Invasion Of SGC-7901 Cells Research Articles

ERK1/2 signalling pathway is involved in CD147-mediated gastric cancer cell line SGC7901 proliferation and invasion

This study aimed to investigate the role of CD147 in the progression of gastric cancer and the signalling pathway involved in CD147-mediated gastric cancer cell line SGC7901 proliferation and invasion. Short hairpin RNA (shRNA) expression vectors targeting CD147 were constructed to silence CD147, and the expression of CD147 was monitored by quantitative realtime reverse transcriptase polymerase chain reaction and Western blot and further confirmed by immunohistochemistry invivo. Cell proliferation was determined by Cell Counting Kit-8 assay, the activities of matrix metalloproteinase (MMP)-2 and MMP-9 were determined by gelatin zymography, and the invasion of SGC7901 was determined by invasion assay. The phosphorylation and non-phosphorylation of the mitogen-activated protein kinases, extracellular signal-regulated kinase1/2 (ERK1/2), P38 and c-Jun NH2-terminal kinase were examined by Western blot. Additionally, the ERK1/2 inhibitor U0126 were used to confirm the signalling pathway involved in CD147-mediated SGC7901 progression. The BALB/c nude mice were used to study tumour progression invivo. The results revealed that CD147 silencing inhibited the proliferation and invasion of SGC7901 cells, and down-regulated the activities of MMP-2 and MMP-9 and the phosphorylation of the ERK1/2 in SGC7901 cells. ERK1/2 inhibitor U0126 decreased the proliferation, and invasion of SGC7901 cells, and down-regulated the MMP-2 and MMP-9 activities. In a nude mouse model of subcutaneous xenografts, the tumour volume was significantly smaller in the SGC7901/shRNA group compared to the SGC7901 and SGC7901/snc-RNA group. Immunohistochemistry analysis showed that CD147 and p-ERK1/2 protein expressions were down-regulated in the SGC7901/shRNA2 group compared to the SGC7901 and SGC7901/snc-RNA group. These results suggest that ERK1/2 pathway involves in CD147-mediated gastric cancer growth and invasion. These findings further highlight the importance of CD147 in cancer progression, indicating that CD147 would be an attractive therapeutic target for gastric cancer.

Effect of shRNA-mediated inhibition of Nanog gene expression on the behavior of human gastric cancer cells

The aim of the present study was to employ RNA interference (RNAi) technology to construct and select shRNA-Nanog recombinant plasmids for the inhibition of Nanog gene expression and transfer these plasmids into the human gastric cancer cell line, SGC-7901, as well as to detect the expression of Nanog and the effects on the proliferation, migration, invasion, cell cycle and apoptosis of SGC-7901 cells. The pshRNA-Nanog interference plasmids were constructed and used to transfect SGC-7901 cells using lipofectamine. The expression of the Nanog gene was detected by fluorescence microscopy, RT-PCR and western blotting, and the most markedly inhibited group was identified. The SGC-7901 cells were transfected with recombinant shRNA-Nanog plasmids from the most markedly inhibited group using lipofectamine and the effect on proliferation was determined by CCK-8 assay. The migration and invasion of the SGC-7901 cells was determined by Transwell assays, while the cell cycle and apoptosis were analyzed by flow cytometry. The group with the highest inhibition rate was successfully constructed and identified. It was observed that the proliferation, invasion and migration capacity of the cells was reduced, that the cell cycle was arrested at the S phase and that apoptosis was significantly increased. The Nanog gene in gastric cancer cells is closely associated with cell proliferation, the cell cycle, apoptosis and migration and invasion abilities. The present study establishes the foundations for a novel approach for the genetic treatment of gastric cancer.

