Immunoblot assays demonstrated time-dependent inhibition of G2/M regulatory proteins. Moreover, asperolide A significantly activated MAP kinases  by phosphorylation, and only the inhibition of ERK activation by PD98059 reversed downregulation of G2/M regulatory proteins CDC2, and suppressed upregulation of p21 and p-p53 levels. Transfection of cells with dominant-negative Ras (RasN17) mutant genes up-regulated asperolide A-induced the decrease of cyclin B1 and CDC2, suppressed Raf, ERK activity and p53-p21 expression, and at last, abolished G2/M arrest. This study indicates that asperolide A-induced G2/M arrest in human NCI-H460 lung carcinoma cells relys on the participation of the Ras/Raf/MEK/ERK signaling pathway in p53-p21 stabilization. An in vivo study with asperolide A illustrated a marked inhibition of tumor growth, and little toxcity compared to Cisplatin therapy. Overall, these findings provide potential effectiveness and a theoretical basis for the therapeutic use of asperolide A in the treatment of malignancies.Here we first demonstrate that asperolide A, a very recently reported marine-derived tetranorditerpenoid, leads to the inhibition of NCI-H460 lung carcinoma cell proliferation by G2/M arrest with the activation of the Ras/Raf/MEK/ERK signaling and p53-dependent p21 pathway. Treatment with 35 μM asperolide A (2 × IC Lung cancer remains one of the leading causes of cancer-related deaths worldwide . In addiin vitro during the past few years, e.g., cisplatin [in vitro findings indicated that balance between gemcitabine-induced and AG1478 (one of EGFR inhibitors)-inhibited ERK phosphorylation may have effects [The Ras family, containing H-(or Ha-) Ras, K-(or Ki-) Ras, and N-Ras, which transmits extracellular signals to the interior of the cells via switching the inactive GDP-bound state to the active GTP-bound state, are commonly thought of as oncogenes; the Ras/Raf/MEK/MAPK pathway is thought to be functional downstream of EGFR , which has been associated with a more aggressive disease or poor prognosis in variety of cancer systems including lung cancer ,9. Activisplatin , paclitaisplatin  and etopisplatin . Further effects . There aAspergillus wentii EN-48 led to the isolation of three new tetranorlabdane diterpenoids and five related derivatives. The structures of asperolide A were herein established based on spectroscopic interpretation, and confirmed by X-ray crystallographic analysis. The absolute configuration of asperolide A was determined by application of the modified Mosher’s method.Very recently, three new tetranorditerpenoids, asperolides A–C, were isolated and reported from a marine-derived endophytic fungus, Asperolides wenti EN-48. The results from preliminary biological evaluation demonstrated cytotoxicity of these compounds . In addiin vitro and the anti-tumor effect in vivo. We found that in vitro, like the Cisplatin, it activated the Ras/Raf/MEK/ERK signaling pathway. In addition, cell cycle arrest was a p53-p21 dependent event with modulation by the Ras/Raf/MEK/ERK signaling pathway. In in vivo study, it was effective in inhibition of tumor xenograft growth and safer than Cisplatin in the dosage of 5 mg/kg. All of these present a potential use of asperolide A to treat NSCLC.Because of its activity against various tumor cell lines (data not shown), especially NCI-H460 cells, we test the effect of asperolide A on cell cycle progression and apoptosis in NCI-H460 cells. The results showed that there is progressive arrest in the G2-M phase, which ensures proliferation inhibition of asperolide A against NCI-H460 cells. Then we further explored the molecular mechanism of asperolide A 50 value of asperolide A was 17.71 ± 3.56 μM (5.10 ± 1.02 μg/mL) for NCI-H460 cells.The MTT assay is used to investigate the inhibitory effect of asperolide A on proliferation of NCI-H460 cells. Cells were incubated in the absence or presence of various concentrations of asperolide A (0–56 μM) for 48 h. In addition, as shown in 50 concentration) for 12 h, 24 h and 48 h, respectively. The results showed that cells treated with asperolide A accumulated progressively in G2/M phase , p38 MAPK, and extra-cellular signal-regulated kinase (ERK). Over the last decade, extensive work have demonstrated that MAPK signaling pathways played critical roles in the regulation of a wide variety of cellular processes including cell growth, migration, proliferation, differentiation, development, apoptosis, and cell growth arrest [The mitogen activated protein kinase (MAPK) family contains c-Jun h arrest .To clarify whether asperolide A exerted its antitumor effects by the changes in MAPK pathway, ERK1/2, JNK and p38 MAP kinase assays were performed. The results of these experiments indicated that ERK1/2, JNK and p38 MAP kinases were significantly activated by asperolide A which increased the amount of phosphorylated ERK1/2, JNK and p38 MAP kinase B. To conAs described above, the examination is essential to check whether Ras-an activator of the ERK1/2 signaling pathway-is involved in asperolide A-induced G2/M-phase arrest. Therefore, protein level of the Ras-actived form Ras-GTP was analyzed by Western blotting. As expected, asperolide A induced the activation of Ras in NCI-H460 cells, while unchanged the expression level of Ras. And c-Raf (Raf-1), the main effector recruited by GTP-bound Ras to activate the MEK-MAP kinase pathway, was also enhanced activity by phosphorylation at Ser338 A. Then, Above all, these results suggest that the Ras/Raf/MEK/ERK signaling pathway may be involved in asperolide A-induced p53-p21-mediated G2/M-phase arrest and growth inhibition in NCI-H460 cells.in vivo, equal numbers of NCI-H460 cells were injected s.c. into the right armpit of six-week old BALB/c male athymic mice. Vehicle-treated control mice (1% DMSO) or Cisplatin-treated mice (2.5 mg/kg) were used as negative or positive control group to assess effect and toxicity of asperolide A. In this study, we adopted 5 mg/kg asperolide A i.v. according to the preliminary experiments, and tumor growth inhibition was very obvious in asperolide A treated group  Ras, K-(or Ki-) Ras, and N-Ras. Guanylyl-imidodiphosphate (GMP-PNP) of GTP-Ras may interact specifically with c-Raf ,34. Raf in vitro , we studIn order to further explore whether the Ras/Raf/MEK/ERK pathway is required by G2/M phase cell cycle arrest induced by asperolide A in NCI-H460 cells, cells were transfected with a dominant negative Ras (RasN17) and then treated with asperolide A for 48 h. The percentage of G2/M phase cells came to normal level and asperolide A-induced increase of p-ERK, p-p53, p21 and decrease of CDC2, cyclin B1 were completely reversed by transfection with RasN17.All above strongly suggest it is the Ras/Raf/MEK/ERK signaling pathway that is participating in p53-p21 stabilization in order to induce G2/M arrest in human NCI-H460 lung carcinoma cells. We characterize the probable effect of asperolide A on Ras/Raf/MEK/ERK signaling pathway in In vitro, cisplatin inhibits DNA synthesis by causing double-strand breaks, leading to apoptosis at the G2/M transition [In vivo, cisplatin (cis-diamminedichloroplatinum (II)) is a chemotherapeutic agent used in the treatment of a wide range of human malignancies, including NSCLC. Though Cisplatin combination chemotherapy is the cornerstone of treatment of many cancers, cellular drug resistance together with a narrow therapeutic range limit its use [ansition . A link ansition ,41. In v its use ,43. In a its use ; the weiAsperolide A was isolated from a marine-derived endophytic fungus Asperolide wentii EN-48 . MagnoloThe human large cell lung carcinoma cell line NCI-H460 was obtained from the Keygen Biotech Co., Ltd. . The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and antibiotics (100 μg/mL streptomycin and 100 U/mL penicillin). Cultures were maintained at 37 °C in an atmosphere containing 5% CO2. All the experiments were performed on logarithmically growing cells. Asperolide A was dissolved in DMSO and further diluted in PBS. The final DMSO concentration was 0.1%.NCI-H460 cells were seeded into 96-well plates at 4 × 104 cells/mL, incubated for 24 h, and then treated with the indicated concentrations of asperolide A for 48 h. Cell viability was determined using 3--2,5-diphenyltetrazolium bromide (MTT) assay.6 cells/mL. After adding 5 μL Annexin V-FITC and 5 μL PI, cells were mixed and incubated at room temperature in the dark for 5–15 min. The samples were analyzed with a FACScalibur flow cytometer and results were calculated by CellQuest software.Cell apoptosis was determined by Annexin V-FITC/PI assay with Annexin V-FITC kit . NCI-H460 cells seeded in 6-well plates were treated with different concentrations of asperolide A for 48 h. Then, the cells were collected and re-suspended with 500 μL binding buffer at a concentration of 10Cells were seeded in six-well plates for 24 h and treated with asperolide A for 48 h. Then they were harvested, washed twice with PBS, fixed in 70% ethanol and stored at 4 °C overnight. Cells then were washed with PBS, incubated with RNase at 37 °C for 30 min, and then stained with PI (1 mg/mL) for at least 30 min. Cell cycle phase analysis was performed by using a FACScalibur flow cytometer. g centrifugation for 15 min, the protein content of supernatant was determined by BCA protein assay . Equal amounts of the total protein samples were separated by 12% SDS-PAGE, and transferred to nitrocellulose membranes using an electro-blotting apparatus . The membranes were blocked in blocking buffer (TBST plus 5% non-fat dry milk), and incubated with primary antibodies overnight at 4 °C. Then the membranes were washed with TBST and incubated with HRP (horseradish peroxidase)-conjugated secondary antibodies for 1.5 h at 4 °C. Antibody-reactive proteins were developed by the ECL system .Cells were lysed in Western blotting lysis buffer  at 4 °C for 30 min. After 12,000× TM Transfection Reagent  according to the manufacturer’s instructions. Then stable transformants were selected using G418 . After 2 months selection, the transfected cells were used for subsequent experiments. The pCMV vector encoding dominant negative Ras (RasN17) were purchased from Clontech . Cells were transfected with vector in 6-well plates at 5 μg final quantities per well using Xfect6 NCI-H460 cells s.c. into the right armpit of six-week old BALB/c male athymic mice . The mice were randomized into vehicle control and treatment groups of nine animals when xenografts were palpable with an average size of ~100 mm3. Vehicle (1% DMSO) or drugs (2.5 mg/kg Cisplatin or 5 mg/kg asperolide A) was administered i.v. every other day until sacrifice. Body weight and tumor size was measured and recorded every two days from the fourth day. The tumor size was measured using electronic caliper, and tumor volumes were calculated using the formula: length × width2/2. 20 days post inoculation, the mice were sacrificed, the tumors collected, weighed, and photographed. The tumor inhibition effect was calculated using the following equation:T is the average tumor weight of the treated group and C is that of the control group.The mouse xenograft model was established by injection of 5 × 10t-test analysis. P-values < 0.01 were considered to be statistically significant.Data were given as means ± SD and Statistical comparisons were made by Student’s in vivo studies with asperolide A showed a marked inhibition of tumor growth and little toxcity.In summary, our data indicate that: (a) asperolide A inhibit cell-cycle progression at the G2/M phase by decreasing the levels of CDC2, cdc25c and cyclinB; (b) asperolide A-induced G2/M arrest is mediated by p53-dependent p21 induction which is regulated by Ras/Raf/MEK/ERK signaling pathway; and (c)


Knowledge about the role of specific HLA class I alleles, CD8+ T cell responses, and viral escape on viral control have not been well characterized in clade CRF01_AE and Asian ethnics.195 naïve HIV-1 CRF01_AE infected Thai individuals were screened for HIV-1 specific CD8+ T cell responses with a set of 413 OLPs of the HIV-1 proteome by IFN-gamma ELISpot assay. Novel epitopes were characterized by HLA restriction and fine epitope mapping. The association of epitopes and/or HLA alleles with low VL level and/or viral escape was analyzed.Thirty-three OLPs were identified as potential novel epitopes. A viral control epitope, RI10  was found restricted by HLA-A*0203 in Thais. Interestingly, HLA-A*0203+ve patients with RI10 responders had a significantly lower VL than non-responders (p = 0.0167). This data may support the low VL from loss of viral fitness of RI10. Moreover, the patients exhibiting mutations in RI10 showed no ELISpot responses. Another known HLA-A*1101 epitope, AK11 (in Gag-p24) was also identified as a viral control epitope in this study. Of note, there was no significant mutation found in patients expressing A*1101. We also found a novel immunodominant epitope (29% response rate) restricted by HLA-Cw*0102: YI9 (in Gag-p24) which was associated with viral escape. Mutations at P2 (S278X), P4 (V280X) and P5 (S281G) impaired the Elispot responses, however the P2 anchor S278K mutation had the highest negative impact (p = 0.0002).In HIV-1 CRF01_AE infected Thais we characterized three CD8 epitopes  restricted by HLA-A*0203, -A*1101 and -Cw*0102, respectively. RI10 and AK11, but not YI9, were associated with lower VL and possible control of HIV. Further characterization of those possible novel epitopes is warranted.


