Gastric Cancer Cell Line SGC-7901 Research Articles

Long non-coding RNA LINC01314 represses cell migration, invasion, and angiogenesis in gastric cancer via the Wnt/\u03b2-catenin signaling pathway by down-regulating KLK4

BackgroundIn recent years, gastric cancer (GC) has become a major cause of mortality among various malignancies worldwide with high incidence rates. Long non-coding RNA (lncRNAs) may serve as oncogenes and tumor suppressors in cancers. Therefore, we investigated the effect of LINC01314 on the development of GC cells in relation to the Wnt/β-catenin signaling pathway.MethodsMicroarray data analysis was conducted to screen GC-related differentially expressed lncRNAs, followed by determination of the binding interaction between LINC01314 and kallikrein 4 (KLK4). Human GC cell line SGC-7901 was treated with over-expressed or silenced LINC01314 or KLK4 to investigate the mechanism LINC01314 affecting GC cellular activities. The levels of KLK4, Wnt-1, β-catenin, cyclin D1, N-cadherin and E-cadherin were measured, and cell invasion and migration were evaluated. Next, the tumor weight, micro-vessel density (MVD) and the expression of VEGF-C and VEGFR-3 in transplanted tumors were measured.ResultsLINC01314 was poorly expressed in GC cells and KLK4 was revealed to be a direct target gene of LINC01314. Overexpressed LINC01314 or silencing of KLK4 led to inhibited GC cell migration and invasion, corresponding to decreased Wnt-1, β-catenin, cyclin D1 and N-cadherin while increased E-cadherin. Also, in response to over-expression of LINC01314 or silencing of KLK4, tumor weight and the MVD of transplanted tumors were reduced and angiogenesis was suppressed, which was indicated by down-regulated positive expression of VEGF-C and VEGFR-3.ConclusionThe findings indicated that over-expression of LINC01314 down-regulated KLK4 to inhibit the activation of the Wnt/β-catenin signaling pathway, thus suppressing migration, invasion, and angiogenesis in GC cells, which provides new insight for the treatment of GC.

Effects of neddylation modification inhibitor MLN4924 on proliferation and migration of human gastric cancer cells

Objective To explore the effects of NEDD8 activating enzyme (NAE) inhibitor MLN4924 on gastric cancer cell proliferation and migration. Methods Human gastric cancer cell lines NCI-N87 and SGC-7901 were treated with different concentrations of MLN4924, and the inhibitory concentrations were detected by cell counting kit-8 (CCK-8) method. 20% maximal inhibitory concentration (IC20) was used to treat gastric cancer cells. The cell proliferation was measured by CCK-8 and clone formation assay, and cell migration was determined by cell scratch test. Results When human gastric cancer cell lines NCI-N87 and SGC-7901 were treated with 0.528 and 0.375 μmol/L respectively, the inhibitory rate reached to 20%. CCK-8 method showed that MLN4924 inhibited the proliferation of human gastric cells in a dose- and time-dependent manner. In the clone formation assay, the clone number of NCI-N87 cells was 71.00±10.19 in MLN4924 treated group and 215.33±9.56 in the control group. The clone number of SGC-7901 cells was 23.66±18.66 in MLN4924-treated group, and 32.33±34.12 in the control group.In vitro wound scratch assay revealed that the healing rate of MLN4924-treated NCI-N87 cells was (5.30±0.78)%, and (51.05±6.35)% in the control group, and that of MLN4924-treated SGC-7901 cells was (3.12±0.52)%, and (23.05±2.01)% in the control group. Conclusion Neddylation modification inhibitor MLN4924 suppresses the growth and migration of gastric cancer cells. Key words: Gastric cancer; Neddylation modification inhibitor; Proliferation; Migration

Relationship between microRNA-95 and proliferation, apoptosis of gastric cancer cells and underlying mechanism