Expression of microRNA-100 in human gastric cancer

To investigate the expression of microRNA-100 (miR-100) in human gastric cancer cells and its role of miR-100 in migration, invasion and proliferation in gastric cancer cell line SGC7901. Total RNAs were extracted from formalin-fixed and paraffin-embedded tissue samples of SGC7901 cells, gastric cancer (50 cases), non-tumor (18 cases) and lymph nodes with metastases (18 cases). The expression of miR-100 was examined by reverse transcription (RT)-qPCR. Additionally, SGC7901 cells were transfected with Pre-miR-100 and negative control constructs, and then their ability of migration, invasion and proliferation in vitro was documented after 48 hours. RT-qPCR showed that although miR-100 expressed in all samples, compared to non-tumour tissues, the expression was lower both in SGC7901 cells and gastric cancer tissues (P = 0.0077, P < 0.01). SGC7901 cells and primary gastric cancer tissues with lymph nodes metastasis had lower miR-100 expression than those of without lymph node metastasis (P = 0.0361, P = 0.0356). The migration ability and invasion of SGC7901 cells transfeced with pre-miR-100 decreased as compared with control cells (P = 0.0025, P = 0.0028 respectively). However, miR-100 expression had no significant effects on the cell proliferation. Expression of miR-100 inhibits the migration and invasion of gastric cancer cells without significant alteration of proliferation. Therefore, miR-100 may play an inhibitory role in the progression of gastric carcinoma.

Protein inhibitor of activated STAT-1 is downregulated in gastric cancer tissue and involved in cell metastasis

Protein inhibitor of activated STAT-1 (PIAS1) is a novel modulator of the JAK/STAT signaling pathway that negatively regulates the inflammatory response. It has been also reported to be downregulated in a variety of human cancer cell lines. However, the role of PIAS1 in gastric cancer remains unclear. In this study, we investigated the prognostic value of PIAS1 expression and its regulated mechanisms in gastric cancer cell metastasis. Therefore, the expression of PIASI was explored in gastric cancer tissues and adjacent tissues of gastric cancer with 31 cases of patients, and the prognostic value was analyzed. In addition, the growth and invasion in SGC7901 cells were investigated in the restoration of PIAS1 expression with Ad5/F35-PIAS1 or Ad5/F35-vector or PBS treatment, and the activity of P38MAPK, P-P38MAPK, JNK/SAPK, P-JNK/SAPK, ERK and P-ERK, were detected by western blotting. The tumor migratory factors MMP-9, MMP-2 and ICAM-1 were analyzed by western blotting. The results demonstrated that 22 of 31 (70.9%) gastric cancer specimens showed low levels of PIAS1 expression from immunohistochemistry staining using tissue microarrays. Statistical analysis suggested that the downregulation of PIAS1 was significantly correlated with tumor staging. Furthermore, we found that the restoration of PIAS1 expression mediated by Ad5/F35 virus suppressed cell proliferation and invasion accompanied by the inhibition of P38MAPK and ERK protein expression and activity, but not JNK/SAPK protein. Notably, PIAS1 restoration with the transfection of Ad5/F35-PIAS1 robustly decreased the expression of tumor migratory factors including MMP-9, MMP-2 and ICAM-1 compared to Ad5/F35-vector. These data suggest that PIAS1 may function as a tumor suppressor to regulate gastric cancer cell metastasis by targeting the MAPK signaling pathway.

RKIP inhibits the malignant phenotypes of gastric cancer cells

Raf kinase inhibitor protein (RKIP) is first identified as an interacting partner of Raf-1. RKIP expression is low or absent in several established cell lines derived from metastatic breast cancer, prostate cancer and melanoma cells. However, the functional role of RKIP in gastric cancer remains unclear. In this study, we employed human gastric cancer cell line SGC7901 as a model to reconstitute RKIP expression in gastric cancer cells. The growth curve and soft agar assay showed that RKIP inhibited the growth and clonogenicity of SGC7901 cells. Flow cytometry analysis showed that RKIP inhibited the cell cycle progression and induced the apoptosis of SGC7901 cells. Wound healing and transwell invasion assay showed that RKIP inhibited the migration and invasion of SGC7901 cells. Furthermore, we observed that RKIP inhibited the growth of SMGC7901 cells in xenografts in nude mice. Taken together, our in vitro and in vivo data demonstrate that RKIP modulates the proliferation, apoptosis, migration, invasion and tumorigenicity of SGC7901 cells. These results reveal the tumor suppressor role of RKIP in gastric cancer and suggest that RKIP may be new therapeutic target for gastric cancer.