Bacilli, is a good model system for investigating determinants of the oligomeric state. A short segment of C-terminal residues defines whether TRAP forms an 11-mer or 12-mer assembly. To understand which oligomeric state is more stable, we examine the stability of several wild type and mutant TRAP proteins.Subunit number is amongst the most important structural parameters that determine size, symmetry and geometry of a circular protein oligomer. The L-tryptophan biosynthesis regulator, TRAP, present in several B. stearothermophilus, B. halodurans and B. subtilis TRAP, we find that the former is the most stable whilst the latter is the least. Thermal stability of all TRAP is shown to increase with L-tryptophan concentration. We also find that mutant TRAP molecules that are truncated at the C-terminus - and hence induced to form 12-mers, distinct from their 11-mer wild type counterparts - have increased melting temperatures. We show that the same effect can be achieved by a point mutation S72N at a subunit interface, which leads to exclusion of C-terminal residues from the interface. Our findings are supported by dye-based scanning fluorimetry, CD spectroscopy, and by crystal structure and mass spectrometry analysis of the B. subtilis S72N TRAP.Among the wild type We conclude that the oligomeric state of a circular protein can be changed by introducing a point mutation at a subunit interface. Exclusion (or deletion) of the C-terminus from the subunit interface has a major impact on properties of TRAP oligomers, making them more stable, and we argue that the cause of these changes is the altered oligomeric state. The more stable TRAP oligomers could be used in potential applications of TRAP in bionanotechnology. Cyclic protein oligomers pose promise as the basis for engineering molecular machines, including those that operate by rotation of one or more components about a principal axis. Their suitability, owing to their unique geometry, is evident by their frequent occurrence in key bio-mechanical and regulatory roles. Examples include circular protein oligomers found in flagellar motors BacillitrpEDCFBA operon transcript. This modulates terminator hairpin formation and so downregulates L-tryptophan biosynthesis The tryptophan RNA-binding attenuation protein, TRAP, possesses similar geometric features to other circular assemblies, being assembled of multiple subunits and containing a central tunnel. Typically composed of 11 or 12 identical subunits, with a molecular weight of 90.6 to 101.9 kDa B. stearothermohphilus, a hyperthermophile. The presence of L-tryptophan – and hence the bound holo-TRAP state – has been associated with a greater structural rigidity and integrity B. stearothermophilus apo-TRAP crystal structure d in the 2.8 µM to 6.9 µM range, with small variation between TRAP from different species. We explore how the presence of L-tryptophan and its’ binding affects thermal stability in the context of the three wild type and three mutant TRAP molecules. We also explore the influence of the oligomeric state on thermal stability by comparing wild type 11-subunit and mutant 12-subunit molecules from the same species. Finally, we show how the 11-subunit to 12-subunit switch could be induced by the point mutation S72N. The analysis includes crystal structure and native mass spectrometry data on B. subtilis S72N TRAP. We find that the increase in subunit number increases the stability of the assembly.TRAP oligomers have previously been reported to be highly thermostable B. subtilis TRAP assembly B. subtilis S72N TRAP, determined at 2.7 Å, reveals a circular 12-mer assembly . The 12-mer state was identified as the dominant species in the mass spectra , as opposed to the wild type protein which forms 11-mers To understand if the 12-mer state of the S72N TRAP observed in the X-ray structure, predominates in solution, we analyzed this protein by native mass spectrometry (S3) was averaged between four observations and the midpoints of fitted sigmoidal curves were taken as the melting temperatures, Tm. Melting temperatures (Table S1) were then plotted against L-tryptophan concentration  to verify that melting temperatures were not perturbed by interaction with the dye, Figure S2. We note that there is coherence between melting temperatures derived from the two respective techniques.As the fluorescent dye could potentially bind in the tryptophan-binding pocket, we employed CD spectroscopy  becomes apparent. In B. halodurans TRAP, the nature of the last four amino acids dictate that the C-terminal segment is excluded from the subunit-subunit interface, thereby generating favorable interactions at the outer surface of the oligomer. This is considered as the crucial factor in permitting the higher oligomeric state, which is related to the 11-mer state by a 2.7o rigid-body rotation of adjacent subunits Comparing the structure of the 12-mer B. halodurans TRAP, regarding the exclusion of the C-terminus, could logically be mimicked by truncating the 4 or 5 C-terminal residues, removing this segment from the subunit interface. This is observed in the previously determined structures of B. stearothermophilus E71stop TRAP and B. subtilis K71stop TRAP B. stearothermophilus wild type and E71stop is 6°C, from 81.4°C to 87.4°C. The equivalent increase between B. subtilis wild type and K71stop is 18.7°C, 44.3°C to 63.0°C. This hints at an increase in thermal stability in response to incorporation of an additional subunit into the ring. However, this difference in melting temperature could also be a result of the absence of the C-terminal residues themselves. For instance, we note that both B. subtilis and B. stearothermophilus wild type TRAP contain two flexible lysine residues at their C-termini B. subtilis TRAP 11-mer with that of the B. subtilis S72N TRAP 12-mer, which still possesses the C-terminal segment but has it excluded from the subunit interface for steric reasons.Taking our investigation a step further, we focused further studies at determining whether there is a general relationship between the oligomeric state of TRAP and its thermal stability, hence exploring factors that are not species-specific. The scenario found in the 12-mer B. halodurans TRAP. Residue 72 in particular (Ser 72 in B. subtilis TRAP) – which sits at the subunit-subunit interface in both wild type 11-mer structures studied – serves as a critical determinant in the conformation of the C-terminus. This is evident from the crystal structure presented in this study. From the structure of the S72N TRAP we see that substituting serine 72 for a bulkier amino acid, asparagine, does indeed introduce the necessary constraints to exclude the subsequent segment from the subunit interface. The thermal stability of this mutant 12-mer TRAP, S72N, is higher than that of its wild type 11-mer counterpart. For example at 50 µM L-tryptophan the melting temperature of the wild type and mutant oligomers are 44.°C and 54.5°C respectively, yielding a stabilization of 10.2°C. This is significant, albeit it is less compared to the 18.7°C stabilization we see from C-terminal truncation (i.e. B. subtilis K71stop).Exclusion of the C-terminus from the subunit interface can be achieved by introducing steric strain at a key region towards the C-terminus that causes the segment to pivot out. In this respect, exclusion of the C-terminus in S72N is achieved in a manner more similar to how exclusion is achieved in in vivo.We conclude that by inducing an expansion in the oligomeric state from 11 to 12 subunits the thermal stability of the oligomer is increased. This increase is predominantly a result of the oligomeric state transition, and to a much lesser extent the influence of C-terminal or other specific residues. A potential, general relationship in TRAP oligomers therefore exists, in which melting temperature depends on subunit number. Further studies should be focused on evaluation of influence of the oligomeric state of TRAP on its activity B. subtilis wild type and S72N mutant TRAP, the 11-mer to 12-mer transition does coincide with a slight increase in affinity for L-tryptophan, these changes are only twofold, implying there are other more significant influences.To explore whether the stabilization by L-tryptophan was directly related to the affinity of TRAP oligomers to the binding of L-tryptophan, dissociation constants were measured. Results showed no obvious correlation with thermal stability or subunit number Table 2.B. subtilis wild type TRAP however, over the L-tryptophan concentration range studied. Most concentrations in the monitored range represent many times the typical Kd value, hence L-tryptophan binding-sites in TRAP are expected to be saturated. This therefore suggests a degree of non-specific binding of L-tryptophan to TRAP as the principal cause of the stabilization, over a thermodynamic or kinetic influence involving the L-tryptophan binding sites. It is likely that non-specific binding, beyond the 1∶1 stoichiometry, would hence increase with increasing concentration of L-tryptophan.For most TRAP oligomers studied we observe an increase in melting temperature in response to an increase in L-tryptophan concentration from 0 µM to 100 µM added. The trend does not appear in in vitro. In the case of TRAP, used in our studies, an increase in subunit number increases the stability for the oligomeric assembly. Predictably, this stability is dependent on solvent conditions, specifically L-tryptophan concentration. We conclude that a similarly subtle approach could be used during protein engineering of other ring-like structures, for stabilizing a particular oligomeric state thereby modifying dimensions, symmetry and stability.This study demonstrates how the oligomeric state of a circular protein can be changed by introducing a point mutation at a subunit interface. Importantly, we demonstrate that a change in subunit number changes the stability of a circular assembly B. subtilis S72N mutant TRAP was generated using the QuikChange kit  and a pET9a plasmid containing the wild type gene. S72N TRAP proteins were produced and purified as described previously Figure S1) and X-ray crystallographic studies.The B. subtilis S72N TRAP was transferred into solution containing 10 mM triethanolamine (pH 8.0), 100 mM NaCl, 15 mM L-tryptophan and concentrated to 33.5 mg/ml. The reservoir contained 100 mM Bis-Tris propane (pH 8.5), 200 mM KSCN and 13% PEG 3350 (v/v). Protein crystals were frozen using solutions containing all the crystallization ingredients with addition of 20% glycerol (v/v). The X-ray data were collected at 120 K at I04 beamline at the Diamond Light Source. Data were processed using HKL2000 Crystallization was carried out at 18°C by hanging drop vapor diffusion. For crystallization, B. stearothermophilus TRAP as a search model. Refinement was performed by REFMAC o| - |Fc| and |Fo| - |Fc| electron density maps. Molecular contacts between adjacent monomers of TRAP were examined by CONTACT All crystallographic calculations were carried out using the CCP4 program package 2, with variable L-tryptophan concentration, in 30 kDa cut-off Vivaspin concentrators, and diluting to 1 mg/ml, monitoring concentration by the Comassie-blue Bradford method. L-tryptophan concentration was varied from 0 to 0.1 mM . Samples were transferred to 96-well thin-walled clear PCR plates. Each well contained 30 µl of solution, consisting of 15 µl of the 1 mg/ml protein , 1/1000 diluted SYPRO Orange (Sigma-Aldrich) and 50 mM Tris pH 8 with a specified L-tryptophan concentration. A Stratagene Mx3005P QPCR instrument was programmed to increase temperature in 1°C increments at 30 second intervals, from 25°C to 95°C with 470 nm excitation, measuring fluorescence at 570 nm.Dye-based scanning fluorimetry, or the Thermofluor method, was performed as previously described L-tryptophan binding to TRAP was measured using a fluorescence assay based on competition between L-tryptophan and 1-anilionaphthalene-8-sulfonic acid (ANS) as described previously B. subtilis S72N TRAP and structure factors have been deposited with the Protein Data Bank under accession code 4B27.Refined coordinates of the Figure S1Native mass (Nanoflow electrospray) spectrum of B. subtilis S72N TRAP. m/z corresponds to the mass-to-charge ratio. Red hexagons correspond to 12-mer TRAP species without bound tryptophan.