Objective To investigate the role of microRNA (miRNA, miR)-95 in the proliferation and apoptosis of gastric cancer cells and the possible mechanism. Methods Real-time quantitative polymerase chain reaction (Real-time PCR) was used to detect the expression of miR-95 in tumor tissues and gastric mucosal tissues from 70 patients with gastric cancer. The patient’s clinicopathological features were recorded and the relationship between miR-95 and these clinicopathologic features was analyzed. The human gastric cancer cell line BGC823 was transfected with anti-miR-95 and the cell viability, cell cycle and apoptosis rate were examined before and after transfection. Simultaneously, Real-time PCR and Western blotting were used to detect the expression of proliferation- and apoptosis-related genes proliferating cell nuclear antigen (PCNA), Cyclin D1, p21, Survivin, Livin and Bad. Results The miR-95 level in gastric cancer was significantly higher than that in gastric mucosal tissues (t=-3.399, P<0.05). Its expression was related to the degree of tumor differentiation and lymph node metastasis. The level of miR-95 in poorly differentiated tumors and in tumors with lymph node metastasis was significantly higher than that in well-differentiated tumors and without lymph node metastasis (t=-3.021, P<0.05; t=3.348, P<0.05). In gastric cancer cell lines AGS, SGC7901, MKN45 and BGC823, and gastric epithelial cell line GES-1, the miR-95 expression was higher in gastric cancer cell lines than GES-1, and highest in BGC823 cells (F=5.998, P<0.05). After transfection of anti-miR-95, the activity of the cells significantly decreased, and the ratio of G0/G1 phase increased (F=39.758, P<0.01), but the ratio of G2/M (F=10.349, P<0.05) and S phases decreased (F=15.118, P<0.01); apoptosis rate increased significantly after anti-miR-95 transfection (F=25.182, P<0.01). The mRNA and protein expression levels of PCNA, Cyclin D1 and Survivin significantly decreased, and those of p21 and Bad increased (P<0.05). Conclusion The up-regulated expression of miR-95 in gastric cancer is associated with tumor differentiation and lymph node metastasis, which may be involved in the progression of gastric cancer by regulating the expression of some proliferative and apoptotic genes. Key words: Gastric cancer; MicroRNA-95; Proliferation; Apoptosis; Clinicopathological features

NEDD9 Facilitates Hypoxia-Induced Gastric Cancer Cell Migration via MICAL1 Related Rac1 Activation.

Aims and Hypothesis: NEDD9 is highly expressed in gastric cancer and has a significant involvement in its pathogenesis. However, the mechanism behind hypoxia-promoted cancer cell migration and its regulation because of NEDD9 is still unknown. The aim of this study is to investigate the involvement of NEDD9 in gastric cancer cell migration under hypoxia and explore the underlying potential molecular mechanisms.MethodsCell motility was measured by wound healing and transwell assay. NEDD9 and MICAL1 expressions were examined by western blot analysis. Interaction between NEDD9 and MICAL1 was assessed by immunohistochemistry and co-immunoprecipitation assay, respectively. Cells were transfected with plasmids or siRNA to upregulate or downregulate the expression of NEDD9 and MICAL1. Rac1, Cdc42, and RhoA activation was assessed by pulldown assay.ResultsThe mRNA and protein level of NEDD9 increased as a result of hypoxia in gastric cancer cell lines BGC-823 and SGC-7901 while decreased levels of NEDD9 caused reduced cell migratory potential in response to hypoxia. Hypoxia also caused the enhancement of MICAL1 expression. Furthermore, it was revealed that there is a positive correlation between NEDD9 and MICAL1 protein while hypoxia played role in increasing their interaction. Under hypoxic conditions, silencing of NEDD9 caused reduction in the stability of MICAL1 protein, while depletion of MICAL1 also inhibited the migration of NEDD9-overexpressing gastric cancer cells. In addition, silencing of NEDD9 or MICAL1 expression reversed the increased GTP forms of Rac1 and Cdc42 in hypoxic cells. However, only the upregulation of Rac1-GTP level was observed in gastric cancer cells that were already overexpressed by MICAL1.ConclusionIn all, it is concluded that MICAL1 is regulated by NEDD9 that facilitates hypoxia-induced gastric cancer cell migration via Rac1-dependent manner.

A new Schiff base coordinated copper(II) compound induces apoptosis and inhibits tumor growth in gastric cancer

BackgroundGastric cancer, as a multifactorial disorders, shows cytological and architectural heterogeneity compared to other gastrointestinal cancers, making it therapeutically challenging. Cisplatin is generally used in clinic for gastric cancer treatment but with toxic side effects and develops resistance. Anti-tumor properties of copper and its coordinated compounds have been explored intensively in recent years.MethodsIn this study, we synthesized a novel Schiff base copper coordinated compound (SBCCC) and examined its antitumor effects in two gastric cancer cell lines SGC-7901 and BGC-823 as well as a mouse model of gastric cancer.ResultsThe results show that SBCCC can significantly inhibit the proliferation of gastric cancer cells in a dose- and time-dependent manner. The IC50 of SBCCC in SGC-7901 and BGC-823 cells is 1 μM, which is much less than cisplatin’s IC50. SBCCC induces apoptosis and causes cell cycle arrest at the G1 phase. SBCCC induces apoptosis via multiple pathways including inhibition of NF-κB, ROS production and autophagy.ConclusionsThe synthesized SBCCC induced cancer cell death via inhibition of NF-κB, ROS production and autophagy. The multiple cell-killing mechanisms were important to overcome therapeutic failure because of multidrug-resistance of cancer cells. SBCCC, with a lower IC50 compared to cisplatin, could render it the potential to overcome the side-effect for clinical application.

