Normal Human Gastric Epithelial Cells Research Articles

MiR-635 targets KIFC1 to inhibit the progression of gastric cancer

Accumulating studies have shown that the dysregulation of microRNAs is related to the carcinogenesis and development of gastric cancer (GC), and the role of miR-635 in GC remains largely unknown....

Pyrroline-5-carboxylate reductase 1 (PYCR1) upregulation contributes to gastric cancer progression and indicates poor survival outcome.

BackgroundProline levels are significantly increased in tumor specimens and urine samples from gastric cancer (GC) patients, and we previously showed that intracellular proline levels significantly differ between human GC cell lines and normal gastric epithelial cells. Pyrroline-5-carboxylate reductase 1 (PYCR1) is the key enzyme in intracellular proline synthesis, but its role in GC remains largely unknown.MethodsBioinformatic analysis and immunohistochemical (IHC) staining with a tissue microarray were conducted to assess the association between PYCR1 expression and clinical parameters. PYCR1 downregulation and overexpression were then established in two GC cell lines (AGS and MKN28 cells) to determine whether PYCR1 promotes malignant behavior in GC. Gene set enrichment analysis (GSEA) was further performed to investigate the pathway regulating PYCR1 in GC.ResultsPYCR1 expression was up-regulated in different GC cohorts. High PYCR1 protein expression was correlated with advanced tumor stage, aggressive histological type and high Ki-67 index. High PYCR1 expression in GC tissues was an indicator of poor outcome in GC patients. In vitro, PYCR1 knockdown markedly attenuated GC cells growth and promoted apoptosis, while overexpression produced the opposite effects. GSEA analysis indicated PI3K/Akt axis was strongly correlated with PYCR1 expression and that PIK3CB and AKT1 mRNA expression was positively associated with PYCR1 in GC tissues. PI3K inhibition further significantly reduced PYCR1 mRNA and protein expression. Moreover, as PYCR1 is a mitochondrial endomembrane protein, nutrient stress induced by glucose deprivation also regulated PYCR1 expression.ConclusionsPYCR1 is highly expressed in GC and acts as a mitochondrial oncogene to induce cancer progression by enhancing tumor proliferation and responding to metabolic stress. PYCR1 is a novel prognostic marker and a potential therapeutic target in GC.

Carbonic anhydrase IV inhibits cell proliferation in gastric cancer by regulating the cell cycle.

Carbonic anhydrase IV (CA4) is silenced in colorectal cancer. However, the effect of CA4 on the development of gastric cancer (GC) is poorly understood. The present study aimed to determine the role of CA4 in GC tumorigenesis and its underlying molecular mechanism. The levels of CA4 in GC cells and tissues were evaluated by reverse transcription-quantitative PCR and immunohistochemistry. CA4 expression was suppressed in GC cells and tissues compared with adjacent healthy tissues and normal human gastric epithelial cells, respectively. This reduced expression was significantly associated with tumor size, invasion and differentiation. Analyses with a real-time cell analyzer and clonogenic assays were conducted to validate the impact of CA4 on GC cell lines (AGS and HGC-27) and normal human gastric epithelial cell line (GES-1) proliferation. The effects of CA4 on the cell cycle in GC cells were determined by flow cytometry. The levels of CA4 and cell cycle-associated proteins were confirmed by western blotting. CA4 overexpression inhibited GC cell proliferation and reduced colony-forming ability, arrested the cell cycle in the G2/M phase, inhibited cyclin B1 and cyclin-dependent kinase 2 expression and induced p21 expression. These results indicate that CA4 may serve an important role in GC tumorigenesis by inhibiting cellular proliferation via regulating the expression of cell cycle-associated proteins. CA4 may serve as a diagnostic biomarker and a potential therapeutic target in GC.

MiR-502-5p inhibits the proliferation, migration and invasion of gastric cancer cells by targeting SP1.

Gastric cancer (GC) is the third most common cause of cancer-associated mortality in China. Aberrant microRNA (miR) expression can occur through multiple biological processes and has been implicated in cancer development. However, to the best of our knowledge, the function of miR-502-5p in GC is currently unclear. In the present study, the expression and function of miR-502-5p in GC was evaluated. Reverse transcription-quantitative (RT-q) PCR was used to measure the expression levels of miR-502-5p in GC tissues, normal adjacent tissues, a normal human gastric epithelial cell line (GES-1) and two GC cell lines. miR-502-5p expression levels were significantly lower in GC tissues and GC cell lines compared with those in adjacent normal tissues and GES-1 cells, respectively. Subsequently, the target genes of miR-502-5p were predicted, and it was demonstrated that the transcription factor SP1 was a direct target. SP1 expression, cell viability, migration and invasion, and SP1 protein levels were examined using RT-qPCR, an MTT assay, Transwell assay and western blotting, respectively. Human GC cells were then transfected with an miR-502-5p mimic to emulate miR-502-5p overexpression, resulting in inhibition of the proliferation, migration and invasion capacities of human GC cells. Compared with the negative control, cells overexpressing miR-502-5p had decreased levels of SP1 mRNA and protein. These data suggest that miR-502-5p serves as a tumor suppressor gene by targeting SP1 to regulate the proliferation, migration and invasion of GC cells.