(DOCX)Click here for additional data file.Figure S2Melting curves of B. halodurans and B. stearothermophilus wild type TRAP. Normalised fluorescence data (A) B. halodurans wild type TRAP (12-mer). (B) B. stearothermophilus wild type TRAP (11-mer). Sigmoidal curves were fitted to CD spectroscopy data (red) for comparison against fluorescence data. The melting temperatures represented by the midpoints of these curves were found to be 78.5°C and 81.6°C for (A) and (B) respectively. These compare well with the melting temperatures derived from curves fitted to the fluorescence data, 75.6°C and 81.4°C respectively (ectively .(DOCX)Click here for additional data file.Figure S3A) Overlays of 8 normalised fluorescence versus temperature curves of B. stearothermophilus E71Stop TRAP, with concentration of L-tryptophan increasing left to right with lightening shades of green: 0, 4, 8, 20, 25, 50, 75, 100 µM. (B)Curves derived from four replicates at two concentrations of L-tryptophan, for B. stearothermophilus E71Stop TRAP: 0 µM L-tryptophan , 50 µM L-tryptophan , normalised to the mean curve, showing equivalent midpoint and plateau positions within replicates.((DOCX)Click here for additional data file.Table S1Melting temperatures for TRAP oligomers over 0 to 100 mM L-tryptophan concentration.B. halo. ; B. stearo. (B.stearothermophilus); B. sub. (B. subtilis); WT (wild type). Standard deviations across four repeated measurements are shown in the parentheses.(DOCX)Click here for additional data file.Text S1Materials and Methods - Native Mass Spectrometry.(DOCX)Click here for additional data file.Text S2Materials and Methods - Circular Dichroism spectroscopy.(DOCX)Click here for additional data file.


MicroRNAs (miRNAs) are small noncoding RNAs that negatively regulate gene expression post-transcriptionally. They play a critical role in developmental and physiological processes and have been implicated in the pathogenesis of several diseases including cancer. To identify miRNA signatures associated with different stages of neoplastic development, we examined the expression profile of 776 primate miRNAs in VERO cells , progenitor primary African green monkey kidney (pAGMK) cells, and VERO cell derivatives: spontaneously immortalized, non-tumorigenic, low-passage VERO cells (10-87 LP); tumorigenic, high-passage VERO cells (10-87 HP); and a cell line (10-87 T) derived from a 10-87 HP cell tumor xenograft in athymic nude mice. When compared with pAGMK cells, the majority of miRNAs were expressed at lower levels in 10-87 LP, 10-87 HP, and 10-87 T cells. We identified 10 up-regulated miRNAs whose level of expression correlated with VERO cell evolution from a non-tumorigenic phenotype to a tumorigenic phenotype. The overexpression of miR-376a and the polycistronic cluster of miR-376a, miR-376b and miR-376c conferred phenotypic changes to the non-tumorigenic 10-87 LP cells that mimic the tumorigenic 10-87 HP cells. Thirty percent of miRNAs that were components of the identified miRNAs in our spontaneously transformed AGMK cell model are also dysregulated in a variety of human tumors. These results may prove to be relevant to the biology of neoplastic development. In addition, one or more of these miRNAs could be biomarkers for the expression of a tumorigenic phenotype. It is well documented that repeated passage of cells in culture can result in their immortalization and can lead to the acquisition of the capacity to form tumors when injected into animals in vitro passage in vitro.Cells from non-human mammalian species, in the absence of known carcinogenic stimuli, can spontaneously undergo processes of neoplastic transformation during culture in vivo.Recently, it has become evident that, in addition to mutation of, and/or changes in, the expression of protein-coding genes, alterations in the expression of non-coding microRNAs (miRNAs) can also contribute to cancer pathogenesis in vitro in kidney cells from this non-human primate.In this report, we performed high-throughput miRNA profiling to examine the expression level of miRNAs in pAGMK cells and in VERO cells at non-tumorigenic and tumorigenic stages of neoplastic development. The analysis involved (1) pAGMK cells, (2) non-tumorigenic 10-87 low-passage VERO cells (10-87 LP), (3) tumorigenic, high-passage VERO cells (10-87 HP), and (4) a cell line (10-87 T) derived from a 10-87 HP cell tumor xenograft in athymic nude mice. Our data revealed that, although some miRNA dysregulation occurred during the immortalization of the AGMK cells that established the VERO cell line, the majority of miRNA dysregulation occurred in VERO cells during the transition from a non-tumorigenic to a tumorigenic phenotype. We identified the up-regulation of specific miRNAs that correlated with, and were perhaps involved in, the evolution of the neoplastic phenotype that occurred during serial passage of VERO cells in culture. Indeed, the overexpression of miR-376a and the polycistronic cluster of miR-376a, miR-376b and miR-376c in non-invasive 10-87 LP cells were able to recapitulate the migration and invasion phenotypes of 10-87 HP cells. Cumulatively, these data suggest that the patterns of dysregulation of the miRNAs identified in this study might be a feature of neoplastic development The 10-87 VERO cell lines used in this study were derived from the World Health Organization (WHO) VERO cell bank (10-87) after serial passage in tissue culture from p140 to p256 in vitro. Survival, proliferation, invasion, and migration are among the common functions acquired by cancer cells during neoplastic development in vivo.The above results suggested: first, 10-87 LP cells had already attained the proliferative potential expressed by 10-87 HP and 10-87 T cells ; second,p<0.01 were selected and transformed to z-intensity values for inclusion in the heat-maps. These miRNAs were regarded as significant and were considered to be differentially expressed  differences in fluorescence intensity between the means of two groups were >500 arbitrary units. The stringency was set at ≥4 fold with the intent of identifying the smallest number of miRNAs with the highest probability of contributing to VERO cell neoplastic development. Using these criteria, we identified 10 upregulated and 17 downregulated miRNAs in 10-87 HP cells  fold change was ≥4 relative to levels observed in pAGMK cells; (2) HP cells . The difHP cells . The magIn order to validate the miRNA microarray data for VERO cells at the passage levels tested, we performed qRT-PCR analysis to quantify the expression level of selected miRNAs from a list of differentially expressed miRNAs. The data are presented as fold over pAGMK miRNA levels . qRT-PCRin vitro assays was compared, a close similarity with the behavior of 10-87 HP cells was observed in the migration assay (p<0.01). As found for 10-87 HP cells and 10-87 T cells, SF-VERO cells and A4497 VERO cells expressed increased levels of miR-376a, miR-654-3p, miR-543, and miR-382 over the levels found in pAGMK cells  cassette, and introducing them into the non-tumorigenic 10-87 LP cells. We confirmed the expression of each of the miRNAs in their stable cell lines by qRT-PCR. The consequence of miRNA over-expression on the phenotype of the 10-87 LP cells was assessed in the wound-healing and the Matrigel-invasion assays. In the former assay, photomicrographs were taken of wounded cell-culture monolayers at time 0 and at every 3 h up to 15 h to determine the effect of overexpression of the miRNAs on the rate of migration. Only photomicrographs at 0 and 12 h are shown . The celUsing the Sanger miRNA database, we examined the chromosomal distribution of miRNAs that were altered in 10-87 LP and 10-87 HP cells. miRNA genes are frequently located in cancer-associated regions of the human genome in vivo neoplastic development in a mouse pancreatic tumor model in vivo .Dysregulation of miRNA expression has been linked to the formation and progression of many types of cancers in vitro passage of VERO cells selected for the expansion of cells bearing alterations in the expression of specific miRNAs. These findings lead us to propose that the miRNA signatures identified in early-passage VERO cells (10-87 LP) and late-passage VERO cells (10-87 HP and 10-87 T cells) were indicative of the progressive changes in VERO cells that were undergoing neoplastic transformation during serial passage in cell culture.Reported here are the results of the first miRNA expression-profiling study of non-human primate cells that transformed spontaneously during serial passage in cell culture. We evaluated patterns of miRNA expression in pAGMK cells and in derivatives of the 10-87 VERO cell line  in an attempt to identify the miRNAs whose altered expression might correlate with, and perhaps be involved in, the evolution of the neoplastic phenotypes that occurred during passage of these AGMK cells in tissue culture. Hierarchical clustering of miRNA expression profiles revealed that many miRNAs were differentially expressed in the tumorigenic 10-87 HP cells and tumor-derived 10-87 T cells when compared with pAGMK cells. In contrast, fewer miRNAs were differentially expressed when the non-tumorigenic 10-87 LP cells were compared with pAGMK cells. These results suggested that the dysregulation of the majority of miRNAs occurred in the 10-87 cell lineage during the transition from a non-tumorigenic to a tumorigenic phenotype. Furthermore, when the expression levels of the up-regulated miRNAs were organized according to their cell line-determined expression patterns, a correlation emerged between the relative expression (compared with levels in pAGMK cells) of representative miRNAs and the evolution of the 10-87 VERO cells into cells that could form tumors in nude mice . From thTo determine whether the overexpression of the identified signature miRNAs could increase the levels of cell migration or invasion – phenotypes that correlated with the capacity to form tumors in animals – stable cell lines expressing the pre-miRNA sequences of some miRNAs were generated from the non-tumorigenic 10-87 LP cells. These experiments demonstrated that miR-376a and miR-376abc were sufficient to confer the cell migration and invasion phenotype. No change in migration and invasion phenotypes was observed in cells expressing miR-382 or miR-299-5p. Recently, a target and function of miR-376a has been identified. A human cytomegalovirus (HCMV) miRNA acts synergistically with a cellular miR-376a to suppress one of the major histocompatibility complex class I polypeptide-related sequences (MICB) during HCMV infection in vitro model of neoplastic development in VERO cells, which were derived from the kidney of African green monkey, and tumors of the kidney that originated in vivo in humans, the finding of similar patterns of dysregulation of miRNAs in both systems suggests that similar molecular processes may be underway during neoplastic development in cells from both species irrespective of their microenvironments.When we compared experimental results from several published studies on the differential expression of