Objective To observe the proliferation and migration of endothelial cells after 30% total burn surface area (TBSA) of deep partial thickness scald, and the effect of basic fibroblast growth factor (bFGF) on angiogenesis during wound healing.Methods A total of 133 male Wistar ratswere divided randomly into normal control (n=7), injured control group (n=42), bFGF group (n=42) andanti-c-fos group (n=42). The apoptosis expression of fibroblasts was determinedwith in situ hybridization and the changes of proliferation cell nuclear antigen(PCNA), focal adhesion rinase(FAK), c-fos and extracellular signalregulated kinase(ERK) proteins expression were detected with immunohistochemistry staining technique after 3 hours, 6 hours, 1 day, 3 days, 7 days, 14 days and 21 days of scald.Results In injured control group and bFGF group, theproliferation rate of the vascular endothelial had evident changes 7 days and14 days after scald； the expression of FAK was increased 14 days after scald. ERK proteins expression was different between injury control group and bFGF group at initial stage after scald. Stimulation of ERKs by bFGF led to up-regulation of c-fos and b expression of FAK. Conclusion Exogenous bFGF extended the influence on wound healing process by ERK signaling pathway, affecting migration cascade of vascular endothelial cell. The oncogene proteins play an important role on accelerating angiogenesis duringwound healing.
ObjectiveTo observe the effect of interleukin-8 (IL-8) on the adhesion and migration of retinal vascular endothelial cells (RCEC). MethodsA cell experiment. Human RCEC (hRCEC) was divided into normal control group (N group), advanced glycation end product (AGE) treatment group (AGE group), and AGE-induced combined IL-8 antagonist SB225002 treatment group (AGE+SB group). The effect of AGE on IL-8 expression in hRCEC was observed by Western blot. The effect of SB225002 on hRCEC migration was observed by cell scratch assay. The effects of SB225002 on leukocyte adhesion and reactive oxygen species (ROS) on hRCEC were detected by flow cytometry. Student-t test was performed between the two groups. One-way analysis of variance was performed among the three groups. ResultsCompared with group N, the expression level of IL-8 in cells of AGE group was significantly increased, with statistical significance (t=25.661, P＜0.001). Compared with N group and AGE+SB group, cell mobility in AGE group was significantly increased (F=29.776), leukocyte adhesion number was significantly increased (F=38.159, 38.556), ROS expression level was significantly increased (F=22.336), and the differences were statistically significant (P＜0.05). ConclusionIL-8 antagonist SB225002 may down-regulate hRCEC adhesion and migration by inhibiting ROS expression.
ObjectiveTo investigate the influence of hypoxia on pro-metastasis induced by epithelial-mesenchymal transition (EMT) of human lung adenocarcinoma.
MethodsThe human lung cancer cell line H460 was cultured in hypoxic condition and the morphologic changes of the cells were observed under the microscope. The EMT-related markers including E-cadherin and vimentin were detected by Western blot. Transwell migration assay and transwell invasion assay were employed to detect the migratory and invasive activity of cancer cells.
ResultsHypoxic induced morphological changes were consistent with the mesenchymal phenotype, such as an elongated fibroblastic morphology, and conversion from a tightly packed epithelial cobblestone pattern to a loosely packed scattered phenotype. Compared with the control group, hypoxic attenuated the quantity of E-cadhenrin, but increased vimentin, which resulted in promotion of migration and invasion of H460.
ConclusionHypoxia induces EMT in H460 and enhances the invasive and migratory abilities of lung cancer cells.
ObjectiveTo investigate the effect of up-regulation of microRNA-31(miR-31) on the biological behaviour in AGS cell of gastric cancer and on the expression of liver receptor homolog-1(LRH-1), and to analyze the possible mechanisms of miR-31 on initiation and development of gastric cancer.
MethodsAGS cells were divided into 3 groups, receiving miR-31 transfection(MT group), empty liposomes transfection(NC group), and treatment of PBS (BC group). Then the cells' proliferation was determined by cell counting kit-8(CCK-8), the apoptosis situation was determined by flow cytometer, the migration was determined by Transwell test, the expression of LRH-1 protein was tested by Western blot method, and the target of miR-31 was tested by luciferase reporter assay.
