Proliferation Of HGC-27 Cells Research Articles

Long noncoding RNA NNT-AS1 promotes gastric cancer proliferation and invasion by regulating microRNA-363 expression.

Increasing studies showed that long noncoding RNAs (lncRNAs) had crucial regulatory roles in various tumors, including gastric cancer (GC). Recent studies demonstrated that lncRNA nicotinamide nucleotide transhydrogenase-antisense RNA1 (NNT-AS1) played an important role in several tumors. However, the role and expression of NNT-AS1 in GC progression remain unknown. In our study, we indicated that NNT-AS1 expression was upregulated in GC samples compared with the nontumor tissues. We also showed that NNT-AS1 expression was upregulated in the GC cell lines. Ectopic expression of NNT-AS1 promoted GC cell line HGC-27 cell proliferation, cell cycle progression, and invasion. In addition, we showed that NNT-AS1 acted as a sponge competing endogenous RNA for microRNA-363 (miR-363), which was downregulated in the GC samples and cell lines. miR-363 expression was negatively related with NNT-AS1 expression in GC samples. Upregulated expression of miR-363 suppressed GC cell growth, cycle, and invasion. Furthermore, we reported that elevated expression of NNT-AS1 promoted GC cell proliferation, cycle, and invasion partly by suppressing miR-363 expression. These results indicated that lncRNA NNT-AS1 acted as an oncogene in the development of GC partly by inhibiting miR-363 expression.

Effects of recombinant adenovirus Ad-miR-29b2c on HGC-27 cell proliferation and migration

We constructed recombinant adenoviruses expressing miR-29b2c (Ad-miR29b2c), and analyzed their effects on the proliferation and migration of HGC-27 and MGC-803 cells. miR-29b2c gene was amplified by PCR from genomic DNA and cloned into the pAdTrack-CMV vector to create the shuttle plasmid pAdT-29b2c. The recombinant plasmid was verified by restriction enzyme digestion and sequencing. The linearized shuttle vector was mixed with an adenoviral backbone plasmid (pAdEasy-1), followed by cotransformation into competent BJ5183 cells to generate the recombinant plasmid pAd-miR-29b2c. Finally, recombinant adenoviral vectors were generated by transfecting the recombinant plasmid into 293A packaging cell line. HGC-27 and MGC-803 cells were infected with the recombinant adenoviruses expressing pAd-miR-29b2c, then MTT and wound-healing assay were used to analyze the effects of pAd-miR-29b2c on the proliferation and migration of HGC-27 and MGC-803 cells. The miR-29b and miR-29c levels were significantly increased in HGC-27 cells after infected with pAd-miR-29b2c. MTT and wound-healing analysis also revealed a significant decrease in proliferation and migration of HGC-27 and MGC-803 cells compared to the control Ad-GFP-infected cells. Furthermore, western blotting results demonstrated that the protein expression level of δ-catenin was reduced in pAd-miR-29b2c transfected HGC-27 and MGC-803 cells. Taken together, the recombinant adenoviral vector was generated, and it can significantly inhibit the proliferation and migration of HGC-27 and MGC-803 cells.

Overexpression of eukaryotic translation initiation factor 5A2 (EIF5A2) correlates with cell aggressiveness and poor survival in gastric cancer.