miRNA in different cancers with our results, 30% (18 miRNAs) of the dysregulated miRNAs identified in our study  have an The other observation was the chromosomal clustering of the genes for some of the miRNAs up-regulated in 10-87 HP cells. Importantly, all of the genes for the 10 up-regulated miRNAs in 10-87 HP cells are clustered in a homologous chromosome region of rhesus monkeys and humans. The genes for the 10 miRNAs highly expressed in 10-87 HP cells and 10-87 T cells are spread over 47.6 kb on rhesus chromosome 7. This highly conserved chromosomal region is homologous to human 14q32.31. Interestingly, specific up-regulation of some miRNAs whose genes are located in the human 14q32 imprinted domain has been reported in acute myeloid leukemia in vivo or in vitro, are thought to represent the accumulation of a series of genetic and epigenetic alterations that disrupt the normal processes of cell division and tissue integrity in vitro, at some point producing cells that form invasive tumors or cells that possess the capacity to form tumors when injected into animals. In the VERO cell model, we have found that miRNA down-regulation occurs during the initial stages of neoplastic transformation that are associated with immortalization and some yet-to-be defined forms of neoplastic progression that are not reflected in a currently detectable phenotype by VERO cells at p140. miRNA up-regulation occurs as the immortalized cells are undergoing further neoplastic progression through the conversion, during 116 passages (from p140 to p256) in cell culture, to the expression of a tumorigenic phenotype. We have identified miR-376a and the polycistronic miR-376abc as having a functional role in establishing the enhanced cell migration and cell invasion phenotypes, which are correlated with the expression of the VERO cell tumorigenic phenotype. Studies are underway to determine whether one or more of these over-expressed miRNAs can confer on non-tumorigenic VERO cells the capacity to form tumors in animals and consequently can be used as biomarkers for the expression of the VERO-cell tumorigenic phenotype.The phenomena of neoplastic development and neoplastic transformation, whether they occur in vitro and in vivo have similarities. If further evidence for these similarities is forthcoming, the VERO-cell model may provide a format for the systematic comparison of neoplastic development in vivo and neoplastic transformation in vitro across tissues, organs, and species.The associations between neoplastic processes and miRNA dysregulation in the VERO-cell model presented in this report provide additional support for the concept that neoplastic development in vivo is summarized in Cells from the World Health Organization (WHO) VERO cell bank 10–87 (ATCC 10–87 VERO cells) were serially passaged in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS) (DMEM-10) from p134 to p250 by sub-culturing VERO cells before they reached confluence 4 cells in DMEM-10. The cells were trypsinized and counted in triplicate (using trypan-blue exclusion to determine the number of viable cells) after 16, 24, 48, and 72 h in culture using a Cellometer . Growth curves of the VERO cells were plotted from successive cell counts as described Dishes (60 mm) were seeded with 5×10Total RNA from pAGMK cells and the cell lines was extracted and purified using the miRNeasy mini kit according to the manufacturer's procedures . Taqman miRNA assays  were used to quantify mature miRNA expression as described http://microrna.sanger.ac.uk/). Total RNA (4 to 8 µg) was size fractionated using a YM-100 Microcon centrifugal filter , and the small RNAs (<300 nucleotides) were isolated and 3′ extended with a poly(A) tail using poly(A) polymerase. An oligonucleotide tag was then ligated to the poly (A) tail for later fluorescent-dye staining. Hybridization was performed overnight on a µParaflo microfluidic chip using a micro-circulation pump . The hybridization buffer was 6xSSPE  containing 25% formamide at 34°C. On the microfluidic chip, each detection probe consisted of a chemically modified nucleotide-coding segment complementary to target miRNA (http://microrna.sanger.ac.uk/sequences/) and a spacer segment of polyethylene glycol to extend the coding segment away from the substrate. The detection probes were made by in situ synthesis using photogenerated reagent (PGR) chemistry. The hybridization-melting temperatures were balanced by chemical modifications of the detection probes. After RNA hybridization, tag-conjugating Cy3 and Cy5 dyes were circulated through the microfluidic chip for dye staining. Fluorescence images were collected using a laser scanner  and digitized using Array-Pro image analysis software . Data were analyzed by first subtracting the background and then normalizing the signals using a LOWESS filter  vs. the pAGMK cells  and p-values of the t-test were calculated; differentially detected signals were those with p<0.01. The expression patterns of unfiltered data (the raw data from 654 miRNA data sets) were assessed using unsupervised hierarchical clustering of samples based on average linkage and Euclidian distance. When comparing two groups, the findings were considered significant if: 1) the fold change was ≥4, 2) the t-test, p-value was <0.01, and 3) the difference in fluorescence intensity between the two group means were >500 arbitrary units. ANOVA was done between the four groups of cell lines. All significantly expressed miRNAs with mean intensity value more than 500 (arbitrary units) in at least in one of the four groups and a p<0.01 were selected and transformed to z-intensity values for inclusion in the heat-maps. The clustered heat-map was created in TM4 microarray software (http://www.tm4.org) using the z-transformed miRNA expression. The dendrogram along the rows was calculated using 1-Pearson correlation for the distance measure and complete linkage for the clustering algorithm. Microarray data comply with MIAME requirements and have been deposited at Gene Expression Omnibus data base (accession number GPL10649).The microarray-expression assay was performed using a service provider . Because the genome sequence of the African green monkey is not available, there are no commercially available African green monkey miRNA chips. A primate miRNA chip that contained all human and non-human primate miRNA transcripts in the Sanger miRBase Release 10.0 was used (5 cells/dish). When the cultures reached 90% confluence, they were serum starved for 8 h. After scratching the monolayer with a pipette tip, cells were washed with PBS, cultured in DMEM-10, and photographed under a 10× objective every 3 h for the first 15 h and then at 24 h.The wound-healing assay was carried out as described 4) in DMEM with 1% FBS (DMEM-1) were placed in the upper chamber of a Biocoat Matrigel-invasion chamber , and DMEM-10 was used as the chemoattractant in the lower chamber. After incubation for 18 h at 37°C, the filters were stained with Diff-Quick stain set , and the number of cells that penetrated the Matrigel and the control insert were counted with a microscope using a 20× objective (5 randomly selected fields).The assay was performed as described 5′-tgctGTAAAAGGTAGATTCTCCTTCTATGAGTACATTATTTATGATTAATCATAGAGGAAAATCCACGTTTTC-3′ and miR-376a anti-sense 5′- cctgGAAAACGTGGATTTTCCTCTATGATTAATCATAAATAATGTACTCATAGAAGGAGAATCTACCTTTTAC-3′. miR-299-5p sense, 5′- tgctGAAGAAATGGTTTACCGTCCCACATACATTTTGAATATGTATGTGGGATGGTAAACCGCTTCTT-3′ and miR-299-5p anti-sense 5′-cctgAAGAAGCGGTTTACCATCCCACATACATATTCAAAATGTATGTGGGACGGTAAACCATTTCTTC-3′, miR-382 sense 5′- tgctGTACTTGAAGAGAAGTTGTTCGTGGTGGATTCGCTTTACTTATGACGAATCATTCACGGACAACACTTTTTTCAGTA-3′ and miR-382 anti-sense 5′-cctgTACTGAAAAAAGTGTTGTCCGTGAATGATTCGTCATAAGTAAAGCGAATCCACCACGAACAACTTCTCTTCAAGTAC-3′. An expression plasmid for the polycistronic gene cluster for miR-376a, miR-376b and miR-376c was generated by amplifying 200 bp up-stream and 200 bp down-stream of the genes (termed as miR-376abc) in VERO cell DNA using primers 5′-CCCTCGACGAGAGTGATGGAAGGTGAATC-3′ and 5′-CCAGATCTATACTGAGAACACAGCCTTGT-3′. This PCR product was digested with Sal I and Bgl II and cloned into the Sal I and Bgl II sites of the digested pcDNA6.2-GW/EmGFP-miR expression vector in the 3′ UTR region of GFP protein. The plasmids were verified by sequencing across the insert. The expression constructs were introduced into the non-tumorigenic 10-87 LP cells using Lipofectamine 2000 . Stable cell lines that constitutively expressed the specific miRNAs were selected using blasticidin (8 µg/mL). Blasticidin-resistant colonies were identified after 15 days of selection. The stable cell lines were sorted for high GFP expression using a BD FACSAria II  and tracked using fluorescence microscopy.The double-stranded oligonucleotides encoding the pre-miRNA sequences of miR-376a, miR-299, miR-382 or irrelevant miRNA  were cloned into the pcDNA6.2-GW/EmGFP-miR expression vector such that the pre-miRNA insertion site was in the 3′ untranslated (3′UTR) region of the green fluorescent protein (GFP) gene as per the instruction manual . The following oligonucleotides were used to generate the pre-miRNAs: miR-376a sense,


Caenorhabditis elegans oocytes that are revealed through the activity of an endogenous nuclease ('endocleavage’).Germ cells in animals are highly specialized to preserve the genome. A distinct set of chromatin structures must be properly established in germ cells to maintain cell fate and genome integrity. We describe DNA-surface interactions in activated C. elegans oocytes can be recovered in fragments of approximately 500 base pairs or shorter, cleaved at regular intervals (10 to 11 nt) along the DNA helix. In some areas of the genome, DNA cleavage patterns in these endoreduplicated oocytes appear consistent from cell-to-cell, indicating coherent rotational positioning of the DNA in chromatin. Particularly striking in this analysis are arrays of sensitive sites with a periodicity of approximately 10 bp that persist for several hundred base pairs of genomic DNA, longer than a single nucleosome core. Genomic regions with a strong bias toward a 10-nt periodic occurrence of A(n)/T(n)  appear to exhibit a high degree of rotational constraint in endocleavage phasing, with a strong tendency for the periodic A(n)/T(n) sites to remain on the face of the helix protected from nuclease digestion.Our analysis began with an unexpected observation that a majority (>50%) of DNA from ovulated but unfertilized C. elegans oocytes in which genomic DNA and associated protein structures are coherently linked.The present analysis provides evidence for an unusual structure in  Caenorhabditis elegans, a number of groups have been applying combinations of genomics, biochemistry, and genetics .C. elegans oocytes arrest at the diakinesis stage of meiotic prophase I. Oocyte chromosomes at this stage are highly condensed, giving rise to the characteristic appearance of six discrete bivalents [, in mutant C. elegans that are fully feminized), oocytes usually arrest at the diakinesis stage. However, in certain mutant strains that produce defective sperm, oocytes continuously mature and ovulate, endoreduplicating their DNA and resulting in a large number of unfertilized polyploid oocytes accumulating in the uterus. In this study, we use an endogenous nuclease activity present in these oocytes to identify an unusual chromatin structure.ivalents . Oocyte ivalents -6. A mativalents , and theC. elegans germline development [fer-1(b232) oocyte samples was present in fragments of <500 bp  young adult animals). Likewise the pattern was not observed in MNase-digested chromatin from L1 larvae or adult N2 animals [Additional file C. elegans embryos, but with a