ResultsThe cell's proliferation results showed that the mean of A450 value in MT, NC, and BC groups were 1.31, 2.26, and 2.14 respectively on the 4 days after transfection, which lower in MT group(P<0.01).Results of flow cytometer experiment showed that the mean of apoptosis ratio of MT, NC, and BC groups were 39.5%, 9.3%, and 10.0% respectively, the mean of proportion of cell in G1+S stage were 92.54%, 73.23%, and 74.58% respectively, which both lower in MT group (P<0.05).Results of Transwell experiment showed that the mean of number of migrated cells in MT group was lower (P<0.05).Results of Western blot experiment demonstrated that the expression level of LRH-1 protein in MT group was lower than those of BC group and NC group(P<0.01).
ConclusionsUp-regulation of miR-31 can obviously inhibit the proliferation of AGS cell, promoting its apoptosis and depressing its migration ability. On the other side, the up-regulation of miR-31 can also inhibit the expression level of LRH-1 protein, which indirectly induces the inhibition of proliferation of AGS cell. So miR-31 may be an important regulator in the initiation and development of gastric cancer through regulating LRH-1 gene.
ObjectiveTo observe the effects of A549 cells under hypoxicconditions on the migration of human umbilical vein endothelial cells (HUVECs) and microvascular formation.
MethodsAfter cultured for 24 h in normoxia condition(21% O2),hypoxia condition (2% O2),and anaerobic condition (0% O2),respectively,morphology of A549 cells was observed with inverted phase contrast microscope,proliferation was detected by MTT assay,and intracellular hypoxia-inducible factor-1α (HIF-1α) protein was detected by immunocyto-chemical technique,for determining whether the hypoxia model is successful. Then A549 cells' supernatant in the normoxic group,the hypoxia group and HUVECs culture medium were taken to intervene HUVECs. The migration of HUVECs was observed with cell scratch test,pseudopodia formation of HUVECs was observed with microfilament green fluorescent staining method,and blood vessel formation was observed with three-dimensional culture techniques in vitro.
ResultsCompared with the normoxic group,the growth of A549 cells was better in the hypoxia group with more proliferation,and was poor in the anaerobic group with decreased number of cells. A549 cells in the hypoxia group and the anaerobic group both expressed HIF-1α protein,which was more obvious in the anaerobic group. Compared with the HUVECs supernatant intervention group,the hypoxia supernatant intervention group and the normoxic supernatant intervention group both had varying degrees of migration,pseudopodia structure formation and vascular lumen sample structure formation,which were more obvious in the former group.
ConclusionA549 cells in hypoxic environment grow very well,proliferated significantly,but anaerobic environment is not conducive to the growth of A549 cells which found to be apoptosis. A549 cells in hypoxic environment can promote HUVECs migration,pseudopodia formation and angiogenesis.
ObjectiveTo identify the expression functions of human NF-κBp65 nuclear localization signals' deletion mutant plasmids(namely pcDNA3.1(+)-NF-κBp65ΔNLS, NF-κBp65ΔNLS, for short) and the changes of proliferation, migration and adhesion ability of A549 lung cancer cells with low expression of NF-κBp65 (namely A549/NF-κBp65 shRNA cells).
MethodsHuman A549/NF-κBp65 shRNA cells were cultivated and divided into a control group, a transfection pcDNA3.1 (+) group, and a transfection NF-κBp65ΔNLS group. Indirect immunofluorescence, real-time fluorescent quantitative PCR and Western blot techniques were used to detect the NF-κBp65 intracellular localization and the change of NF-κBp65 mRNA and protein expression level. MTT, Transwell and cell adhesion experiments were used to analyze the changes of proliferation, migration and adhesion ability of A549/NF-κBp65 shRNA cells.
ResultsThe human NF-κBp65ΔNLS eukaryotic expression plasmid was successfully constructed. Compared with the control group and the transfection pcDNA3.1(+) group, NF-κBp65 mRNA expression level in A549/NF-κBp65 shRNA cells was increased in the transfection NF-κBp65ΔNLS group(10.63±0.84 vs. 1.04±0.21 and 1.23±0.22, P < 0.01) and NF-κBp65 protein expression level was also increased (1.07±0.06 vs. 0.53±0.02 and 0.59±0.04, P < 0.01). NF-κBp65 protein mainly located in the cytoplasm, and did not significantly transferred into the nucleus after stimulated by TNF-α. At the same time, A549/NF-κBp65 shRNA cells' proliferation, migration and adhesion ability were enhanced compared with the control group and the transfection pcDNA3.1(+) group.