Silenced PITX1 promotes chemotherapeutic resistance to 5-fluorocytosine and cisplatin in gastric cancer cells.

Resistance to chemotherapeutic drugs leads to a poor prognosis in gastric cancer (GC). The present study aimed to assess the association between pituitary homeobox paired homeodomain transcription 1 (PITX1) expression and the sensitivity of GC cells to the chemotherapeutic drugs 5-fluorouracil (5-FU) and cisplatin (CDDP). In the present study, the gastric cancer cell lines GES-1, AGS, BGC-823, MCG-803 and SGC-7901 were used. The expression of PITX1 was determined via reverse transcription-quantitative polymerase chain reaction in GC cell lines. AGS and BGC-823 cells, which exhibit a decreased PITX1 expression, were transfected with a PITX1 cDNA construct and its control vector. MCG-803 and SGC-7901 cells, which exhibit an increased PITX1 expression, were transfected with siRNA against PITX1 and its control scramble sequence. A Cell Counting kit-8 assay was performed to determine the impact of PITX1 expression on the sensitivity of GC cells to 5-FU and CDDP. The Cancer Genome Atlas database was used to analyze the expression of PITX1 with GC prognosis in the Asian population and to assess the potential mechanism of PITX1 in 5-FU and CDDP resistance. The results revealed that the overexpression of PIXT1 increased the sensitivity of GC cells to 5-FU/CDDP. The combination of 5-FU/CDDP and PITX1 overexpression also reduced the proliferation of GC cells. Additionally, PIXT1 knockdown decreased the sensitivity of GC cells to 5-FU/CDDP. TCGA data revealed that a lower expression of PITX1 is exhibited in Asian GC patients than in normal individuals. GC patients with a lower expression of PITX1 had a poor prognosis. The expression of PITX1 affected the sensitivity of GC cells to 5-FU/CDDP, indicating that PITX1 may increase the efficacy of treatment in GC patients.

MicroRNA-574-3p regulates epithelial mesenchymal transition and cisplatin resistance via targeting ZEB1 in human gastric carcinoma cells

MicroRNA-574-3p (miR-574-3p) has different roles in different cancer types. However, the exact regulation mechanism of miR-574-3p in gastric cancer (GC) progression remains unclear. Thus, we aimed to evaluate the role of miR-574-3p in GC metastasis. We investigated the mechanism via which miR-574-3p regulated cancer cell migration and invasion to determine the relationship between epithelial mesenchymal transition (EMT) and drug resistance. Our results indicated that human GC cell line SGC7901 cells were more sensitive to cisplatin (DDP), but SGC7901 cisplatin-resistant cells (SGC7901/DDP) were more resistant to DDP and had mesenchymal characteristics. In addition, miR-574-3p overexpression up-regulated E-cadherin expression, and concomitantly down-regulated the expression of vimentin. We also identified zinc finger E-box binding homeobox transcription factor 1 (ZEB1), a crucial EMT inducer gene, as a new target of miR-574-3p. In fact, miR-574-3p bound the 3′ untranslated region (3′-UTR) of ZEB1, regulating expression of this transcription factor at both the mRNA and protein levels. Furthermore, miR-574-3p overexpression reduced the migratory and invasive properties of the SGC7901/DDP cells and inhibited cisplatin (DDP) resistance in vitro and in vivo. In conclusion, the results indicated that miR-574-3p inhibited the EMT and enhanced cisplatin sensitivity in GC cells by suppressing ZEB1. These results provide further evidence for the critical roles of miR-574-3p and ZEB1 in invasion and migration regulation characteristics of GC cells.

Expression of pleomorphic adenoma gene like-2 in gastric carcinoma and its possible mechanism on invasion and epithelial-mesenchymal transition of gastric carcinoma