considerably lower concentration relative to the total DNA present . With a direct comparison of the approximately 10-nt periodic ladder pattern between embryos and activated oocytes  strain or the oocyte isolation procedure used. An approximately 10-nt ladder pattern similar to that observed from purified fer-1(b232) was observed for DNA extracted from whole animals from a different fer-1 mutant stock (fer-1(hc13)) raised at the restrictive temperature of 25°C  [spe-26(hc138) [fer-1 mutant oocytes  ,12 (requ (fer-1hc) raised C. elegans embryos depends on programmed cell death machinery (the caspase-homolog CED-3) and an associated type-II DNase NUC-1 [Chromosome fragmentation is a hallmark of apoptotic cells in mammals, as well as in lower metazoans (reviewed in ). Aruscase NUC-1 ,15-17.fer-1(hc13);ced-3(n717) and fer-1(hc13);nuc-1(e1392), growing each at the restrictive temperature (25°C) to allow oocyte accumulation. DNA from fer-1(hc13);ced-3(n717) double mutant animals yields an approximately 10-base periodic ladder similar to that observed in apoptosis-competent fer- and spe-strains, indicating that CED-3 activity  is not required for the observed oocyte endocleavage. In contrast, the fer-1(hc13);nuc-1(e1392) double mutant fails to yield an evident 10-nt ladder pattern  oocyte DNA from different size ranges  was isolated by using an agarose gel. The majority of these DNA fragments were double-stranded with a 2 to 3 nt overhang at the 3′ ends (see later section and Methods). Size-selected DNA fragments were treated with T4 DNA polymerase to produce blunt ends, followed by linker ligation. The resulting libraries were analyzed by paired-end sequencing (Illumina HiSeq 2000 sequencing system), allowing both ends to be mapped. With the criterion that paired reads match perfectly to a unique position of the reference genome, approximately 6.1, 8.0, 15.6, and 15.5 million alignments were obtained by this process for the 20 to 80, 40 to 130, 80 to 230, and 130 to 430 nt size ranges, respectively. We also performed single-end Illumina sequencing for endocleaved oocyte DNA in the 40 to 90, 90 to 140, and 140 to 240 nt size ranges.To understand the relation between fragmentation activities and the arrangement of DNA packaged in oocyte chromatin, we set out to characterize the precise sites of DNA cleavage. Fragmented , peaks at 178, 189, 199 nt) [Additional file The size of captured DNA fragments can be assessed by the distance between paired reads. The distributions of the distances between paired reads show approximately a 10-bp periodicity  and which face is accessible. At any given locus in the genome, the accessible face can be random among individual cells  or, in the other extreme, a particular face can be consistently accessible in all cells . The 10-nt ladder appearance of the fragmented DNA in activated fer-1(b232) oocytes indicates that the endogenous DNA cleavage events tend to occur periodically for every helical turn in the DNA double helix.The detailed structure of chromatin in a region can determine which face of the DNA double helix is protected by an underlying surface  -20. The , peaks at 10, 21, 31, 41, 51, 62, 72, and 82 nt, etcetera)  with th oocyte chromatin, the coincidence numbers oscillate with additional periodicity that corresponds to an approximately 178-nt nucleosome-like repeat length   oocyte endo-cleaved DNA fragments in the size range of 1 to 100 nt (and approximately 7-fold in the size range of 101 to 200 nt)  oocytes argues for differentiating features of oocyte chromatin that produce greater long-range periodicity and greater cell-to-cell rotational consistency than was observed in the somatic embryo tissue.When the auto-correlation analysis was performed for the endo-cleavage DNA fragments from wild type embryos, the degree of non-random rotational positioning is approximately 5-fold lower than that observed for fer-1b22 oocyte In summary, the prominent approximately 10-nt periodic signals in the oocyte auto-correlation analyses indicate that a particular face of the activated oocyte DNA in a large fraction of the genome is preferentially cleaved by the endogenous DNase activity. For this portion of the genome, we can infer that the activated oocyte DNA has been operationally constrained in its rotational positioning relative to an underlying protective surface., H3.3) [, H3K4me2/3) [Genome-wide nucleosome mapping studies from a number of model organisms have shown nucleosome positioning that appears to be variable for a substantial fraction of the genome ,28. A sm, H3.3)  and postK4me2/3) .C. elegans genes by mapping nucleosomes that are enriched for H3K4me2/3 [C. elegans are highly over-represented in this set of H3K4me2/3-anchored genes. To evaluate the expression status of these genes in oocytes, we used serial analysis of gene expression (SAGE) data from purified oocytes [Previously, we assigned the plus-one nucleosomes for 3903 3K4me2/3 . House-k oocytes . Out of Two additional features of the endo-cleavage in activated oocyte chromatin were revealed upon detailed examination of the plots in Figure 1. The ends of the activated oocyte DNA fragments tend to be within regions that are packaged into nucleosome cores in mixed-tissue preparations. For example, at the plus-one nucleosome position . We note the possibility that the underlying structure in these regions may be non-nucleosomal in some or all oocytes : whether the surface of interaction being revealed by these cleavages is indeed a pair of nucleosomes remains to be determined.in vitro[. A significant fraction of the C. elegans genome has a strong approximately 10-nt periodic A(n)/T(n) signal [in vitro[DNA with short A(n)/T(n) sequences that are distributed along one face of the DNA double helix exhibits unusual curvature and a tendency for the A(n)/T(n) sequence to face inward towards the nucleosome, at least in vitro-40. A si) signal -43, with) signal . Althoug[in vitro  t-2N-1|N1N2). Regardless of the sequence composition at the cleavage site, we observed a consistent bias for the A(n)/T(n)-rich face to be protected from cleavage in the activated oocyte DNA /T(n) signal is associated with the rotational positioning in activated oocytes, each position in the PATC regions was assigned an integer phase /T(n) face  are consistently organized on a surface that constrains accessibility of one helical face. That these organized segments are larger than individual nucleosomes argues either (i) for a stereotyped multi-nucleosome structure that might allow an uninterrupted approximately 10-bp periodicity, (ii) for a larger 'mega’ nucleosome-like structure that might accommodate several hundred base pairs of DNA, or (iii) for a large non-nucleosomal surface that might organize DNA. We consider each of the three models to be potentially valid hypotheses for further study.The patterns of DNA fragmentation in activated A number of previous structural discussions  could account for a periodicity as we have observed; candidate surfaces might include nuclear lamina and envelope structures, meiotic condensation cores, and  yet-to-be-discovered protein-DNA interfaces.C. elegans[C. elegans) that the chromosome organization described here would have been present and functionally relevant on a sufficient evolutionary timescale to influence the underlying sequence, either through selection at the organismal level or through mutational biases introduced by the anisotropic activity.Whatever their structural basis, the biochemical patterns revealed by our analysis match features associated with promoter organization and periodic nucleotide sequence composition in germline-expressed . elegans genes, sC. elegans oocytes were purified from the fer-1(b232) mutant worms grown at the restrictive temperature of 25-26°C as described [2. For micrococcal nuclease (MNase) digestion, 0.1 ml oocyte crude lysate was first pre-warmed to 37°C for one minute, then treated with 0.002, 0.02, or 0.1 U/l MNase (Roche). The reaction mix was incubated at 37°C for two minutes and digestion was then stopped by adding EGTA to a final concentration of 20 mM. DNA was isolated by incubating the worm sample with 400 μl lysis buffer  at 65°C for 1 hour, followed by organic extraction and DNA precipitation as above.escribed . OocytesDNA was treated with 0.5 U/μl T4 polynucleotide kinase (NEB) in 1x T4 DNA ligase buffer  at 37°C for 1 hour to phosphorylate 5′ ends and dephosphorylate 3′ ends. Ends were then blunted in a reaction containing 0.75 U/μl of T4 DNA polymerase (NEB), dNTP , and 1x NEB buffer 1 at 12°C for 15 min. DNA was then ligated to previously annealed DNA oligonucleotides AF-SG-133 (5′ Pi-AGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG-OH 3′) and AF-SG-134 (5′ OH-CCCTACACGACGCTCTTCCGATCT-OH 3′) (NEB Quick Ligation Kit). DNA captured by linker oligonucleotides was resolved using 6% polyacrylamide (acrylamide:bis-acrylamide = 19:1) gels containing 8 M urea, followed by PCR amplification with primers AF-SG-135 (5′ OH-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-OH 3′) and AF-SG-137 (5′ OH-CAAGCAGAAGACGGCATACGAGCT-OH 3′). We carefully selected cycle numbers for which product levels have not saturated ; this protocol ensures that the majority of amplified segments are still annealed to a true complement, avoiding reannealing distortion in the resulting sequence libraries . After sAll sequencing data used in this study have been deposited in GEO ( GSE46499).fer-1(b232) oocyte DNA by using T4 polynucleotide kinase and ATP gamma [that is, two nearby cleavage events on the opposite strands are staggered by 2 to 3 nt).We radioactively labeled 5′-ends of fer-1b2 oocyte DC. elegans Gene Expression Consortium http://elegans.bcgsc.bc.ca/.SAGE data were obtained from the Genome BC PATCs: periodic A(n)/T(n) clusters; SAGE: serial analysis of gene expression.The authors declare that they have no competing interests.fer-1 Oocytes were prepared by B. G. and J.D.M. Experiments and data analyses for Figure Experiments were conceived in discussions among all authors. Figure S1. DNA extracted from MNase digested chromatin and naked DNA treated with. MNase digestion was resolved on a 3% agarose gel (a) or a denaturing 12% polyacrylamide gel (8 M urea) (b).Figure S2. Size distributions of fragmented fer-1(b232) oocyte DNA determined by paired-end Illumina sequencing for each of the four libraries prepared from fragmented DNA in different size ranges . Figure S3. Characterization of the ends of fragmented fer-1(b232) oocyte DNA. Denaturing PAGE analysis of fragmented fer-1(b232) oocyte DNA treated with T4 DNA polymerase and a full set of dNTP. Figure S4. A positional correlation analysis of endo-cleaved DNA fragments from fer-1(b232) C. elegans oocyte for each chromosome (A) A positional correlation analysis of endo-cleaved DNA fragments from fer-1(b232) C. elegans oocyte (B) and wild type (N2) embryo (C) chromatin using endo-cleavage ends that fall in intronic or exonic sequence. Figure S5. (A) Coverage by endo-cleaved DNA fragments (left panels) or MNasegenerated nucleosome DNA (right panels) from fer-1(b232) C. elegans oocytes was plotted as a function of position for each of the C. elegans six chromosomes. (B) A density scatter plot comparing the coverage of endo-cleaved DNA fragments and coverage of MNase-generated nucleosome DNA from fer-1(b232) C. elegans oocyte for each one-kb window throughout the genome. The density of dots are indicated by color. (C) Aggregate coverages of endo-cleaved DNA fragments or MNase-generated nucleosome DNA from fer-1(b232) C. elegans oocyte were plotted as a function of positions at the 5′ end and 3′ ends of annotated C. elegans genes. Genes are grouped as high, median, and low based on the levels of oocyte expression.Click here for file