Cinnamic hydroxamic acid inhibits the proliferation of gastric cancer cells via upregulation of miR 145 expression and down-regulation of P13K/Akt signaling pathway

Purpose: To investigate the anti-proliferative effect of cinnamic hydroxamic acid (CHA) on gastric cancer (GC) cells, and its mechanism of action.Methods: Two GC cell lines (SGC-7901 and MKN1) and normal human gastric epithelial cells (GES1) were used for this study. The GC cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin solution at 37 °C for 24 h in a humidified atmosphere of 5 % CO2 and 95 % air. GES1 cells were cultured in RPMI medium supplemented with 10 % FBS only. Cell viability and apoptosis were determined using 3 (4,5 dimethyl thiazol 2 yl) 2,5 diphenyl 2H tetrazolium bromide (MTT), and flow cytometric assays, respectively. The level of expression of microRNA-145 (miR-145) was determined using real-time quantitative polymerase chain reaction (qRT-PCR). Protein expressions of c-Myc, p-AKT, PI3K, p21, and matrix metalloproteinase (MMP)-2 and MMP-9were determined using Western blotting.Results: Treatment of GC cells with CHA for 72 h led to significant and dose-dependent reduction in their viability, and significant and dose-dependent increase in the number of apoptotic cells (p < 0.05). It also significantly arrested GC cell cycle at G1 phase (p < 0.05). The treatment significantly and dosedependently decreased SGC-7901 and MKN1 cell migration and invasion, and upregulated miR-145 mRNA expression (p < 0.05). The expression of miR-145 mRNA was significantly higher in MKN1 cells than in SGC-7901cells (p < 0.05). Treatment of SGC-7901 and MKN1 cells with CHA significantly downregulated protein expressions of c-Myc, MMP-2/9, PI3K and p-AKT, but upregulated p21 protein expression (p< 0.05).Conclusion: These results show that CHA inhibits the proliferation of GC cells via upregulation of miR-145 expression and down-regulation of P13K/Akt signaling pathway. Therefore, CHA has a good potential as a therapeutic agent for the management of gastric cancer
 Keywords: Apoptosis, Cinnamic hydroxamic acid, Gastric cancer, Metastasis, Proliferation

Overexpressed circ-RPL15 predicts poor survival and promotes the progression of gastric cancer via regulating miR-502-3p/OLFM4/STAT3 pathway

BackgroundIncreasing studies have revealed that circular RNAs (circRNAs) contribute to gastric cancer (GC) progression. The circular RNA ribosomal protein L15 (circ-RPL15) is involved in chronic lymphocytic leukemia. However, its expression and functions in GC remain elusive. MethodsThe expression of circ-RPL15 in human GC tissues and adjacent normal tissues, human gastric cancer cell lines (MGC-803, BGC-823, MGN-28, SGC-7901, AGS) and normal gastric mucosal epithelial cell line (GES-1) were detected by RT-PCR. The relationship between circ-RPL15 level and clinical-pathological indicators were also analyzed. Gain- of function experiments of circ-RPL15 and miR-502-3p were conducted to verify their roles in mediating GC cell proliferation, apoptosis and metastasis. Also, the downstream mechanisms of circ-RPL15 were predicted by bioinformatics analysis, and the interactions between circ-RPL15 and miR-502-3p, miR-502-3p and OLFM4 were verified by dual luciferase reporter gene assay and RNA FISH. Resultscirc-RPL15 was upregulated in GC tissues and cell lines, and the overexpressed circ-RPL15 was correlated with poorer survival of GC patients. Functionally, circ-RPL15 upregulation distinctly promoted the proliferation, migration and invasion of GC cells and inhibited apoptosis. Mechanistically, circ-RPL15 functioned as a competitive endogenous RNA via sponging miR-502-3p and activated OLFM4/STAT3 pathway. Conclusioncirc-RPL15 promotes GC progression and predicts poor prognosis of GC patients, and regulates the malignant phenotypes of GC cells by mediating the miR-502-3p/OLFM4/STAT3 axis.