Objective To detect the expression of pleomorphic adenoma gene like-2 (PLAGL2) in human gastric cancer and cell lines, and its role in cell invasion and epithelial-mesenchymal transition (EMT) and the molecular mechanism involved. Methods Fifteen cases of pathologically confirmed gastric cancer tissues and their adjacent normal tissues were collected during operation in our hospital from Jan. 10, 2016 to Mar. 20, 2018. Gastric cancer cell lines MKN-28, BGC-823, NCI-N87 and SGC-7901 were cultured. The expression levels of PLAGL2 in gastric cancer tissue samples and MKN-28, BGC-823, NCI-N87 and SGC-7901 cells were determined by real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting. After successful transfection with PLAGL2-small interfering RNA (siRNA) in SGC-7901 cells, the expression levels of PLAGL2 mRNA and protein were detected by Real-time PCR and Western blotting respectively. Cell invasion was detected by Transwell assay. The expression levels of N-cadherin, Vimentin and E-cadherin, which were EMT related markers, and Wnt/β-catenin related proteins were detected by Western blotting. After PLAGL2-siRNA SGC-7901 cells were treated with Wnt pathway agonist SKL2001 and inhibitor XAV939, the changes of cell invasion ability were retested by Transwell assay. Results The expression levels of PLAGL2 mRNA (1.11±0.27) and protein (0.98±0.26) in gastric cancer tissues were significantly higher than those in the adjacent tissues (mRNA: 0.37±0.10; protein: 0.28±0.11; t=-20.941, P<0.01; t=-20.186, P<0.01). The expression levels of PLAGL2 mRNA and protein in MKN-28, BGC-823, NCI-N87 and SGC-7901 were significantly higher than GES-1 cells (mRNA: t=-12.327, P<0.01; t=-19.812, P<0.01; t=-13.080, P<0.01; t=-15.218, P<0.01; protein: t=-11.816, P<0.01; t=-21.162, P<0.01; t=-14.517, P<0.01; t=-15.899, P<0.01). The expression levels of PLAGL2 mRNA and protein in SGC-7901 cells were significantly decreased after PLAGL2-siRNA transfection (mRNA: 2.10±0.29; protein: 1.96±0.27) as compared with control siRNA group (mRNA: 6.61±0.73; protein: 6.46±0.71; t=-1.000, P<0.01; t=-1.000, P<0.01). The number of invasive cells at 24, 48, 72 hin PLAGL2-siRNA transfection group [(30.75±5.74), (48.75±6.90) and (64.50±4.80) cells] was significantly less than in control siRNA group [(51.00±4.97), ( 75.75±4.92) and (132.50±5.57) cells; t=-1.000, P<0.05; t=-1.000, P<0.01; t=-1.000, P<0.01]. The expression of N-cadherin, Vimentin, β-catenin, matrix metalloproteinase (MMP)-7 and c-Myc proteins was significantly down-regulated, and that of E-cadherin protein was significantly up-regulated (t=-1.000, P<0.01; t=-1.000, P<0.01; t=-1.000, P<0.01; t=-1.000, P<0.01; t=-1.000, P<0.01; t=-8.205, P<0.01). SKL2001 could reverse the inhibitory effect on EMT and invasion of SGC-7901 cells after PLAGL2-siRNA transfection, while XAV939 enhanced the above effects of PLAGL2-siRNA. Conclusion PLAGL2 is highly-expressed in gastric cancer. Inhibition of PLAGL2 gene by siRNA can inhibit the invasion of gastric cancer cells probably by down-regulating the Wnt/β-catenin signaling pathway. Key words: Gastric cancer; Invasion; Pleomorphic adenoma gene like-2; Epithelial-mesenchymal transition; Wnt/β-cateninn signaling pathway

An ssDNA aptamer selected by Cell-SELEX for the targeted imaging of poorly differentiated gastric cancer tissue

Gastric cancer (GC) is associated with high morbidity and mortality rates worldwide. Poorly differentiated GC predicts a poor prognosis and is related to patients’ response to chemotherapy and targeted therapy. Therefore, it is very important to accurately evaluate the tumour differentiation status for the treatment of poorly differentiated GC. To develop a molecular probe to analyse poorly differentiated GC, we selected aptamers against poorly differentiated GC by subtractive Cell-SELEX using the poorly differentiated GC cell line BGC-823 as the target and the moderately differentiated GC cell line SGC-7901 as the negative control. After 15 rounds of selection, aptamer PDGC21 exhibited the highest affinity, and the Kd value of the truncated aptamer PDGC21-T was 35.2 ± 1.1 nM. Aptamer PDGC21-T not only specifically bound to the target cells but also bound to other poorly differentiated GC cells. When combined with fluorescent nanoparticle quantum dots (QDs), the PDGC21-T-QD probe could distinguish poorly differentiated GC cells in mixed culture cells and clinical specimens. Furthermore, in a tissue microarray containing 15 cases from patients, there was a higher positive rate in GC tissues compared with adjacent normal tissues; in poorly differentiated tissues, in particular, the fluorescence signal was significantly higher than that in well/moderately differentiated tissues. Therefore, aptamer PDGC21-T holds great potential for use as a molecular imaging probe for the detection of poorly differentiated GC, which is of great significance for diagnosis and treatment.

1,25-Dihydroxyvitamin D3 affects gastric cancer progression by repressing BMP3 promoter methylation.