Therefore, C646 may be a potential candidate for treating AE-positive AML.AML1-ETO fusion protein (AE) is generated by t chromosomal translocation, which is one of the most frequently observed structural abnormalities in acute myeloid leukemia (AML) and displays a pivotal role in leukemogenesis. The histone acetyltransferase p300 promotes self-renewal of leukemia cells by acetylating AE and facilitating its downstream gene expression as a transcriptional coactivator, suggesting that p300 may be a potential therapeutic target for AE-positive AML. However, the effects of p300 inhibitors on leukemia cells and the underlying mechanisms have not been extensively investigated. In the current study, we analyzed the anti-leukemia effects of C646, a selective and competitive p300 inhibitor, on AML cells. Results showed that C646 inhibited cellular proliferation, reduced colony formation, evoked partial cell cycle arrest in G1 phase, and induced apoptosis in AE-positive AML cell lines and primary blasts isolated from leukemic mice and AML patients. Nevertheless, no significant inhibitory effects were observed in granulocyte colony-stimulating factor-mobilized normal peripheral blood stem cells. Notably, AE-positive AML cells were more sensitive to lower C646 doses than AE-negative ones. And C646-induced growth inhibition on AE-positive AML cells was associated with reduced global histone H3 acetylation and declined  Leukemogenesis involves a variety of recurrent chromosomal abnormalities. t translocation is the most common chromosomal aberration identified in AML, which occurs in 40% of patients with French-American-British (FAB) M2 subtype and constitutes 12% of all newly-diagnosed cases p300 protein is a transcriptional co-activator with intrinsic histone acetyltransferase (HAT) activity, and it plays a crucial role in cell cycle progression, differentiation and apoptosis c-kit and bcl-2 levels. These results suggest a remarkable potential of C646 for treating AE-positive AML.Despite that anti-tumor activity of p300 inhibitors in other cancers has been reported 6 viable cryopreserved primary leukemia cells from AML1-ETO9a (AE9a) transgenic leukemia mice 6 viable cells/mouse. Animals were maintained in a room at 22–25°C under a constant day/night rhythm and given food and water ad libitum. All animal experiments were carried out in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee at our hospital.Female C57BL/6 mice  were supplied by the experimental animal center of our hospital. A total of 1×10The mononuclear cells from bone marrow samples with more than 70% blasts from 2 untreated AML patients, and granulocyte colony-stimulating factor-mobilized PBSCs from a healthy donor were obtained from Department of Hematology in our hospital, and prepared by Ficoll-Hypaque density gradient centrifugation . The FAB subtype and cytogenetic characteristics of the primary AML samples were as follows: patient #1, FAB M2, t; patient #2, FAB M2, t; patient #3, FAB M2, normal karyotype. This study was carried out in accordance with the principles of Declaration of Helsinki, and was approved by the Human Subject Ethics Committee in our hospital. Signed informed consent was obtained from each subject.2+-inducible mouse MT1 promoter 4 for 8 h to induce AE expression.The above cell lines were maintained in RPMI-1640 medium  supplemented with 10% fetal bovine serum. For SKNO-1 cells, 10 µg/L granulocyte-macrophage colony-stimulating factor  was added to the medium. Primary AML blasts isolated from the spleen of transplanted AE9a leukemia mice or the bone marrow sample of AML patients, as well as normal PBSCs isolated from the healthy donor were cultured in Iscove's Modified Dulbecco's Medium  supplemented with 20% fetal bovine serum, 100 µg/L stem cell factor, 10 µg/L interleukin-3, 10 µg/L interleukin-6 and 10 µg/L granulocyte-macrophage colony-stimulating factor .Kasumi-1, SKNO-1, HL60, NB4, HEL, K562 and U937 cell lines were obtained from American Type Culture Collection . U937-AE cell line was a gift from Dr. Clara Nerv . This cell line was obtained by electroporation into U937 wild-type cells of an HA-tagged AE cDNA subcloned into a vector carrying the Zn6 cells/well. C646 stocks (10 mM in anhydrous DMSO) were directly added to culture media at desired concentrations. DMSO concentration was kept constant at 0.1% among different treatments. After treatment with C646 or DMSO for 24 h, cells were harvested and subjected to the following assays. The number of viable cells was assessed by Cell Counting Kit-8 . For cell cycle assay, the cells were washed twice with ice cold PBS and fixed in 70% ethanol at 4°C overnight, followed by incubation with 10 µg/mL Ribonuclease A  at 37°C for 30 min. The cells were then incubated with 50 µg/mL propidium iodide . Flow cytometry analysis of DNA content was performed on a FACScalibur flow cytometer . ModFit LT software  was used for subsequent analysis. For apoptosis assay, cells were stained with Annexin V-FITC  or Annexin V-Alexa Fluor 647 , and analyzed by flow cytometer. FlowJo software  was used for subsequent analysis.Cells were seeded in 6-well plates at 1×103 cells/mL. For primary leukemia cells, 100 µg/L stem cell factor, 10 µg/L interleukin-3, 10 µg/L interleukin-6 and 10 µg/L granulocyte-macrophage colony-stimulating factor were added to the methylcellulose medium and the cells were seeded at a concentration of 1×105 cells/mL. Colony formation was assessed 7 to 14 d later The frequency of colony forming units (CFU) was calculated as number of colonies counted/number of cells plated.Cells were treated with C646 or DMSO as described above. After 24 h, cells were harvested and subjected to colony formation assay by using Methocult H4230 . For cell lines, they were plated in methylcellulose at a concentration of 1×10Cells were treated with C646 or DMSO as described above. In some experiments, the pan-caspase inhibitor Q-VD-OPH  was added at 50 µM 1 h prior to addition of C646. Total protein was extracted from cells using radio immunoprecipitation assay buffer  in the presence of proteinase inhibitor cocktail . Polyacrylamide gel electrophoresis, tank-based transfer to Immobilon Hybond-C membranes (Amersham Biosciences) and immunodetection were performed with standard techniques. The following antibodies were used: caspase-9 (Asp330) antibody, cleaved caspase-8 (Asp391) (18C8) antibody, cleaved caspase-3 (Asp175) (5A1E) antibody , AML1/RHD domain (Ab-2) antibody , c-kit (C-19) antibody , bcl-2 antibody , histone H3 antibody  and acetylated H3 antibody . β-actin antibody  was used to normalize the amount of analyzed samples. Signals were visualized using Immobilon Western Chemiluminescent HRP substrate  by exposure to films.15. Quantitative real-time PCR (qRT-PCR) was carried out in an ABI Prism 7500 Fast Real-time PCR System using TaqMan master mix  according to the protocol. All data were normalized using the endogenous control (ABL). The sequences for the primers and probes were described in RNA was isolated from cells using TRIzol reagent  and cDNA was synthesized from 1 µg of total RNA using oligo(dT)t test was applied to compare C646-induced changes to respective controls. The survival data were presented in a Kaplan-Meier format showing the percentage of mouse survival at various time-points post-transplantation. The overall survival comparisons among subtypes were performed by Mantel-Haenszel log-rank test. A P value of less than 0.05 was chosen as a threshold for statistical significance.SPSS 15.0 software  was used to process the data. Student's vs 2.5 µM), the apoptotic percentage expressed a gradual increase over time. To further analyze the mechanism of C646-induced apoptosis, the dose- and time-course caspase cleavage were investigated by Western blot. C646 induced cleavage of caspases 3, 8, and 9 in Kasumi-1 cells. Increased cleavage occurred with increasing the concerntration or exposure time of C646  showed only a marginal increase in the percentage of cells in G1 phase . Even tron of AE , were mope cells . Togethein vitro treatment of C646 induced a cell cycle arrest in G1 phase and a dramatic elevation in apoptotic percentage in the AML blasts isolated from the spleens of leukemia mice. The number and size of colonies formed in vitro were also markedly reduced upon C646 treatment (vs 30 d), indicating that C646 could suppress in vivo growth of transplanted leukemia blasts  of the ETO gene, encodes a C-terminally truncated AE protein and is expressed in the majority of t patients reatment . Notablya blasts .Next, we assessed the effects of C646 on human primary leukemia blasts isolated from AE-positive and -negative AML patients and normal hematopoietic stem cells isolated from granulocyte colony-stimulating factor-mobilized PBSCs of 2 healthy donors. As shown in c-kit proto-oncogene and bcl-2 anti-apoptotic gene appear to be abnormal activation and closely related to apoptosis, cell cycle and proliferation in AE-positive AML cells c-kit and bcl-2 by Western blot and qRT-PCR, respectively. Consistent with the induction of cell cycle arrest and apoptosis, a significant decrease of c-kit and bcl-2 protein levels were observed in Kasumi-1 and SKNO-1 cells treated with 10 µM of C646  AML patients are considered as a favorable risk group, for more than 80% of younger cases can reach a complete remission. However, 40–50% of patients relapse and the long-term disease-free survival rate is around 60% In addition to growth arrest, p300 is required for orderly G1/S transition in human cancer cells and inhibition of p300 induces block of progression into the S-phase of cell cycle and apoptosis c-kit and bcl-2 in Kasumi-1 and SKNO-1 cell lines, which characterized by harboring c-kit mutation/overexpression, as well as bcl-2 overexpression. Corresponding to the growth-inhibitory effects of C646 on AE-positive leukemia cells, there was a dose-dependent reduction in global histone H3 acetylation. It has been confirmed that aberrant activation of c-kit promotes cell cycle progression and contributes to abnormal cell proliferation by altering the tyrosine kinase signaling c-kit mutation cooperates with AE to induce leukemogenesis bcl-2bcl-2, and down-regulation of c-kit and bcl-2 might involve in C646-mediated growth inhibition, cell cycle arrest and apoptosis in AE-positive AML cells. Therefore, the suppressive activity of C646 on aberrant expression of c-kit and bcl-2 explains the high selectivity and sensitivity of AE-positive cells to C646. Certainly, the effects of C646 on AE-positive AML cells reflect a collective suppression of histone acetylation, bcl-2, c-kit and other factors. Identification of these uncharted factors and their roles in AML cells remains the subject of future investigations.The ability of p300 to acetylate cellular proteins is critical for their functions in growth control. The HAT activity endows p300 the capacity to influence chromatin activity by modulating histones and several non-histone proteins c-kit and bcl-2 levels. The credible selectivity for AE-positive AML cells but not AE-negative ones, and the comparative safety for normal PBSCs provide C646 a nice perspective in the clinics. Further investigating the in vivo effects of C646 will undoubtedly promote its clinical application for relevant patients.In conclusion, C646 exerts anti-leukemia effects on AE-positive AML cells. C646 inhibits cellular proliferation, reduces colony formation, evokes partial cell cycle arrest in G1 phase, and induces apoptosis in AE-positive AML cells, with reduced histone H3 acetylation and declined Table S1Sequences of the primers used in this study.(DOC)Click here for additional data file.