ConclusionsThrough gene mutation technology to build the human NF-κBp65ΔNLS eukaryotic expression plasmid and transfect into A549/NF-κBp65 shRNA lung cancer cell lines, both mRNA and protein expression levels of NF-κBp65 were increased significantly, and NF-κBp65 protein mainly located in the cytoplasm. The overexpressed NF-κBp65 in cytoplasm can obviously enhance the A549/NF-κBp65 shRNA cell's proliferation, migration and adhesion ability. It suggests that NF-κBp65 stranded in the cytoplasm can still regulate biological behavior of lung cancer cells by influencing the NF-κB signaling pathway related proteins.
Objective To observe the effect of bone forming protein 4 (BMP4) on the proliferation and migration of human retinal pigment epithelium (RPE) cells under oxidative stress, and to preliminarily explore its effect on epithelial-mesenchymal transition (EMT) of RPE cells. MethodsHuman RPE cells cultured in vitro were divided into normal group, pure 4-hydroxynonenal (HNE) group (4-HNE group), 4-HNE+NC group and 4-HNE+ small interfering BMP (siBMP4) group. The effect of 4-HNE on the proliferation of RPE cells was detected by thiazole blue colorimetry. The effects of 4-HNE and BMP4 on cell migration were determined by cell scratch test. The expression of BMP4 was detected by immunofluorescence staining, Western blot and real-time quantitative polymerase chain reaction. The transfection efficiency of siBMP4 was observed by fluorescence microscopy. Mitochondrial reactive oxygen species (MitoSOX) were detected by flow cytometry. The expression of EMT markers E-cadherin and Fibronection were detected by immunofluorescence assay. t-test was used for comparison between the two groups, and one-way analysis of variance was used for comparison between the three groups. ResultsCompared with normal group, cell proliferation and migration ability of 4-HNE group were significantly enhanced, with statistical significance (t=21.619, 24.469; P＜0.05). The expression of BMP4 in cells was significantly increased, and the difference was statistically significant (t=19.441, P＜0.05). The relative expression levels of BMP4 mRNA and protein were also significantly increased, with statistical significance (t=26.163, 37.163; P＜0.05). After transfection with siBMP4 for 24 h, the transfection efficiency of BMP4 in RPE cells was＞90%. Compared with 4-HNE group and 4-HNE+NC group, the relative expression levels of BMP4 protein (F=27.241), mRNA (F=36.943), cell mobility (F=46.723) and MitoSOX expression levels (F=39.721) in normal group and 4-HNE+siBMP4 group were significantly decreased. The differences were statistically significant (P＜0.05). The epithelial marker E-cadherin increased significantly, while the mesenchymal marker Fibronection decreased significantly, with statistical significance (F= 51.722, 45.153; P＜0.05). ConclusionsBMP4 inhibits RPE proliferation and migration under oxidative stress. BMP4 is involved in inducing EMT in RPE cells.
ObjectiveTo explore the effects of silencing the expression of ubiquitin-conjugating enzyme E2T (UBE2T) gene on proliferation, apoptosis, migration and invasion abilities of A549 cells.MethodsA549 cells were cultured in vitro. Three sets of shRNA-UBE2T plasmid vectors (UBE2T-shRNA1 group, UBE2T-shRNA2 group, UBE2T-shRNA3 group) and shRNA-NC (shRNA-NC group) were constructed, respectively. A549 cells were transfected with lipofection transfection. The cells transfected with empty vector were enrolled as control (control group). The transfection efficiency was detected by RT-PCR. The effects of silencing the expression of UBE2T gene on biological behaviors (proliferation, apoptosis, migration, and invasion) of lung cancer A549 cells were detected by clone formation assay, flow cytometry, Transwell assay and scratch test. The expression of proliferation and apoptosis related proteins, and expression of PI3K/AKT signaling pathway proteins were detected by Western blot. ResultsAfter transfection, expression level of UBE2T mRNA in UBE2T-shRNA1 group, UBE2T-shRNA2 group and UBE2T-shRNA3 group was significantly down-regulated (all P<0.05), whose down-regulation was the most significant in UBE2T-shRNA3 group (P<0.05). Compared with control group and shRNA-NC group, clone formation rate, number of invasion A549 cells, scratch healing rate, Ki67 expression, PCNA expression, p-PI3K/PI3K ratio and p-AKT/AKT ratio were significantly decreased in UBE2T-shRNA3 group (P<0.05), while A549 apoptosis rate, Bax/Bcl-2 ratio and cleaved caspase-3/caspase-3 ratio were significantly increased (P<0.05). There were no significant differences in the above indexes between control group and shRNA-NC group (P>0.05). ConclusionsThe shRNA interfering with UBE2T is reliable to construct the model of A549 cells with stable low-expression UBE2T. Down-regulation of UBE2T expression can promote apoptosis of A549 cells, inhibit their proliferation, invasion and migration abilities. The mechanism may be related to inhibiting the activation of PI3K/AKT signaling pathway.