Eukaryotic translation initiation factor 5A2 (EIF5A2) plays an important role in tumor progression and prognosis evaluation. However, little information is available about its potential role in gastric cancer. This study aimed to investigate the function of EIF5A2 in tumor progression and its potential mechanisms. EIF5A2 expression was measured in human gastric cancer cell lines, the immortalized gastric mucosal epithelial cell line (GES-1) and human gastric cancer tissues and knocked down by RNA interference or upregulated by EIF5A2 plasmid transfection. Cell proliferation, migration and invasion were assessed in vitro. The downstream targets of EIF5A2 were examined by western blotting. EIF5A2 and its potential target metastasis-associated protein 1 (MTA1) expression were examined in 160 pairs of human gastric cancer and adjacent non-tumor specimens using immunohistochemistry (IHC) staining, and its correlation with clinicopathological features and survival was investigated. Knockdown of EIF5A2 or MTA1 caused an apparent suppression of HGC27 cell proliferation, migration and invasion. After knockdown of EIF5A2 in HGC27 cells, E-cadherin levels were upregulated and vimentin, cyclin D1, cyclin D3, C-MYC and MTA1 levels were downregulated. Upregulation of EIF5A2 in MKN45 cells resulted in the converse. IHC results showed a positive correlation between EIF5A2 and MTA1 expression in gastric cancers (P<0.001). Both EIF5A2 and MTA1 overexpression were correlated with pT stage (P=0.018 and P=0.042), pN stage (P=0.037 and P=0.020) and lymphovascular invasion (P=0.016 and P=0.044). EIF5A2 or MTA1 overexpression was significantly associated with poor overall survival and disease-free survival (All P<0.05). Multivariate analyses identified EIF5A2 as an independent predictor for both overall survival (P=0.012) and disease-free survival (P=0.008) in gastric cancer patients. Our findings indicate that EIF5A2 upregulation plays an important oncogenic role in gastric cancer. EIF5A2 may represent a new predictor for poor survival and is a potential therapeutic target for gastric cancer.

Expression and regulatory function of miRNA-34a in targeting survivin in gastric cancer cells

We aimed to investigate the expression of microRNA-34a (miR-34a) in human gastric cancer cells and to evaluate the effects of miR-34a, acting via its gene survivin, on gastric cancer cell HGC-27 to provide potential new strategies for treating gastric cancer. In vitro cultures of the human gastric cancer cell lines MGC80-3, HGC-27, NCI-N87, and SGC-7901 and the normal human gastric epithelial cell line GES-1 were established. The expression of miR-34a in each gastric cancer cell line and GES-1 normal human gastric epithelial cell line was detected using quantitative real-time polymerase chain reaction (qRT-PCR). After the HGC-27 cells were transfected with a miR-34a mimic for 48 h, the changes in the expression levels of miR-34a were detected using qRT-PCR. The effect of miR-34a on HGC-27 cell viability was measured using a tetrazolium-based colorimetric [-(4,5)-dimethylthiahiazo-(-z-y1)-3,5-di-phenytetrazoliumromide (MTT)] assay. Flow cytometry was used to analyze the effects of miR-34a on HGC-27 cell proliferation. Annexin V/propidium iodide double staining and flow cytometry were used to analyze the effects of miR-34a on HGC-27 cell apoptosis. A Transwell invasion chamber was used to detect the effects of miR-34a on HGC-27 cell invasion. Finally, western blotting was used to analyze the effects of miR-34a on survivin protein expression. The qRT-PCR test determined that miR-34a expression in gastric cancer cells was significantly reduced compared to the normal gastric epithelial cell line GES-1 (p < 0.01). Compared to the control group, cellular miR-34a expression levels were significantly increased in HGC-27 human gastric carcinoma cells after transfection with a miR-34a mimic for 48 h (p < 0.01). The MTT assay demonstrated that after overexpressing miR-34a in HGC-27 cells, cellular viability was significantly reduced (p < 0.05). Flow cytometry analysis determined that upon miR-34a overexpression, the proliferation index decreased significantly (p < 0.05), and cellular apoptosis was significantly increased (p < 0.01). The Transwell invasion chamber assay illustrated that after increasing the expression of miR-34a, the number of cells passing through the Transwell chamber was significantly reduced (p < 0.01). Based on western blotting, compared with the control group, survivin protein expression levels were significantly decreased in the HGC-27 cells transfected with the miR-34a mimic for 48 h (p < 0.01). In conclusion, the expression level of miR-34a was downregulated in human gastric cancer cell lines. miR-34a can negatively regulate survivin protein expression and inhibit gastric cancer cell proliferation and invasion. Therapeutically enhancing miR-34a expression or silencing the survivin gene may benefit patients with gastric cancer.