To explore the key regulatory genes associated with lung cancer in order to reduce its occurrence and progress through silencing these key genes.To identify the key regulatory genes involved in lung cancer, we performed a combination of gene array and bioinformatics analyses to compare gene transcription profiles in 3 monoclonal cell strains with high, medium or low metastatic abilities, which were separated from the SPC-A-1sci and SPC-A-1 cell lines by limiting dilution monoclone assay. We then analyzed those genes’ biological activities by knocking down their expression in SPC-A-1sci cells using siRNA and lenti-viral shRNA vectors, followed by determinations of the invasion and migration capabilities of the resulting cell lines in vitro as well as their potential for inducing occurrence and metastasis of lung cancer in vivo. To examine the clinical relevance of these findings, we analyzed the expression levels of the identified genes in human lung cancer tissues (n = 135) and matched adjacent normal tissues by immunohistochemical (IHC) staining.. Clinical sample analyses indicated that osteopontin, LAMB3 and ITGB1 protein expression levels were higher in lung cancer patients, compared to non-cancerous adjacent tissues, and correlated with lymphatic metastasis.Three monoclonal cell strains characterized with high, medium or low metastatic abilities were successfully selected. Gene array and bioinformatics analyses implied that osteopontin, LAMB3 and ITGB1 were key genes involved in lung cancer. Knockdown of these genes suppressed human lung cancer cell invasion and metastasis in vitro and in vivoWe confirmed that osteopontin, LAMB3 and ITGB1 played important roles in the occurrence and metastasis of lung cancer, thus provided important clues to understanding the molecular mechanism of metastasis and contributing to the therapeutic treatment of lung cancer. The bioinformatics analysis revealed more details about the significant functions, pathways, expressive tendencies and signal flows of these different genes. The resulting graph from the signal-flow model indicated key regulatory roles for SPP1 (or osteopontin), LAMB3, MAPK1, and JUN in the metastasis of the SPC-A-1sci cell line.Previously, it has been reported that cells change their cell-cell adhesion properties, rearrange their extracellular matrix (ECM) environment, and reorganize their cytoskeletons to facilitate migration and invasion.A lot of evidences have indicated that osteopontin facilitated the attachment of cells to the ECM through binding to several types of integrins (INTs), enhanced the expression and activity of MMP-2, and promoted tumor growth and metastasis through the activation of survival pathways All animal experiment protocols used in this study were approved by the Shanghai Medical Experimental Animal Care Commission at Shanghai Jiaotong University .2.The human lung adeno-carcinoma cell line SPC-A-1 was obtained from Cellular Institute of Chinese Academy of Science . This cell line was originally isolated from the surgical specimens of a Chinese man with advanced lung adeno-carcinoma by Shanghai Chest Hospital and Cellular Institute of Chinese Academy of Science in 1980 SPC-A-1sci cells were trypsinized and resuspended in DMEM at a concentration of 100 cells per 10 mL, of which 0.1 mL was added to each well of 96-well plates already containing 0.1 mL of 10% FBS DMEM. Two weeks later, each well was observed under a microscope, and 20 monoclonal strains of SPC-A-1sci cells were selected and propagated.Total RNA from each cell line was harvested using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. Total RNA was sent to the Shanghai Biotechnology Co., Ltd . Total RNA was hybridized onto the Affymetrix U133 plus 2.0 array  and processed according to Affymetrix technical protocols. Statistical algorithms were used in the analysis of GeneChip expression data.2 test were used to classify the GO category, and the false discovery rate (FDR) was calculated to correct the P-value, with a smaller FDR corresponding to a smaller error in judging the P-value. The FDR was defined as κrefers to the number of Fisher’s test P-values less than the χ2 test P-values. We computed P-values for the GO classifications of each differentially expressed gene. Enrichment provides a measure of the significance of the function: as the enrichment increases, the corresponding function is more specific, which helps us to identify those GOs in the experiment with more concrete functional descriptions. Within each significant category, the enrichment (Re) was given by: f is the number of differential genes within the particular category, n is the total number of genes within the same category, fN is the number of differentially expressed genes in the entire microarray, and N is the total number of genes in the microarray GO analysis was applied to analyze the main functions of the differentially expressed genes according to Gene Ontology, the key functional classification of the NCBI. Generally, Fisher’s exact test and χThe Go-map, an interaction network of the significant GOs of the differentially expressed genes, was built according to the Gene Ontology classifications to identify interactions among the significant GOs directly and systemically. This map potentially summarizes the functional interactions of the differentially expressed genes under disease conditions 2 test to select the significant pathways, and the threshold of significance was defined by the P-value and FDR. The enrichment was calculated according to the equation described above Similarly, pathway analysis was used to determine the significant pathways of the differentially expressed genes according to KEGG, Biocarta and Reatome. Again, we utilized the Fisher’s exact test and χA Path-Net was built to directly and systemically identify interactions among the significant pathways of the differentially expressed genes according to the interactions among pathways of the KEGG database. It provided a summary of the pathway interactions of the differentially expressed genes under disease conditions and assisted in determining why a certain pathway was activated  denotes the weight of gene j into gene i, j, i expression value at the t time point. Using this genetic algorithm, we estimated model parameters External cellular stimulus affects cellular behavior and is reflected in protein interaction and gene expression kinetics. We therefore inferred a dynamic gene regulatory network, calculated according to the fold expression of genes and their interactions in pathways. The relationships of the gene expression data were inferred using a continuous time recurrent neural network (CTRNN) as an abstract dynamic model for the gene regulatory network mediating the cellular decision to migrate upon an external stimulus. The model describes the mutual influence of genes and their stimulus response as dynamic elements, regardless of how such an interaction or stimulation is realized in concrete biological terms. The CTRNN model is generally described as 5 cells/well were plated into 6-well plates, cultured overnight to achieve 50–70% confluence, then washed with DMEM without serum and antibiotics. LipofectAMINE 2000 reagent and siRNA were mixed and added to the cells at final concentrations of 5 µL/well and 2.5 µg/well, respectively, followed by incubation at 37°C for 48 h.siRNA oligonucleotides with the following sequences were synthesized by company: negative control siRNA, sense: 5′-UUC UCC GAA CGU GUC ACG Utt-3′ and antisense: 5′-ACG UGA CAC GUU CGG AGA Att-3′; osteopontin siRNA, sense: 5′-CCA UUC UGA UGA AUC UGA U-3′ and antisense: 5′-AUC AGA UUC AUC AGA AUG G-3′; LAMB3 siRNA, sense: 5′- CCA UUC UGA UGA AUC UGA U-3′ and antisense: 5′-AUC AGA UUC AUC AGA AUG G-3′; ITGB1 siRNA, sense: 5′-CCA UUC UGA UGA AUC UGA U-3′ and antisense: 5′-AUC AGA UUC AUC AGA AUG G-3′. Delivery of siRNAs was achieved using LipofectAMINE 2000 reagent  according to the manufacturer’s instructions. Briefly, 3×10Proteins from the cultured SPC-A-1sci cell lines that received siRNA treatments were harvested and pooled from 10 wells of two 6-well plates. All protein extraction procedures were performed on ice. Briefly, cells were rinsed with cold PBS, scraped by cell scraper, then transferred to 1.5 mL tubes and lysed with 200 µL of ice-cold lysis buffer per tube. After a 40-min incubation on ice, the homogenates were centrifuged at 12,000 g for 10 min at 4°C. The supernatants were collected after centrifugation.All protein extracts were stored at −20°C.Using the total protein isolated above. SDS-PAGE (12%) gels were run and subsequently transferred onto nitrocellulose membranes at 4?C. The membranes were blocked in PBS containing 5% milk for 2 hours, followed by the addition of primary antibodies against osteopontin (1∶400), ITGB1 (1∶500), LAMB3 (1∶500), and β-actin (1∶15000) Primary antibodies were incubated overnight at 4°C, followed by incubation with appropriate HRP-conjugated secondary antibodies at room temperature for 1.5 hours. The membranes were then washed with PBST , and specific proteins were detected using the HRP system.5′-GACAGCCAGGACTCCATT-3′; osteopontin reverse: 5′-GATGTCAGGTCTGCGAAA -3′; LAMB3 forward: 5′-AGGCAAGAACTGTGAGCG-3′; LAMB3 reverse: 5′-GGGTTGGCGTAGGTGAGT-3′; ITGB1 forward: 5′-AATGTAACCAACCGTAGC-3′; ITGB1 reverse: 5′-CAGGTCCATAAGGTAGTAGA-3′; β-actin forward: 5′-AGTGTGACGTGGACATCCGCAAAG-3′; β-actin reverse: 5′-ATCCACATCTGCTGGAAGGTGGAC-3′. Gene expression was normalized to β-actin. All reactions were run in triplicate.Total RNA from the cultured SPC-A-1sci cell lines that received siRNA treatments was harvested using Trizol reagent according to the manufacturer’s instructions. Reverse transcription was achieved using Reverse Transcription Reagents (Takara) and following the manufacturer’s instructions. Real-time PCR analysis was performed on a 7300 Real-Time PCR System with SDS RQ Study software (Applied Biosystems) according to the manufacturer’s instructions. cDNA templates were combined with SYBR Green premix containing Rox (Takara) to perform the quantitative PCR reactions. Primers used for quantitative PCR were as follows: osteopontin forward: The scratch-would healing assay was performed with slight modification from that previously described Cell migration and invasion assays were performed using 6.5-mm trans-well chambers  as described previously, with some modifications MluI/ClaI sites of the pLVTHM vector (Addgene) using the following oligonucleotides: 5′-CGCGTCGTAGCGACTAAACACATCAATTttc aagagaAATTGATGTGTTTAGTCGCTATTTTTTGGAAT-3′ and 5′-CGATTCCAAAAAATAGCGACTAAACACATCAATTtctcttgaaAATTGATGTGTTTAGTCGCTACGA-3′ for the negative control, CAAGAGAAATCGTCCATGTGGTCATGGCTTTTTTGGAAT-3′5′-CGTCGGCCATGACCACATGGACGATTTT and CTCTTGAAAATCGTCCATGTGGTCATGGCCGA-3′5′-CGATTCCAAAAAAGCCATGACCACATGGACGATTT for osteopontin, TTCAAGAGATCTTTGCATCTAGGATCCGGGTTTTTTGGAAT-3′5′- CGCGTCGCCCGGATCCTAGATGCAAAGA and TCTCTTGAATCTTTGCATCTAGGATCCGGGCGA-3′5′- CGATTCCAAAAAACCCGGATCCTAGATGCAAAGA for LAMB3 and TCAAGAGATGAAACTTCGGATCTGTACACTTTTTTGGAAT-3′5′-CGCGTCGGTGTACAGATCCGAAGTTTCAT and CTCTTGAATGAAACTTCGGATCTGTACACCGA -3′5′- CGATTCCAAAAAAGTGTACAGATCCGAAGTTTCAT for ITGB1. Lenti-virus generation and infection of SPC-A-1sci cells were performed as described above.The lenti-viral shRNA vector was constructed as described previously 6 SPC-A-1sci cells were injected into the tail veins of nude mice. Ten weeks later, all mice were sacrificed, and the lungs were removed and processed for standard histological studies by fixation in 10% formalin. The fixed samples were embedded in paraffin, and three non-sequential serial sections were obtained