MicroRNA‑383‑5p inhibits the proliferation and promotes the apoptosis of gastric cancer cells bytargeting cancerous inhibitor of PP2A.

The aberrant expression of microRNA (miRNAor miR)‑383‑5p has been found in numerous types of cancer. However, the function of miR‑383‑5p in gastric cancer (GC) remains elusive and requires further investigation. In the present study, the level of miR‑383‑5p and cancerous inhibitor of PP2A (CIP2A) in GCcell lines was determined by reverse transcription‑quantitative PCR analysis. GCcell proliferation, apoptosis and cell cycle distribution were determined by the MTTassay and flow cytometry, respectively. The mRNA target of miR‑383‑5p was identified by dual luciferase activity assay. It was observed that the expression of miR‑383‑5p was lower and that of CIP2A was higher in GCcells compared with the GES‑1 normal human gastric epithelial cell line. Transfectoin with miR‑383‑5pmimic significantly inhibited GCcell proliferation, while it promoted cell apoptosis and G0/G1arrest by targeting CIP2A. Taken together, the findings of the present study demonstrate that miR‑383‑5p inhibits GCcell proliferation and promotes apoptosis and G0/G1arrest by targeting CIP2A, indicating that targeting miR‑383‑5p may hold promise as a future therapeutic strategy for patients with GC.

MicroRNA-92a promotes proliferation and invasiveness of gastric cancer cell by targeting FOXO1 gene

The aim of this study is to investigate the effect of miRNA-92a on GC cell proliferation, migration and invasiveness, and the mechanism(s) involved. Four GC cell lines (SGC-7901, BGC-823, MKN45 and HGC-27) and normal human gastric epithelial cells (GES1) were used in this study. MicroRNA-92a mimics or inhibitor were transfected into the cells. The results of transfection were assessed using real-time quantitative polymerase chain reaction (qRT-PCR). Cell proliferation, migration, invasiveness and apoptosis were determined using cell counting kit 8 (CCK-8), scratch test, Transwell invasion assay, and flow cytometric analysis, respectively. The protein target of miRNA-92a was predicted using Bioinformatics. The expression of FOXO1 protein was measured using Western blotting. The expression of miRNA-92a was significantly upregulated in GC cells, relative to normal gastric epithelial cells (p < 0.05). Overexpression of miRNA-92a significantly promoted the proliferation, migration and invasiveness of GC cells, but significantly inhibited their apoptosis (p < 0.05). MicroRNA-92a directly targeted FOXO1 gene, and significantly reduced its protein expression. Overexpression of miRNA-92a promotes the proliferation, migration and invasiveness of GC cells, and plays a role similar to that of oncogene. It directly targets FOXO1 gene by inhibiting its protein expression.

Long non-coding RNA FTX promotes gastric cancer progression by targeting miR-215.

Gastric cancer (GC) is one of the most common malignant tumors in the world, which is seriously harmful to people's health. The increasing number of studies have shown that long non-coding RNA (lncRNA) is related to the occurrence of gastric cancer. In this study, we aimed at investigating the role of lnc FTX in the occurrence of gastric cancer. The expression of FTX in gastric cancer patients and gastric cancer cell lines was detected by RT-qPCR. Univariate Kaplan-Meier method was used to analyze the relationship between FTX expression level, clinicopathological parameters and overall survival rate (OS). After transferring si-FTX and overexpression FTX plasmids into MGC-803 and SGC-7901, the expression of miR-215-3p was detected by RT-qPCR, and the changes of cell proliferation and cell cycle were detected by CCK-8 and flow cytometry. In addition, luciferase activity was used to detect whether miR-215-3p combined with FTX and SIVA1. Finally, Western blot (WB) was used to detect the change of SIVA1 protein expression by miR-215 mimic. We found that the expression of FTX in tumor tissues of 71 GC patients was higher than that in paracancerous tissues, and the prognosis of patients with high FTX was poor. The expression of FTX in gastric cancer cells was higher than that in normal human gastric epithelial cells (GES-1). Transferring overexpression plasmid of FTX into gastric cancer cells (MGC-803 and SGC-7901) promoted cell proliferation and the ratio of cells in G0-G1 phase was decreased. Transferring si-FTX to MGC-803 and SGC-7901 led to opposite results. There was a negative correlation between the expression of mi215-3p and FTX in MGC-803 and SGC-7901 gastric cancer cells, and luciferase results showed that mi215-3p could directly bind to FTX and regulate cell growth and cell cycle changes. In addition, luciferase results showed that mi215-3p could bind directly to SIVA1. What's more, RT-qPCR and WB results showed that mi215 mimic could promote the expression of MGC-803, SGC-7901 SIVA1mRNA and protein. According to these results, this study revealed that the previously neglected FTX-miR2153p-SIVA1 regulatory axis for the regulation of gastric cancer progression, which may be a potential target for the treatment of gastric cancer.