BackgroundVitamin D3 has been known to have an anticancer effect, but the mechanisms underlying this is poorly explored. The present study aimed to investigate the antitumor role of vitamin D3 on gastric cancer and mechanisms.MethodsThe Roche Elecsys platform was applied in retrospective studies to detect the role of 25-hydroxylvitamin D3 in adenocarcinoma and colony formation assay was conducted to verify the effect of 1, 25-dihydroxyvitamin D3 on the proliferation of gastric cancer cells. After the identification of hypermethylation of BMP3 CpG islands by bisulfite genomic sequencing (BGS), we further investigated the relationship of BMP3 expression and gastric carcinogenesis by Western blot analysis and gel electrophoresis mobility shift assay (EMSA).ResultsHere we show that low concentration of 1, 25-dihydroxyvitamin D3 links to can-cerization and significantly inhibits proliferation of undifferentiated gastric cancer cell lines SGC-7901 and BGC-823. BMP3 promoter hypermethylation was highly correlated with gastric tumor. Moreover, BMP3 expression was regulated by its promoter methylation in gastric cells. The further exploration of the relationship between 1, 25-dihydroxyvitamin D3 and BMP3 by EMSA results that 1, 25-dihydroxyvitamin D3 stimulates BMP3 expression by the inhibition of BMP3 promoter methylation in gastric tumor cells.ConclusionIn combination with the data from clinical research, bioinformatics analysis and experimental verification, we propose that 1, 25-hydroxylvitamin D3 affects gastric cancer progression by repressing BMP3 promoter methylation.

MiR-181a, a new regulator of TGF-β signaling, can promote cell migration and proliferation in gastric cancer.

Transforming growth factor-beta (TGF-β) signaling pathway plays pivotal roles in various types of cancer. TGF-β receptor 2 (TGFβR2) contains a kinase domain that phosphorylates and activates the downstream of the TGF-β signaling pathway. Our previous microarray analysis revealed marked changes in miR-181a expression in gastric cancers, and the bioinformatics analysis suggested that miR-181a negatively regulated TGFβR2. In order to verify the effect of miR-181a on TGFβR2 and clarify the influence of miR-181a on the migration and proliferation of gastric cancer, studies in gastric cancer cell lines and xenograft mouse models were carried out. We found that a reduced expression of TGFβR2 and an increased expression miR-181a in gastric cancer tissues compared to adjacent noncancerous tissues. A luciferase reporter assay confirmed that TGFβR2 was a target of miR-181a. In addition, we found that miR-181a mimics, which increased the level of miR-181a, downregulated the expression of TGFβR2 in the gastric cancer cell line SGC-7901. Moreover, both the overexpression of miR-181a and the downregulation of TGFβR2 promoted the migration and proliferation of SGC-7901 cells. Conversely, SGC-7901 cell migration and proliferation were inhibited by the downregulation of miR-181a and the overexpression of TGFβR2. Furthermore, the increased expression of miR-181a and the decreased expression of TGFβR2 also enhanced the tumor growth in mice bearing gastric cancer. Our results herein indicated that miR-181a promoted the migration and proliferation of gastric cancer cells by downregulating TGFβR2 at the posttranscriptional level. The present study suggests that miR-181a is a novel negative regulator of TGFβR2 in the TGF-β signaling pathway and thus represents a potential new therapeutic target for gastric cancer.

The nucleosome binding protein 1 promotes the growth of gastric cancer cells.

Nucleosome binding protein 1 (NSBP1) is identified as a new member of HMGN family and is abnormally overexpressed in a variety of tumors. However, it remains unclear whether NSBP1 is overexpressed and promotes gastric cancer. In this study we employed RNAi mediated knockdown of NSBP1 to investigate potential oncogenic role of NSBP1 in gastric cancer. In BGC823 and SGC7901 gastric cancer cell lines, we showed that NSBP1 knockdown decreased cell proliferation while increased apoptosis in vitro. Western blot analysis showed that NSBP1 knockdown decreased the levels of anti-apoptotic protein Bcl-2 while increased the levels of pro-apoptotic protein Bax. In addition, NSBP1 knockdown inhibited the growth and increased the apoptosis of SGC7901 cells xenografted in nude mice. In conclusion, this study provides the first evidence that NSBP1 enhances the proliferation while inhibits the apoptosis of gastric cancer cells, and this is related to the regulation of the expression of apoptosis related proteins by NSBP1. These data suggest that NSBP1 plays oncogenic role in gastric cancer.

Curcumin inhibits the lymphangiogenesis of gastric cancer cells by inhibiton of HMGB1/VEGF-D signaling.