Oridonin induces apoptosis in gastric cancer through Apaf-1, cytochrome c and caspase-3 signaling pathway

To investigate the effect and mechanism of oridonin on the gastric cancer cell line HGC-27 in vitro. The inhibitory effect of oridonin on HGC-27 cells was detected using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. After treatment with 10 μg/mL oridonin for 24 h and 48 h, the cells were stained with acridine orange/ethidium bromide. The morphologic changes were observed under an inverted fluorescence microscope. DNA fragmentation (a hallmark of apoptosis) and lactate dehydrogenase activity were examined using DNA ladder assay and lactate dehydrogenase-release assay. After treated with oridonin (0, 1.25, 2.5, 5 and 10 μg/mL), HGC-27 cells were collected for anexin V-phycoerythrin and 7-amino-actinomycin D double staining and tested by flow cytometric analysis, and oridonin- induced apoptosis in HGC-27 cells was detected. After treatment with oridonin for 24 h, the effects of oridonin on expression of Apaf-1, Bcl-2, Bax, caspase-3 and cytochrome c were also analyzed using reverse-transcript polymerase chain reaction (RT-PCR) and Western blotting. Oridonin significantly inhibited the proliferation of HGC-27 cells in a dose- and time-dependent manner. The inhibition rates of HGC-27 treated with four different concentrations of oridonin for 24 h (1.25, 2.5, 5 and 10 μg/mL) were 1.78% ± 0.36%, 4.96% ± 1.59%, 10.35% ± 2.76% and 41.6% ± 4.29%, respectively, which showed a significant difference (P < 0.05). The inhibition rates of HGC-27 treated with oridonin at the four concentrations for 48 h were 14.77% ± 4.21%, 21.57% ± 3.75%, 30.31% ± 4.91% and 61.19% ± 5.81%, with a significant difference (P < 0.05). The inhibition rates of HGC-27 treated with oridonin for 72 h at the four concentrations were 25.77% ± 4.85%, 31.86% ± 3.86%, 48.30% ± 4.16% and 81.80% ± 6.72%, with a significant difference (P < 0.05). Cells treated with oridonin showed typical apoptotic features with acridine orange/ethidium bromide staining. After treatment with oridonin, the cells became round, shrank, and developed small buds around the nuclear membrane while forming apoptotic bodies. Lactate dehydrogenase (LDH) release assay showed that after treated with 1.25 μg/mL and 20 μg/mL oridonin for 24 h, LDH release of HGC-27 caused by apoptosis increased from 22.94% ± 3.8% to 52.68% ± 2.4% (P < 0.001). However, the change in the release of LDH caused by necrosis was insignificant, suggesting that the major cause of oridonin-induced HGC-27 cell death was apoptosis. Flow cytometric analysis also revealed that oridonin induced significant apoptosis compared with the controls (P < 0.05). And the apoptosis rates of HGC-27 induced by the four different concentrations of oridonin were 5.3% ± 1.02%, 12.8% ± 2.53%, 28.5% ± 4.23% and 49.6% ± 3.76%, which were in a dose-dependent manner (P < 0.05). After treatment for 24 h, DNA ladder showed that oridonin induced a significant increase in DNA fragmentation in a dose-dependent manner. RT-PCR revealed that mRNA expression levels were up-regulated compared with the controls in caspase-3 (0.917 ± 0.103 vs 0.357 ± 0.019, P < 0.05), cytochrome c (1.429 ± 0.111 vs 1.002 ± 0.014, P < 0.05), Apaf-1 (0.688 ± 0.101 vs 0.242 ± 0.037, P < 0.05) and Bax (0.856 ± 0.101 vs 0.278 ± 0.027, P < 0.05) (P < 0.05), whereas down-regulated in Bcl-2 (0.085 ± 0.012 vs 0.175 ± 0.030, P < 0.05). Western blotting analysis also confirmed this result. Apoptosis of HGC-27 induced by oridonin may be associated with differential expression of Apaf-1, caspase-3 and cytochrome c, which are highly dependent upon the mitochondrial pathway.