per animal. The sections were stained with H&E and analyzed for the presence of metastases.Five- to six-week-old male nude mice were maintained under specific pathogen-free (SPF) conditions. Mice were manipulated and housed according to protocols approved by the Shanghai Medical Experimental Animal Care Commission. To analyze the in vivo metastatic capabilities of the SPC-A-1sci cells with knocked-out osteopontin, Laminin β3 or Integrin β1, 2.0×10Patient samples in this study were obtained following informed consent, according to an established protocol approved by the Ethics Committee of the Shanghai Jiao-Tong University School. The data did not contain any information that may lead to the identification of the patients. Matched pairs (n = 135) of lung cancer tissues and adjacent noncancerous tissues were used for the construction of a tissue microarray  as previously described (34). Immunohistochemical staining was performed to detect the expression of osteopontin, LAMB3, and ITGB1 in the lung cancer tissues and matched non-cancerous tissues. The primary antibodies against osteopontin, LAMB3, and ITGB1 were obtained from Sigma (1∶250), Santa Cruz (1∶50), and Abgent (1∶50). Scoring was measured by the percentage of positive cells with the following staining intensities: less than 5% scored “0”; 5–24% scored “1”; 25–49% scored “2”; 50–74% scored “3”; and more than 74% scored “4”.t-test between two groups . Fisher’s exact test was used to compare qualitative variables. Analyses were performed with SAS 9.0 for Windows.The results are presented as the mean ± SD. Comparisons of quantitative data were analyzed by Student’s In our previous work, a highly metastatic lung cancer cell line (named SPC-A-1sci) was established from the poorly metastatic human lung cancer cell line SPC-A-1 through three cycles of in vivo selection. The significantly higher metastatic potential of the SPC-A-1sci cell line relative to the SPC-A-1 line has been proven by a series of experiments in vivo and in vitro. To explore the causes of the high metastatic potential of cell line SPC-A-1sci, we further obtained the monoclonal cell strains SPC-A-1sci H and SPC-A-1sci M from the SPC-A-1sci cells and the monoclonal SPC-A-1L cell strain from the SPC-A-1 cell line by limiting dilution clonal assay, as described previously, with slight modifications .To explore the metastatic potentials of SPC-A-1sciH, SPC-A-1sciM and SPC-A-1L, scratch-wound healing  and migrFirst, we identified genes differentially expressed by at least twofold in SPC-A-1sciH, SPC-A-1sciM cells respectively compared to SPC-A-1L cells. We found that a total of 4534 genes were differentially expressed as the intersection of SPC-A-1sciH vs. SPC-A-1L and SPC-A-1sciM vs. SPC-A-1L. Of these genes, 2277 genes were upregulated, and 2257 genes were downregulated.P-value <0.0001, from the functional analysis of the up-regulated differentially expressed genes, we found that significant functions were mainly associated with hemidesmosome assembly, positive regulation of erythrocyte differentiation, vesicle organization, synaptogenesis, microtubule and cytoskeleton organization and regulation, cell cycle arrest and regulation, cell adhesion and endocytosis, DNA transcription, inactivation of MAPK activity, protein amino acid dephosphorylation and intracellular protein transport. Cellular proliferation, differentiation and apoptosis were also influenced, accompanied by corresponding immune response and inflammation functions. At the same time, the majority of the down-regulated differentially expressed genes had functions in cell proliferation including: DNA replication and regulation during mitosis, spindle organization, centrosome duplication and cell-cycle checkpoint, nucleotide and negative regulation of epithelial cell proliferation as well as response to DNA damage stimulus through DNA repair and recombination, chromatin assembly or disassembly, carbohydrate, lipid and amino acid metabolism and others.To more precisely identify the key genes related to lung cancer metastasis, bioinformatic analysis was applied to analyze the functional and pathway differences between SPC-A-1sciM vs SPC-A-1L and SPC-A-1sciH vs SPC-A-1L, respectively . The results presented in the Go-map revealed the interactions and attributions for the significant functions of the differentially expressed genes. With a threshold P-value <0.001) .From the pathway analysis and Path-Net, we came to the conclusion that the up-regulated genes mainly participate in the MAPK signaling pathway, Wnt signaling pathway, JAK-STAT signaling pathway, TGF-beta signaling pathway, and cell adhesion. Identified genes also influence the ability of the cell to change its cytoskeleton and polarity and are involved in the occurrence, migration and metabolism of tumors. Meanwhile, the down-regulated genes mainly participated in mismatch repair and homologous recombination or were associated with the PPAR, TGF-beta, and p53 signaling pathways, ECM-receptor interactions and numerous metabolic pathways , a signal-flow model was built. A quantitative regulation network, it is calculated by a neural network using the signal intensities of the differentially expressed genes. Determined from the weight difference between 2 genes and the genes that each gene is connected with, the status of SPP1, LAMB3, MAPK1 and JUN as key regulators was demonstrated . The expTo explore the roles of osteopontin, LAMB3 and ITGB1 in the metastasis of lung cancer, we transfected SPC-A-1sci cells with siRNA-osteopontin, siRNA-LAMB3 or siRNA-ITGB1 and performed migration and invasion assays. RT-PCR and Western blot analysis demonstrated effective knock-down of osteopontin, LAMB3, and ITGB1 in the SPC-A-1sci cells . SPC-A-16 SPC-A-1sci cells transfected with the shRNA-negative control, shRNA-osteopontin, shRNA-LAMB3 or shRNA-ITGB1. Ten weeks later, lungs were collected for immunohistochemical analysis  and matched adjacent normal tissues by immunohistochemical (IHC) staining. Correlations between the osteopontin, LAMB3 and ITGB1 expression levels and clinicopathological characteristics of lung cancer are summarized in Cancer metastasis is a continual, dynamic, complex, multi-step progression with multiple interacting factors that is one of the most important causes of cancer-related death and therapeutic treatment failure. Although confusion and perplexity exist in this field, understanding metastatic progression is significant to inhibiting the development of cancer and key to curing it.. We then performed a combination of gene array and bioinformatics analyses to explore the key genes involved in lung adenocarcinoma metastasis, hoping that new thoughts and clues would be indicated for the research of cancer metastasis. The study of the identified pathways, activities and functions could possibly lead to a cure for cancer.The research in our laboratory has focused on lung cancer metastasis, and we have successfully established a highly node-metastatic mouse model of human lung cancer and obtained a human lung cancer cell line with high metastatic potential that offers us an excellent tool and platform for further research. Recently, increasing evidence has shown that in a primary malignant tumor, there exist only a few highly metastatic cells capable of metastasis and expressing the metastatic phenotype, rather than all of the tumor cells being able to invade and migrate A great deal of evidence has shown that a high level of osteopontin expression is associated with highly metastatic cancer cells Laminins are a family of large glycoproteins present in various types of BM In conclusion, it has become evident that osteopontin, Laminin and Integrin are associated with tumor metastasis and can enhance the migration and invasion of malignant metastatic cells from the primary tumor, mainly through changing adhesion in extracellular matrix. Here, we also provide evidence that osteopontin, LAMB3 and ITGB1 play important roles in regulating the metastatic progression of lung adenocarcinoma. Silencing the expression of each of these genes decreases the migration, invasion and wound-healing abilities of SPC-A-1 sci cells in vitro and suppresses murine lung cancer growth and metastasis in vivo. Furthermore, these 3 genes interact with each other; osteopontin and LAMB3 can both bind to ITGB1, receptors for cellular adhesion to the extracellular matrix and affect adhesion in the microenvironment to enhance the migration and invasion of highly metastatic cells. The growth and development of cancer can be inhibited if the key factors and pathways regulating the progression of cancer metastasis are discovered and blocked using the apposite antagonists. In our experiments, when we inhibit both mRNA and protein expression of osteopontin, LAMB3 and ITGB1 by applying siRNA and shRNA, the growth and metastasis of murine lung cancer decreased in vivo. Furthermore, osteopontin, LAMB3 and ITGB1 expression levels in human lung cancer tissues and matched adjacent normal tissues were examined. There was a significant association between osteopontin, LAMB3 and ITGB1 expression level that positively correlated with lymphatic metastasis in lung cancer patients.Recent preclinical studies have identified the laminin-binding integrin α3β1 as an appealing anti-cancer target Figure S1Go-map for SPC-A-1sciM/SPC-A-1L. A red node indicates the function exists in the up-regulated genes; a blue node indicates the function exists in the down-regulated genes; a yellow node indicates that both the up- and down-regulated genes have the function. The direction of the arrow represents the attribution of the function.(TIF)Click here for additional data file.Figure S2Go-map for SPC-A-1sciH/SPC-A-1L. A red node indicates the function exists in the up-regulated genes; a blue node indicates the function exists in the down-regulated genes; a yellow node indicates that both the up- and down-regulated genes have the function. The direction of the arrow represents the attribution of the function.(TIF)Click here for additional data file.Figure S3Path-Net for SPC-A-1sciH/SPC-A-1L. A red node represents a pathway of the up-regulated genes; a blue node represents a pathway of the down-regulated genes; a yellow node indicates that both the up- and down-regulated genes have the pathway. An arrow represents activation and a T shape represents inhibition.(TIF)Click here for additional data file.Figure S4Path-Net for SPC-A-1sciM/SPC-A-1L. A red node represents a pathway of the up-regulated genes; a blue node represents a pathway of the down-regulated genes; a yellow node indicates that both the up- and down-regulated genes have the pathway. An arrow represents activation and a T shape represents inhibition.(TIF)Click here for additional data file.Figure S5The osteopontin, LAMB3 and ITGB1 mRNA levels were determined by real-time PCR analysis in the SPC-A-1sci H, SPC-A-1sci M and SPC-A-1L cell lines.β-actin served as an internal control.(TIF)Click here for additional data file.Figure S6SPC-A-1sci cells co-transfected with two or more siRNA display low migration and invasion in vitro.  RT-PCR and Western blot were performed and showed the expression of osteopontin, LAMB3 and ITGB1.  Migration and invasion assays of SPC-A-1sci co-transfected with two or more siRNA (400× magnification),sh-O+sh-L represented sh-osteopontin+sh-LAMB3, sh-O+sh-I represented sh-osteopontin +sh-ITGB1, sh-I+sh-L represented sh-ITGB1+sh-LAMB3, sh-O+sh-L+sh-I represented sh-osteopontin +sh-LAMB3+ sh-ITGB1.Three independent experiments were performed; results are expressed as the mean +SD; *P<0.05, **P<0.01, ***P<0.001.(TIF)Click here for additional data file.