Primers used in the cDNA cloning and real-time quantitative PCR.

<p>Primers used in the cDNA cloning and real-time quantitative PCR.</p

The sequence information of the five <i>Lthsp</i> genes and its predicted amino acids.

<p>The sequence information of the five <i>Lthsp</i> genes and its predicted amino acids.</p

Interspecific differences in <i>T</i>_on and <i>T</i>_max between three species of <i>Liriomyza</i> leafminers.

<p>Interspecific differences in <i>T</i>_on and <i>T</i>_max between three species of <i>Liriomyza</i> leafminers.</p

The mRNA expression profiles of genes encoding five HSPs in <i>L</i>. <i>trifolii</i>.

<p>Panels: (A) <i>hsp20</i>; (B) <i>hsp40</i>; (C) <i>hsp60</i>; (D) <i>hsp70</i>; and (E) <i>hsp90</i>. The first temperature where expression was significantly higher than that the control (25°C) was described as the onset temperature (<i>T</i>_on) or the <i>hsp</i>, and the temperature at which the expression level was significantly higher than expression at other temperatures was denoted as <i>T</i>_max. <i>T</i>_on and <i>T</i>_max are marked by arrows (→), and the notable temperature shifts of <i>T</i>_on and <i>T</i>_max are indicated on the curves. The relative level of <i>hsp</i> expression represented the fold increase as compared with the expression in controls. The data were denoted as mean ± SE.</p

Salient features of five genes encoding HSPs in <i>Liriomyza</i> spp.

<p>The amino acid sequences of the deduced protein products of <i>hsp20</i> (A), <i>hsp40</i> (B), <i>hsp60</i> (C), <i>hsp70</i> (D) and <i>hsp90</i> (E) were aligned and conserved motifs or domains are indicated. Dots (.) indicate alignment. Abbreviations: Lt20, <i>L</i>. <i>trifolii hsp20</i>; Lh20, <i>L</i>. <i>huidobrensis hsp20</i> (DQ452370.1); Ls20, <i>L</i>. <i>sativae hsp20</i> (DQ452371.1); Lt40, <i>L</i>. <i>trifolii hsp40</i>; Lh40, <i>L</i>. <i>huidobrensis hsp40</i> (DQ452364.1); Ls40, <i>L</i>. <i>sativae hsp40</i> (DQ452365.1); Lt60, <i>L</i>. <i>trifolii hsp60</i>; Lh60, <i>L</i>. <i>huidobrensis hsp60</i> (AY845949.2); Ls60, <i>L</i>. <i>sativae hsp60</i> (AY851366.2); Lt70, <i>L</i>. <i>trifolii hsp70</i>; Lh70, <i>L</i>. <i>huidobrensis hsp70</i> (AY842476.2); Ls70, <i>L</i>. <i>sativae hsp70</i> (AY842477.2); Lt90, <i>L</i>. <i>trifolii hsp90</i>; Lh90, <i>L</i>. <i>huidobrensis hsp90</i> (AY851367.2); and Ls90, <i>L</i>. <i>sativae hsp90</i> (AY851368.2).</p

Alignment of 5’UTRs of <i>Liriomyza hsp</i> genes.

<p>The TATA-box-like elements are indicated by shading and the dots indicate alignment. Abbreviations are identical to those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181355#pone.0181355.g001" target="_blank">Fig 1</a>. Panels: (A) <i>hsp20</i>; (B) <i>hsp40</i>; (C) <i>hsp60</i>; (D) <i>hsp70</i>; and (E) <i>hsp90</i>.</p

Epidermal Growth Factor-Like Domain-Containing Protein 7 (EGFL7) Enhances EGF Receptor−AKT Signaling, Epithelial−Mesenchymal Transition, and Metastasis of Gastric Cancer Cells

<div><p>Epidermal growth factor-like domain-containing protein 7 (EGFL7) is upregulated in human epithelial tumors and so is a potential biomarker for malignancy. Indeed, previous studies have shown that high EGFL7 expression promotes infiltration and metastasis of gastric carcinoma. The epithelial–mesenchymal transition (EMT) initiates the metastatic cascade and endows cancer cells with invasive and migratory capacity; however, it is not known if EGFL7 promotes metastasis by triggering EMT. We found that EGFL7 was overexpressed in multiple human gastric cancer (GC) cell lines and that overexpression promoted cell invasion and migration as revealed by scratch wound and transwell migration assays. Conversely, shRNA-mediated EGFL7 knockdown reduced invasion and migration. Furthermore, EGFL7-overexpressing cells grew into larger tumors and were more likely to metastasize to the liver compared to underexpressing CG cells following subcutaneous injection in mice. EGFL7 overexpression protected GC cell lines against anoikis, providing a plausible mechanism for this enhanced metastatic capacity. In excised human gastric tumors, expression of EGFL7 was positively correlated with expression levels of the mesenchymal marker vimentin and the EMT-associated transcription repressor Snail, and negatively correlated with expression of the epithelial cell marker E-cadherin. In GC cell lines, EGFL7 knockdown reversed morphological signs of EMT and decreased both vimentin and Snail expression. In addition, EGFL7 overexpression promoted EGF receptor (EGFR) and protein kinase B (AKT) phospho-activation, effects markedly suppressed by the EGFR tyrosine kinase inhibitor AG1478. Moreover, AG1478 also reduced the elevated invasive and migratory capacity of GC cell lines overexpressing EGFL7. Collectively, these results strongly suggest that EGFL7 promotes metastasis by activating EMT through an EGFR−AKT−Snail signaling pathway. Disruption of EGFL7−EGFR−AKT−Snail signaling may a promising therapeutic strategy for gastric cancer.</p></div

EGFL7 promotes Epithelial−Mesenchymal transition (EMT) of GC cells through the EGFR–AKT signaling pathway.