Combination DLL4 with Jagged1-siRNA Can Enhance Inhibition of the Proliferation and Invasiveness Activity of Human Gastric Carcinoma by Notch1/VEGF Pathway

To evaluate the effects of adenovirus- mediated gene transfer of DLL4 and Jagged1 siRNA on proliferation and invasion of SGC7901 cells by Notch/ VEGFR pathway. Plasmid of DLL4 and Jagged1 siRNA were constructed and transfected into SGC7901 cells. siRNA and endostatin (VEGF inhibitor) were designed as the control group. The mRNA and protein expressions of DLL4 and Jagged1 were respectively detected with RT-PCR and western blotting. In order to find out the changes of proliferation and invasion power of SGC7901 cell lines, we analyzed the data by MTT, Boyden chamber and evaluated apoptosis of cell with flow cytometry. We treated BALB/C nude mice with DLL4 and Jagged1-siRNA, and tumor control rate (%) in nude mice was calculated. DLL4 and Jagged1 siRNA transfections specifically down-regulated the corresponding mRNA and protein levels in SGC7901 cells. The experiment of permeated artificial basal membrane showed that the invasion power of SGC7901 cell lines were on the decline after treatment of Ad-DLL4- Jagged1-siRNA (12.23±3.12 vs. 78.38±17.38, p<0.05). The values of 490nm wavelength light absorption were different in the five groups. The number of alive cells in the group of DLL4-Jagged1-siRNA was lower than others in the 6th d (0.77±0.01 vs. 3.00±0.11 p<0.05). The apoptosis rate of transfected DLL4 and Jagged1 group with FACS were 18.07%±0.98±1.78 and there were significant differences between treated and control groups (18.07%±0.98 vs. 1.08%±0.23, p<0.01). The tumor transplantation experiment in BALB/C nude mice showed that intratumoral injection of DLL4 and Jagged1 siRNA could inhibit tumor growth. DLL4 and Jagged1 siRNA gene therapy mediated by adenovirus may be useful for inhibiting growth and invasion of SGC7901 through a Notch/VEGFR pathway. These results provided a novel therapeutic target in preventing gastric cancer cell invasion and metastasis.

MicroRNA-221 and microRNA-222 regulate gastric carcinoma cell proliferation and radioresistance by targeting PTEN

BackgroundMicroRNAs (miRNAs) can function as either oncogenes or tumor suppressor genes via regulation of cell proliferation and/or apoptosis. MiR-221 and miR-222 were discovered to induce cell growth and cell cycle progression via direct targeting of p27 and p57 in various human malignancies. However, the roles of miR-221 and miR-222 have not been reported in human gastric cancer. In this study, we examined the impact of miR-221 and miR-222 on human gastric cancer cells, and identified target genes for miR-221 and miR-222 that might mediate their biology.MethodsThe human gastric cancer cell line SGC7901 was transfected with AS-miR-221/222 or transduced with pMSCV-miR-221/222 to knockdown or restore expression of miR-221 and miR-222, respectively. The effects of miR-221 and miR-222 were then assessed by cell viability, cell cycle analysis, apoptosis, transwell, and clonogenic assay. Potential target genes were identified by Western blot and luciferase reporter assay.ResultsUpregulation of miR-221 and miR-222 induced the malignant phenotype of SGC7901 cells, whereas knockdown of miR-221 and miR-222 reversed this phenotype via induction of PTEN expression. In addition, knockdonwn of miR-221 and miR-222 inhibited cell growth and invasion and increased the radiosensitivity of SGC7901 cells. Notably, the seed sequence of miR-221 and miR-222 matched the 3'UTR of PTEN, and introducing a PTEN cDNA without the 3'UTR into SGC7901 cells abrogated the miR-221 and miR-222-induced malignant phenotype. PTEN-3'UTR luciferase reporter assay confirmed PTEN as a direct target of miR-221 and miR-222.ConclusionThese results demonstrate that miR-221 and miR-222 regulate radiosensitivity, and cell growth and invasion of SGC7901 cells, possibly via direct modulation of PTEN expression. Our study suggests that inhibition of miR-221 and miR-222 might form a novel therapeutic strategy for human gastric cancer.