ObjectiveTo explore the effectiveness of Ovol2 gene for epithelial-mesenchymal transition (EMT) to offer some theory evidences for the targeted therapy in lung adenocarcinoma.
MethodsA549 cells were treated with control and Ovol2 overexpressioned by lentivirus infection. Real-time PCR were performed to test the mRNA level of genes correlated to EMT. Western Blot was performed for protein level of the following makers:E-cadherin, N-cadherin, vimentin, ect. Moreover, we tested the migration and invasion ability of A549 cells by transwell and wound healing experiment.
ResultsAfter treated with Ovol2 overexpressed, the expression level of E-cadherin raised, while the expression level of N-cadherin, vimentin and Twist1 declined in both mRNA and protein expression level. The results of wound healing and transwell experiment indicated that the migration and invasion ability of A549 cells weakened.
ConclusionOverexpression of Ovol2 gene can suppress the distant metastasis ability and invasion ability of A549 cells by inhibiting the EMT.
Objective To investigate the effect of keratin 17 (K-17) on the migration, prol iferation and tube formation of human umbil ical vein endothel ial cell (HUVEC), and to real ize the role of K-17 in angiogenesis. Methods After HUVEC were cultured in DMEM medium supplemented with 10%FBS overnight, K-17-siRNA-mixture (experimental group) and Ncontrol-siRNA-mixture (negative control group) were added into HUVEC, respectively, by Lipofectamine 2000 transfection assay, and the final concentration of the siRNA was 50 nmol/L. Lipofectamine 2000 alone was used as the control. After the cells were cultured for 36 hours, the cell prol iferation abil ity was detected by cell counting. After 30-hour culture, the cell’s abil ities of migration and differentiation to tube were detected by 24-well Mill icell units and the collagen gel assay, respectively. In addition, non-siRNA-treated HUVEC were cultured for 24 hours in DMEM medium supplemented with 10%FBS (group A), 2%FBS (group B) and 2%FBS+10 ng/mL bFGF (group C), respectively, and then the expression of K-17 in HUVEC was detected by RT-PCR and Western blot. Results After the treatment with K-17-siRNA for 36 hours, HUVEC exhibited no significant difference in the prol iferation, compared with both control and negative control groups (P gt; 0.05). After transfected with K-17-siRNA for 30 hours, the number of HUVEC in the experimental group which migrated from the upper chamber to the lower chamber of Mill icell wells within 24 hours (3719.0 ± 319.0) was smaller than both control (7 437.5 ± 212.0) and negative control (7 356.3 ± 795.7) groups, with significant difference (P lt; 0.01). However, there was no significant difference between the control group and the negative control group (P gt; 0.05). After HUVEC were transfected with K-17- siRNA for 30 hours, the number of tubes in the experimental group, the negative control group and the control group in 24 hours was (1.1 ± 0.5), (3.6 ± 0.5) and (3.2 ± 0.6) per field, respectively. The experimental group was significantly different from both control and negative control groups (P lt; 0.01), and there was no significant difference between the negative control group and the control group (P gt; 0.05). The expression of K-17 protein in HUVEC in groups A, B and C was 0.25 ± 0.02, 0.08 ± 0.01 and 0.72 ± 0.03, respectively. There was significant difference among these three groups (P lt; 0.01). Conclusion K-17 has no impact on cell prol iferation, but may augment endothel ial cell migration, which may facil itate angiogenesis.