<p>(A) Immunohistochemistry showing EGFL7, E-cadherin, vimentin, and Snail expression in gastric carcinoma tissues. (B) Cellular morphology of EGFL7-underexpressing cells (BGC2-13, MKN28, and MKN28-NC) was distinct from that of EGFL7-overexpressed cells (BGC823, BGCNC, and MKN28-EGFL7). BGC823, BGCNC, and MKN28-EGFL7 cells exhibited loss of intercellular contacts and typical spindle-shaped mesenchymal cell morphology, whereas BGC2-13, MKN28, and MKN28-NC cells exhibited an epithelial cell-like morphology with small cell size and cobblestone-like shape with tightly arranged intercellular contacts. (C) and (D) Expression levels of EMT-related molecules in cell lines analyzed by qRT-PCR (*<i>P</i><0.05, **<i>P</i><0.05, ***<i>P</i><0.05). (E) Western blot was used to confirm changes in expression of EMT-related molecules. qRT-PCR and Western blot results showed higher E-cadherin mRNA and protein expression levels in BGC2-13 cells and lower mRNA and protein expression levels of the mesenchymal markers vimentin and Snail. GAPDH served as an internal control for qRT-PCR reactions and Western blot. Error bars represent SD of triplicate experiments (*<i>P</i><0.05, **<i>P</i><0.05, ***<i>P</i><0.05 compared to BGC823 and BGC-NC cells). Conversely, qRT-PCR and Western blot results showed lower E-cadherin mRNA and protein expression levels in MKN28-EGFL7 cells and higher mRNA and protein expression levels of mesenchymal markers vimentin and Snail. Error bars represent SD of triplicate experiments (*<i>P</i><0.05, **<i>P</i><0.05, ***<i>P</i><0.05, compared to MKN28 and MKN28-NC cells). (F) Western blots showing both total and phosphorylated levels of EGFR, ERK1/2, and AKT in BGC823, BGC-NC, BGC2-13, MKN28-EGFL7, MKN28-NC, and MKN28 cells. Phosphorylated EGFR and AKT levels (pEGFR and pAKT) were significantly lower in BGC2-13 cells than in BGC823 and BGC-NC cells, while pEGFR and pAKT expression levels were significantly higher in MKN28-EGFL7 cells compared to MKN28 and MKN28-NC cells. Total EGFR and AKT levels did not differ significantly among cell lines. Neither total ERK1/2 nor pERK1/2 differed significantly. Western blots were performed in triplicate.</p

EGFL7 modulates the growth and metastasis of GC xenograft tumors in nude mice.

<p>(A) Subcutaneous xenograft tumors were significantly smaller in BGC2-13 cell-injected mice compared to BGC823 cell- and BGC-NC cell-injected mice, while MKN28-EGFL7 cell-injected mice exhibited significantly larger tumors than MKN28 cell- and MKN28-NC cell-injected mice. Tumor volume was calculated according to tumor volume (mm³) = 0.5×length×width² (*<i>P</i><0.05, **<i>P</i><0.01). (B) Subcutaneous xenograft tumors were analyzed by H&E staining. Liver metastasis was observed in mice injected with high EGFL7-expressing cells (BGC823, BGC-NC, and MKN28-EGFL7). Black arrow, metastatic cancer cells in the liver tissues of nude mice (H&E staining, original magnification×200). (C) Average MVD of tumors was lower in the BGC2-13 group compared to the BGC823 and BGC-NC groups (4±1.2 <i>vs.</i> 15±2 and 13±1, *<i>P</i><0.05), while the average MVD of tumors was higher in the MKN28-EGFL7 group than the MKN28 and MKN28-NC groups (28.7±6.02 <i>vs.</i> 4.3±1.53 and 5.0±2.0, **<i>P</i><0.05).</p

EGFL7 induces anoikis resistance of GC cells in suspension culture.

<p>(A) Effect of EGFL7 underexpression on anoikis resistance. Representative cytograms from flow cytometry analysis of apoptotic cells revealed by Annexin V-PE/7-AAD staining of BGC823, BGC-NC, and BGC2-13 cells after 24 h in suspension culture. A greater percentage of BGC2-13 cells (22.95±1.72%) were apoptotic compared to BGC823 (11.83% ±0.99%) and BGC-NC cells (9.36% ±1.65%). (B) Effect of EGFL7 overexpression on anoikis resistance. Representative cytograms from flow cytometry analysis of apoptotic cells revealed by Annexin V-PE/7-AAD staining of MKN28-EGFL7, MKN28-NC, and MKN28 cells after 24 h of suspension culture. The number of apoptotic MKN28-EGFL7 cells (5.13% ±0.65%) was lower than the number of apoptotic MKN28 (29.53% ±0.68%) and MKN28-NC cells (35.98% ±1.77%). (C) and (D) Flow cytometry results plotted as the mean ± SD of triplicate experiments. *<i>P</i><0.05 considered significant. All experiments were performed in triplicate and repeated at least three times.</p