Mechanism of apoptosis induced by Resveratrol in human gastric cancer HGC27 cells

Objective To investigate the relationship between the expression of VEGF and MMP-9 in PTEN and p-Akt signaling pathway of resveratrol affecting gastric cancer HGC27 cells. Methods The effects of resveratrol on growth inhibition rate and cell cycle of HGC27 cells were examined by CCK-8 test and flow cytometry,respectively.The effects of resveratrol on apoptosis-related PTEN and p-Akt gene in HGC27 cells were detected by Western blotting,and the contents of VEGF and MMP-9 protein in HGC27 cells before and after resveratrol treatment were determined by ELISA. Results The growth inhibition rate of HGC27 cells by resveratrol treatment was concentration-and time-dependent(P0.05).Forty-eight hours after resveratrol treatment,cell cycle analysis indicated that the percent of HGC27 cells with DNA synthesis(S phase) significantly decreased,which exhibited a negative correlation with dosage(r=-0.930,P=0.007).There was a positive relationship between the relative expression of PTEN and Res concentration(r=0.951,P=0.04),while there was a negative relationship between the relative expression of p-Akt,VEGF and MMP-9 and Res concentration(r=-0.969,P=0.03;r=-0.894,P=0.02;r=-0.881,P=0.01). Conclusion Resveratrol could inhibit the proliferation and induce apoptosis of human gastric cancer HGC27 cells,which may be related to the expression up-regulation of PTEN and phosphorylation inhibition of Akt.Resveratrol could also regulate the expression of VEGF and MMP-9 to inhibit the proliferation and apoptosis of HGC27 cells.

Effects of Lactobacillus Rhamnosus GG on the Cell Growth and Polyamine Metabolism in HGC-27 Human Gastric Cancer Cells

Previous in vivo studies have suggested that lactobacilli can exert anti-proliferative effects on the gastric epithelium. However, few data are available on their mechanisms of action. The aim of this study was to investigate the effects of increasing concentrations of Lactobacillus rhamnosus strain GG (L. GG) homogenate on cell growth and proliferation [by 3-(4,5 di-methylthiazol-2-yl)–2,5-diphenyltetrazolium bromide, [3H]-thymidine incorporation and polyamine biosynthesis] and apoptosis processes (by Bax/Bcl-2 mRNA expression) in HGC-27 human gastric cancer cells. To verify which bacterial fraction was involved in the antiproliferative and proapoptotic effects, the cytoplasm and cell wall extracts were tested separately. HGC-27 cells were sensitive to the apoptotic induction and growth inhibition by increased concentrations of bacterial homogenate. HGC-27 cells were resistant to the bacterial cell wall fractions, whereas increasing cytoplasm fraction concentrations induced evident antiproliferative and proapoptotic actions. These data suggest that cytoplasm extracts could be responsible for L. GG action on HGC-27 cell proliferation.

Anti-tumorigenic chalcones.

On the basis of our recent findings that licochalcone A isolated from Xin-jiang licorice showed anti-inflammatory and anti-tumorigenic activities, we synthesized more than 40 chalcone derivatives to examine their anti-tumorigenic activities. In vitro inhibitory activity against phosphorylation of phospholipids promoted by tetradecanoylphorbol-13-acetate (TPA) in HeLa cells was adopted as a screening test for anti-tumor-promoting effect. In vivo experimental mouse skin tumors initiated by dimethylbenz[a]-anthracene (DMBA) and promoted by TPA were used to test the anti-tumor-promoting effect of chalcones. In the results, 3'- and 4'-methyl-3-hydroxychalcone showed the highest potency in inhibiting tumorigenesis. They also showed a remarkable inhibitory effect on the proliferation of HGC-27 cells derived from human gastric cancer. We discuss the structure-activity relationship, including stereo-chemical phototransformation, of some chalcone derivatives with reference to their ultraviolet (UV) and nuclear magnetic resonance (NMR) spectroscopic data.