrosis factor-alpha-induced protein 8-like 2(TNFAIP8L2, TIPE2) in non-small cell lung cancer(NSCLC) growth and metastasis and its underlying mechanism.Methods: The about 555 bp human TIPE2 coding sequence(CDS) fragment was amplified by PCR using pMD19-T/TIPE2 recombinant cloning plasmid as a template and using a human TIPE2-specfic primer pair(TIPE2-F1: 5’-GAA GCT AGC GCC ACC ATG GAG TCC TTC AGC TCA AAG-3’; TIPE2-R1: 5’-ATA GGC GCG CCT CAG AGC TTC CCT TCG TCT AGC AGC-3’) and subsequently subcloned into GFP-labeled pLenti6.3/IRES/GFP lentiviral plasmid between NheI and SgsI restriction enzyme sites to construct a recombinant lentiviral plasmid pLenti6.3/TIPE2/IRES/GFP, which was then identified by PCR, double enzyme digestion and DNA sequencing. Then the pLenti6.3/TIPE2/IRES/GFP or blank control pLenti6.3/IRES/GFP lentiviral plasmid was cotransfected into 293 T human embryonic kidney cells with helper packaging plasmids including pLP1, pLP2 and VSVG by Lipofectamine2000, respectively. The lentivirus expressing TIPE2(LV-TIPE2) or blank lentivirus(LV) was consequently generated and concentrated by ultracentrifugation and the titre was then determined according to green fluorescent protein(GFP) expression by limiting dilution analysis. We quantified TIPE2 mRNA in a panel of cells including A549, SPC-A1, H358, H1299, H292 and H1650 NSCLC cells and HBE normal human bronchial epithelial cells by qRT-PCR analysis using a human TIPE2-specfic primer pair(TIPE2-F2: 5’-TGA CGG CAC TTA GCT TTG GT-3’; TIPE2-R2: 5’-GCA GAC CTG GGT CAG AGA AG-3’). After an optimal dose infection of the H1299 、 H292 NSCLC cells with 25 MOI(multiplicity of infection)LV-TIPE2 or LV followed by selection with 10μg/ml of Blasticidin S(BSD), we obtained the transfectants such as H1299-LV-TIPE2(termed H1299-TIPE2), H1299-LV(termed H1299-mock); H292-LV-TIPE2(termed H292-TIPE2), H292-LV(termed H292-mock). Tofurther detect the lentiviral-mediated GFP and TIPE2 transgene expression, the H1299-TIPE2, H1299-mock, H1299; H292-TIPE2, H292-mock, H292 NSCLC cells were analyzed by fluorescence microscopy, flow cytometry, RT-PCR and Western blot,respectively. The effect of lentiviral-mediated TIPE2 overexpression on human H1299 and H292 NSCLC cell proliferation, cell cycle, apoptosis, migration and invasion in vitro was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay, PI(propidium iodide) cell cycle assay, Annexin V-PE(phycoerythrin-conjugated Annexin V)/7-AAD(7-aminoactinomycin D) apoptosis assay and Transwell chamber migration or invasion assay. The effect of TIPE2 on H1299 and H292 NSCLC cell growth in vivo was monitored by measurement of tumor volume and tumor weight using NSCLC subcutaneously(s.c.) transplanted tumor model in athymic BALB/c nude mouse. The effect of TIPE2 on H1299 and H292 NSCLC cell metastasis to the lung of athymic BALB/c nude mouse in vivo was detected by NSCLC intravenous(i.v.) injection through murine tail vein. The effect of TIPE2 on expression of cell cycle-related proteins such as p21 and p27, apoptosis-related proteins such as Bcl-2, Bcl-XL, Bax and Bak,epithelial-mesenchymal transition(EMT)-related proteins such as E-cadherin, N-cadherin,Vimentin, Snail1 and Snail2(Slug), and kinase-related proteins such as AKT, p-AKT,ERK1/2, p-ERK1/2, GSK3β and p-GSK3β in H1299 and H292 NSCLC cells in vitro was analyzed by Western blot. The GSK3β and proteasome inhibitor(VIII and MG132) assays were further performed to verified whether TIPE2 downregulated Snail1 and Snail2(Slug)via facilitating proteasomal degradation of Snail1 and Snail2(Slug) through regulating AKT-GSK3β-Snail axis and ultimately elucidate an important molecular regulatory mechanism for TIPE2-mediated reversal of EMT and inhibition of metastasis in NSCLC.Results: 1) The recombinant lenitivirus expressing human TIPE2 was successfully constructed; 2) The TIPE2 expression in human NSCLC cells was remarkably downregulated, especially in H1299 and H292 NSCLC cells; 3) The lentivirus-mediated TIPE2-transgenic H1299 and H292 NSCLC cells was successfully established; 4)Lentivirus-mediated TIPE2 overexpression significantly inhibited H1299 and H292 NSCLC cell growth in vitro and in vivo in athymic nude mouse; 5) TIPE2 obviously induces cell cycle G1 phase arrest in H1299 and H292 NSCLC cells via upregulation of cyclin-dependent kinase(CDK) inhibitor p27; 6) TIPE2 induced H1299 and H292 NSCLC cell apoptosis via upregulation of pro-apoptotic proteins(Bcl-2, Bcl-XL)/anti-apoptoticproteins(Bax, Bak) ratio; 7) TIPE2 markedly suppressed H1299 and H292 NSCLC cell migration and invasion in vitro and their metastasis to lung in vivo in athymic nude mouse;8) TIPE2 drastically downregulated Snail1 and Snail2(Slug) EMT-inducing transcription factors(EMT-TFs), N-cadherin and vimentin mesenchymal markers as well as upregulated E-cadherin epithelial marker; 9) TIPE2 notably downregulated the levels of p-AKT 、p-GSK3β in H1299 and H292 NSCLC cells; 10) The inhibition of both GSK3β and proteasome completely attenuated the inhibitory effect of TIPE2 on Snail1 and Snail2(Slug) in H1299 and H292 NSCLC cells.Conclusions: 1) TIPE2 is capable of suppressing NSCLC growth via downregulation of AKT signaling as well as induction of G1 phase arrest through regulating G1 phase checkpoint molecules and activation of intrinsic apoptosis through upregulation of Bcl-2family pro-apoptotic proteins/anti-apoptotic proteins ratio; 2) TIPE2 can reverse NSCLC EMT and inhibit NSCLC metastasis via downregulation of Snail1 and Snail2(Slug)EMT-TFs; 3) TIPE2-AKT-GSK3β-Snail axis-mediated proteasomal degradation of Snail1 and Snail2(Slug) may be an important molecular regulatory mechanism for TIPE2-elicited reversal of EMT and inhibition of metastasis in NSCLC.