Ectopic expression of guanylyl cyclase C in gastric cancer as a potential biomarker and therapeutic target

To investigate the expression of guanylyl cyclase C (GCC) in human gastric cancer (GC) tissues and assess the effect of GCC small interfering RNA (siRNA) on the proliferation and apoptosis of SGC-7901. The expression of GCC in 30 specimens and three human GC cell lines (SGC-7901, AGS, NCI-N87) were detected by RT-PCR for messenger RNA (mRNA) by Western blot and immunofluorescence for proteins. Recombinant plasmids containing GCC siRNA and scrambled siRNA were constructed and transfected into SGC-7901 cells, respectively. A cell counting kit-8, flow cytometry (FCM) and terminal deoxynucleotidyl transferase (TDT)-mediated dUTP-biotin nick end-labeling were used to evaluate cell viability, cell cycle distribution and apoptosis, followed by wound healing assay and cell adherent assay for cell motility and adherent, respectively. The expression of GCC was absent in paracancerous tissues, whereas the GCC mRNA and protein expressions were detected in 20/30 and 19/30 of GC specimens, respectively. Moreover, intestinal GC was statistically different from diffuse GC (P < 0.05). The proliferation of SGC-7901 cells was markedly inhibited by GCC siRNA-3 (P < 0.05) and cell morphological changes including volumetric reduction, karyopyknosis and karyorrhexis were observed. FCM showed that the cell count in the sub-G0/G1 peak increased from 5.47% (48 h after transfection) to 5.63% (72 h after transfection). The wound healing assay and cell adherent assay revealed that GCC gene silencing decreased cell motility and adherent. The over-expression of GCC has been detected in intestinal type GC. GCC siRNA can effectively inhibit the proliferation and invasion of SGC-7901 cells and induce cell apoptosis. GCC might be a novel biomarker and therapeutic target for GC.

Role of histone deacetylase in inhibiting invasion of human gastric carcinoma cell line SGC-7901 by PPAR gamma-mediated pathway

Histone deacetylase (HDAC) can attenuate the function of peroxisome proliferator-activated receptor gamma (PPARgamma) to drive adipocyte differentiation. PPARgamma activation is confirmed to inhibit the development and metastasis of a variety of malignant cells. This study was to investigate the role of HDAC in inhibiting the invasion of human gastric carcinoma SGC-7901 cells through PPARgamma-mediated pathway, and explore potential mechanism. SGC-7901 cells were treated with different concentrations of Trichostatin A (TSA) and Rosiglitazone (ROZ) respectively to select the best combination through assessing cell proliferation by MTT assay. Then cells were randomly divided into control group, TSA group, ROZ group, and combination group. Cell proliferation was detected by MTT assay after 48 h; cell invasion was detected by Boyden chamber invasion test. The mRNA levels of PPARgamma and matrix metalloproteinase-2 (MMP-2) were assessed by reverse transcription-polymerase chain reaction (RT-PCR), and the protein level of MMP-2 was evaluated by Western blot. Both TSA and ROZ inhibited the proliferation of SGC-7901 cells in a dose-dependent manner. A combination of 20 nmol/L TSA and 5 mumol/L ROZ synergistically inhibited the invasion of SGC-7901 cells (q=1.41). ROZ down-regulated the mRNA and protein expression of MMP-2. TSA and ROZ in combination reduced MMP-2 expression more obviously than ROZ alone. TSA up-regulated the expression of PPARgamma mRNA. HDAC suppresses the activation of PPARgamma through a series of molecular mechanisms. The activity of ROZ in inhibiting invasion of human gastric carcinoma cells can be enhanced after the activity of HDAC is inhibited by TSA.

RhoA and RhoC -siRNA inhibit the proliferation and invasiveness activity of human gastric carcinoma by Rho/PI3K/Akt pathway