Accumulating evidence shows that curcumin exerts antitumor activities in a variety of malignancies. High mobility group box 1 (HMGB1) is associated with vascular endothelial growth factor D (VEGF-D)–induced lymphangiogenesis and tumor metastasis in gastric cancer. However, the molecular mechanisms by which curcumin regulates HMGB1-mediated lymphangiogenesis in gastric cancer remain unclear. In this study, the cytotoxic effects of curcumin were investigated in gastric cancer AGS and SGC-7901 cell lines by MTT assay, and curcumin-induced morphological changes and cell apoptosis were assessed by using flow cytometry analysis and caspase-3 activity. The effects of curcumin on HMGB1 and VEGF-D expression were examined by reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis. As a result, we found that curcumin decreased cell viability and caused a dose-dependent cell apoptosis through the activation of caspase-3. The mRNA and protein expression levels of HMGB1 and VEGF-D were significantly eliminated by curcumin administration. Pre-treatment with the recombinant HMGB1 (rHMGB1) markedly abolished curcumin-reduced VEGF-D expression. Our findings suggested that curcumin might exert anti-lymphangiogenesis in gastric cancer by inhibition of HMGB1/VEGF-D signaling.

Up-Regulation of MiR-1915 Inhibits Proliferation, Invasion, and Migration of Helicobacter pylori-Infected Gastric Cancer Cells via Targeting RAGE.

PurposeHelicobacter pylori (HP)-infected gastric cancer (GC) is known to be a fatal malignant tumor, but the molecular mechanisms underlying its proliferation, invasion, and migration remain far from being completely understood. Our aim in this study was to explore miR-1915 expression and its molecular mechanisms in regulating proliferation, invasion, and migration of HP-infected GC cells.Materials and MethodsQuantitative real-time PCR and western blot analysis were performed to determine miR-1915 and receptor for advanced glycation end product (RAGE) expression in HP-infected GC tissues and gastritis tissues, as well as human gastric mucosal cell line GES-1 and human GC cell lines SGC-7901 and MKN45. CCK8 assay and transwell assay were performed to detect the proliferation, invasion, and migration capabilities. MiR-1915 mimics and miR-1915 inhibitor were transfected into GC cells to determine the target relationship between miR-1915 and RAGE.ResultsMiR-1915 was under-expressed, while RAGE was over-expressed in HP-infected GC tissues and GC cells. Over-expressed miR-1915 could attenuate cellular proliferation, invasion, and migration capacities. RAGE was confirmed to be the target gene of miR-1915 by bioinformatics analysis and luciferase reporter assay. Moreover, HP-infected GC cellular proliferation, invasion, and migration were inhibited after treatment with pcDNA-RAGE.ConclusionMiR-1915 exerted tumor-suppressive effects on cellular proliferation, invasion, and migration of HP-infected GC cells via targeting RAGE, which provided an innovative target candidate for treatment of HP-infected GC.

Clinical significance of retinoic acid-induced protein I expression in human gastric cancer

Objective To investigate the clinical significance and regulation of retinoic acid-induced protein I (RIG-I) in human gastric cancer. Methods Immunohistochemistry was used to study RIG-I expression in gastric cancer and normal tissues. We used Wilcoxon test to analyze the difference of RIG-I expression levels in cancer and normal tissues. Chi-square and Log-rank tests were used to analyze the RIG-I expression in cancer tissues in relation to patients’ clinical parameters and prognoses respectively. The gastric cancer cell lines SGC-7901 and AGS were used to construct the stable knockdown expression of RIG-I using RNA interference (RNAi) method. The cell counting kit-8 (CCK-8), wound healing, and Transwell assays were used to examine the cellular function after RIG-I knockdown of these cells. Results RIG-I expression level in human gastric cancer tissues was significantly lower than that in adjacent normal tissues (U=1 490, P=0.001). The ratio of higher RIG-I expression in cancer patients with pathological stage (Ⅲ-Ⅳ) was significantly lower than that in normal tissues (χ2=4.668, P=0.031). The overall survival of the patients with higher RIG-I expression was significantly longer than that with lower RIG-I expression (P=0.023). The COX model showed that the age (P=0.047), TNM stage (P=0.039) and the RIG-I expression (P=0.007) could be used as independent prognostic predictors for cancer patients. CCK-8 assay showed that at the time points of 48 h and 72 h after culture, the A450 value in LV-RIG-I-short hairpin RNA (shRNA) group was significantly higher than that in LV-NC group (for SGC7901: P=0.000 and P=0.000 respectively; for AGS: P=0.002 and P=0.016 respectively). The wound healing assay showed that the cell migration abilities in LV-RIG-I-shRNA group was significantly stronger than LV-NC group (for SGC7901: P=0.001; for AGS: P=0.004). The Transwell assay showed that the cell invasion abilities in LV-RIG-I-shRNA group was significantly stronger than that in LV-NC group (for SGC7901: P=0.032; for AGS: P=0.016). Conclusion RIG-I plays important roles in gastric cancer progression, its knockdown could promote proliferation, migration and invasion of cancer cells, and thus could be used as potential therapeutic target for gastric cancer. Key words: Retinoic acid-induced protein I; Gastric cancer; RNA interference; Immunohistochemistry

MiR‑138‑5p modulates the expression of excision repair cross‑complementing proteins ERCC1 and ERCC4, and regulates the sensitivity of gastric cancer cells to cisplatin.

The microRNA (miR)‑138‑5p affects the chemotherapeutic sensitivity of several human cancer types. In the present study, the expression and regulatory mechanisms of miR‑138‑5p were investigated in the gastric cancer cell line SGC7901 and its cisplatin‑resistant derivative SGC7901/DDP. Gene microarray and reverse transcription‑quantitative polymerase chain reaction analyses revealed that miR‑138‑5p was expressed at significantly lower levels in SGC7901/DDP compared with SGC7901 cells. Using computational predictive algorithms, two proteins involved in the nuclear excision repair pathway were identified, excision repair cross‑complementing (ERCC)1 and ERCC4, as putative miR‑138‑5p target genes. Western blot analysis confirmed that ERCC1 and ERCC4 expression levels were inversely proportional to miR‑138‑5p levels in SGC7901 and SGC7901/DDP cells. Furthermore, ERCC1 and ERCC4 were upregulated in SGC7901 cells expressing miR‑138‑5p‑targeting short hairpin RNA and, conversely, downregulated in SGC7901/DDP cells overexpressing miR‑138‑5p, confirming that this miRNA regulates ERCC protein levels. Notably, miR‑138‑5p silencing enhanced the cisplatin resistance of SGC7901 cells, while miR‑138‑5p overexpression partially reversed the cisplatin resistance of SGC7901/DDP cells. Taken together, these data suggest that miR‑138‑5p regulates the sensitivity of gastric cancer cells to cisplatin, possibly by modulating expression of the DNA repair proteins ERCC1 and ERCC4.

Clinical significance of Stathmin1 expression and epithelial-mesenchymal transition in curatively resected gastric cancer.

In our previous study, it was demonstrated that the Stathmin1 (STMN1) is overexpressed in gastric cancer (GC) and that its high expression level is associated with tumor invasion and metastasis. Epithelial-mesenchymal transition (EMT) has also been shown to be critically involved in GC invasion and metastasis. Certain studies have indicated that STMN1 may serve an important role in the EMT process. However, the association between STMN1 expression and EMT-associated markers, as well as clinicopathological characteristics of patients with GC, remains unclear. The aim of the present study was to investigate the clinicopathological significance and prognostic value of STMN1 and EMT-associated markers in GC. The expression of STMN1 and the EMT-associated proteins E-cadherin (E-Cad) and vimentin (VIM) were analyzed by immunohistochemistry in GC and adjacent non-tumorous tissues. Associations between the expression of these markers and clinicopathological parameters were analyzed. The association between STMN1 expression and EMT-associated markers was investigated in the GC cell lines BGC-803 and SGC-7901. The results revealed that STMN1 was expressed in 63.5% of the 167 GC tissues, which was significantly higher than the percentage observed in the adjacent non-tumorous tissues (P=0.003). The STMN1 expression was demonstrated to be positively associated with the VIM levels (P=0.001) and negatively associated with the E-Cad levels (P=0.022) in GC tissues. The STMN1 expression was associated with Lauren's Classification, invasion depth, lymph node metastasis and pathological Tumor-Node-Metastasis (pTNM) stage (P<0.05). In the univariate analyses, the high E-Cad expression was a positive prognostic indicator for overall survival, whereas the high STMN1 and VIM expression was a negative indicator. COX multiple regression analysis demonstrated that the pTNM stage [hazard ratio (HR) 1.912, 95% confidence interval (CI): 1.282–2.851, P=0.001] and E-Cad expression (HR 0.403, 95% CI: 0.249–0.650, P=0.000) were independent prognostic factors. It was also revealed that the expression level of E-Cad decreased, while the expression level of VIM increased by depleting STMN1 levels in GC cells. The present results suggest that the aberrant expression of STMN1 may promote tumor progression through EMT in GC.

IFT80 Improves Invasion Ability in Gastric Cancer Cell Line via ift80/p75NGFR/MMP9 Signaling.