To evaluate the effects of adenovirus-mediated gene transfer of RhoA siRNA and RhoC siRNA on proliferation and invasion of SGC7901 cells by Rho/PI3K/Akt pathway. Plasmid of RhoA siRNA and RhoC siRNA were constructed and transfected into SGC7901 cells. siRNA and LY294002 (PI3K inhibitor) were designed as the control group. The mRNA and protein expressions of RhoA and RhoC were respectively detected with RT-PCR and western blotting. In order to find out the changes of proliferation and invasion power of SGC7901 cell lines, we analyzed the data by MTT, Boyden chamber and evaluated apoptosis of cell with flow cytometry. We treated BALB/C nude mice with RhoA and RhoC-siRNA, and tumor control rate (%) in nude mice was calculated. RhoA and RhoC siRNA transfections specifically down-regulated the corresponding mRNA and protein levels in SGC7901 Cells. The experiment of permeated artificial basal membrane showed that the invasion power of SGC7901 cell lines are on the decline after treatment of Ad-RhoA and RhoC-siRNA (12.64 +/-3.27 vs 87.38 +/- 17.38, P < 0.05). The values of 490 nm wavelength light absorption were different in the five groups. The number of alive cells in the group of RhoA and RhoC-siRNA was lower than others in the 6(th) d (0.71 +/- 0.01 vs 3.82 +/- 0,11 P < 0.05). The apoptosis rate of transfected RhoA and RhoC-siRNA group with FACS were 19.07% +/- 1.78 and there were significant differences between treated and control groups (19.07 +/- 1.78% vs 1.23 +/- 0.11%, P < 0.01). The tumor transplantation experiment in BALB/C nude mice showed intratumoral injection of RhoA or RhoC siRNA can inhibit tumor growth. RhoA and RhoC siRNA gene therapy mediated by adenovirus may be useful for inhibiting growth and invasion of SGC7901 through a PI3K/Akt pathway. These results provide a novel therapeutic target in preventing gastric cancer cell invasion and metastasis.

Inhibition of PRL-3 gene expression in gastric cancer cell line SGC7901 via microRNA suppressed reduces peritoneal metastasis

High expression of PRL-3, a protein tyrosine phosphatase, is proved to be associated with lymph node metastasis in gastric carcinoma from previous studies. In this paper, we examined the relationship between PRL-3 expression and peritoneal metastasis in gastric carcinoma. We applied the artificial miRNA (pCMV-PRL3miRNA), which is based on the murine miR-155 sequence, to efficiently silence the target gene expression of PRL-3 in SGC7901 gastric cancer cells at both mRNA and protein levels. Then we observed that, in vitro, pCMV-PRL3miRNA significantly depressed the SGC7901 cell invasion and migration independent of cellular proliferation. In vivo, PRL-3 knockdown effectively suppressed the growth of peritoneal metastases and improved the prognosis in nude mice. Therefore, we concluded that artificial miRNA can depress the expression of PRL-3, and that PRL-3 might be a potential therapeutic target for gastric cancer peritoneal metastasis.

Integrinßl asODN - inhibition of cell attachment and invasion of the human gastric cancer cell line SGC7901

Objective To study the inhibition of adhesion and invasion of SGC7901 cells into the ECM by integrinsl antisense oligodeoxynucleotide asODN.

Inhibitory effects of c9, t11-conjugated linoleic acid on invasion of human gastric carcinoma cell line SGC-7901.

To investigate the effect of c9, t11-conjugated linoleic acid (c9, t11-CLA) on the invasion of human gastric carcinoma cell line and its possible mechanism of preventing metastasis. Using reconstituted basement membrane invasion, chemotaxis, adhesion, PAGE substrate zymography and RT-PCR assays, we analyzed the abilities of invasion, direct migration, adhesion of intracellular matrix, as well as the activity of type IV collagenase and expression of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 mRNA in SGC-7901 cells which were treated with gradually increased concentrations (25, 50, 100 and 200 micromol/L) of c9, t11-CLA for 24 h. At the concentrations of 200 micromol/L, 100 micromol/L and 50 micromol/L, c9,t11-CLA suppressed the invasion of SGC-7901 cells into the reconstituted basement membrane by 53.7 %, 40.9 % and 29.3 %, respectively, in comparison with the negative control. Only in the 200 micromol/L c9,t11-CLA group, the chemotaxis of SGC-7901 cells was inhibited by 16.0 % in comparison with the negative control. C9,t11-CLA also could inhibit the adhesion of SGC-7901 cells to laminin, fibronectin and Matrigel, increase the expression of TIMP-1 and TIMP-2 mRNA, and reduce type IV collagenase activities in the serum-free medium supernatant of SGC-7901 cells. c9,t11-CLA can inhibit the invasion of SGC-7901 cells at multiple procedures in tumor metastasis cascade, which may be associated with the induction of TIMP-1 and TIMP-2 mRNA expression.