The assembly and maintenance of cilia depend on intraflagellar transport (IFT) proteins, which play an important role in development and homeostasis. IFT80 is a newly defined IFT protein and partial mutation of IFT80 in humans causes diseases such as Jeune asphyxiating thoracic dystrophy (JATD) and short rib polydactyly (SRP) type III, both characterized by abnormal skeletal development. However, the role and mechanism of IFT80 in the invasion of gastric cancer is unknown. We established SGC-7901 and MKN-45 gastric cancer cell lines that stably overexpressed IFT80, as verified by quantitative reverse transcription-PCR, Western blot, and immunofluorescence. Matrix metalloproteinase-9 (MMP9) plays an important role in tumor invasion, and its expression was assessed by quantitative reverse transcription-PCR, Western blotting, and immunofluorescence. The invasion ability of IFT80 on SGC-7901 and MKN-45 cells was examined by the Matrigel invasion assay. The relationship between p75NGFR, and the p75NGFR antagonists, PD90780 and IFT80, were detected by quantitative reverse transcription-PCR and Western blotting. We first detected an IFT80 expression pattern, and found that IFT80 was highly expressed in gastric cancer clinical samples. Overexpression of IFT80 in the gastric cancer cell lines, SGC-7901 and MKN-45, led to lengthening cilia. Additionally, overexpression of IFT80 significantly improved proliferation and invasion, but inhibited apoptosis, in gastric cancer cells. We further found that overexpression of IFT80 increased p75NGFR and MMP9 mRNA and protein expression. Treatment with the p75NGFR antagonist PD90780 inhibited the increased invasion ability resulting from overexpression of IFT80 in SGC-7901 and MKN-45 gastric cancer cells. Thus, these results suggest that IFT80 plays an important role in invasion of gastric cancer through regulating the ift80/p75NGFR/MMP9 signal pathways.

Down-regulation of E26 transformation-specific homologous factor expression inhibits the growth of gastric cancer cells

Objective To observe the effect of down-regulation of E26 transformation-specific homologous factor (EHF) expression on the growth of gastric cancer cells. Methods Western blotting and real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) were used to detect the expression of EHF protein and mRNA in gastric cancer cell lines BGC823, SGC7901 and gastric mucosal epithelial cell line GES-1, respectively. Using the small interfering RNA (siRNA) interference technology, si-EHF-979 or control siRNA si-NC was transfected into gastric cancer cells BGC823 and SGC7901. The si-EHF-979 transfection group served as experimental group, and si-NC transfection group as control group. The methyl thiazol tetrazolium (MTT) assay was utilized to detect the proliferation of gastric cancer cells. The effect of EHF on the migration and invasion of gastric cancer cells was measured by Transwell assay. The gastric cancer cell BGC823 transfected with si-EHF-979 or si-NC was inoculated into the right axilla of nude mice to establish a nude mouse exenograft model. Results Compared with GES-1 cells, the expression levels of EHF in BGC823 and SGC7901 cells were significantly up-regulated, with the difference being statistically significant (P=0.001). The ability of gastric cancer cells to proliferate, migrate and invade after EHF gene knockdown was significantly inhibited. The nude mouse xenograft model showed that knockdown of EHF expression significantly inhibited tumor growth in vivo, the difference was statistically significant (P=0.001). Conclusion EHF is highly expressed in gastric cancer cell lines. Knockdown of EHF expression can inhibit the proliferation of gastric cancer cells. Key words: Gastric cancer; E26 transformation-specific homologous factor; Cell proliferation; Cell migration

Long Noncoding RNA MRPL39 Inhibits Gastric Cancer Proliferation and Progression by Directly Targeting miR-130.

Gastric cancer (GC) is one of the most prevalent malignant tumors displaying both high incidence and mortality throughout much of the world. Recently, long noncoding RNAs (lncRNAs) have been implicated in the development and progression of GC. In the present study, we investigated the biological function and molecular mechanisms of lncRNA MRPL39 in GC. We found that MRPL39 was significantly downregulated in GC tissues and cell lines and that its expression level was negatively associated with carcinoma size, tumor, lymph node, metastasis (TNM) stage, and lymphatic metastasis. Patients with low MRPL39 expression levels revealed a short overall and disease-free survival period. Over-expression of MRPL39 in the GC cell lines BGC823 and SGC-7901 inhibited cell growth, proliferation, migration, and invasion. MiR-130, a putative target gene of MRPL39, displayed an inverse association with the expression of MRPL39 in GC tissues and cell lines. Moreover, a luciferase assay demonstrated a direct binding between the miR-130 and MRPL39, and the reintroduction of miR-130 abrogated the anti-tumor effect of MRPL39 on GC cells. Taken together, these findings indicate that MRPL39 serves as a tumor suppressor by directly targeting miR-130 in GC, which suggests that it might be a novel biomarker in the diagnosis and prognosis of GC.