MAFG-AS1 is a novel clinical biomarker for clinical progression and unfavorable prognosis in gastric cancer

ABSTRACT MAFG antisense 1 (MAFG-AS1) is recently identified as a novel lncRNA and serves as a tumor promoter in several types of human tumor. However, no prior study has been performed to evaluate the role of MAFG-AS1 in gastric cancer. In our study, we found MAFG-AS1 expression was increased in gastric cancer tissue samples compared with normal gastric mucosa tissue samples, and associated with poor overall survival in gastric cancer patients at The Cancer Genome Atlas database. Furthermore, we confirmed the clinical and prognostic significance of MAFG-AS1 in gastric cancer. We found gastric cancer tissues and cell lines had remarkably increased MAFG-AS1 expression in comparison to normal gastric mucosa tissues and normal human gastric epithelial cell line, and high MAFG-AS1 expression was positively associated with diffuse type, advanced clinical stage, extensive depth of invasion, more lymph node metastasis, and present distant metastasis in gastric cancer patients. Moreover, high MAFG-AS1 expression acted as one of the independent poor prognostic factors for overall survival in gastric cancer patients. The loss-of-function study showed knocking down MAFG-AS1 expression inhibited gastric cancer cell proliferation, migration and invasion in vitro. In conclusion, MAFG-AS1 is probable to be a valuable prognostic biomarker, and a novel potential target for gastric cancer.

Pueraria lobata Root Constituents as Xanthine Oxidase Inhibitors and Protective Agents against Oxidative Stress Induced in GES-1 Cells

Ultrafiltration coupled with liquid chromatography-mass spectrometry (LC-MS) was established to screen xanthine oxidase (XO) inhibitors from Pueraria lobata root extract. Four compounds were screened out and identified as puerarin, daidzin, daidzein and genistein with half-maximal inhibitory concentration (IC50) values of 30.8, 5.31, 14.5 and 3.02 µg mL−1 on XO, respectively. The interactions between these compounds and XO were investigated by fluorescence spectroscopic method. The hydrogen peroxide induced oxidative stress model of human normal gastric epithelial cell lines (GES-1) was used to investigate the protections on injured cell. As a result, four XO inhibitors exhibited protective effects without cytotoxicity. With the increased concentrations of four inhibitors, cell viability was higher with decreased mortality rate, the decrease of superoxide dismutase activity, leakage of lactate dehydrogenase and increase of intracellular superoxide anion production induced by hydrogen peroxide were restrained. It showed that these four XO inhibitors could effectively enhance cell viability and protect injury of GES-1 cells from oxidative stress.

The Long Non-Coding RNA NEAT1 Promotes Gastric Cancer Cell Proliferation and Invasion by Regulating miR-103a/ STAMBPL1 Axis.

Background:Gastric cancer (GC) is a common malignancy with high morbidity. Long non-coding RNAs (LncRNAs) have been demonstrated to be critical post-transcriptional regulators in tumorigenesis. This study aimed to investigate the effect of LncRNA NEAT1 on the proliferation and metastasis of GC.Material and Methods:The expression of LncRNA NEAT1 was examined in clinical samples and GC cell lines. GC cell lines (SGC-7901 and BGC-823) and human normal gastric epithelial cell line (GES-1) were employed. The correlation between NEAT1, miR-103a and STAMBPL1 was determined by luciferase reporter assay. Cell viability was determined by CCK8 assay. Cell invasion capacity was examined by Transwell assay. The protein level of STAMBPL1 was analyzed by western blotting.Results:LncRNA NEAT1 was found to be up-regulated in GC cell lines. Further studies identified LncRNA NEAT1 as a direct target of miR-103a. Moreover, NEAT1 knockdown and miR-103a overexpression inhibited cell proliferation and cell invasion. NEAT1 knockdown and miR-103a overexpression also decreased STAMBPL1 levels.Conclusion:Our study indicated that LncRNA NEAT1 was up-regulated in GC cells and tissues. NEAT1 was targeted and inhibited by miR-103a and acted as an oncogene, which promoted the malignant behavior of GC cells. This regulatory effect of NEAT1 may be associated with STAMBPL1. Therefore, NEAT1 could be used as a biomarker for predicting the progression of GC.

Overexpression of the zinc-α2-glycoprotein accelerates apoptosis and inhibits growth via the mTOR/PTEN signaling pathway in gastric carcinoma cells

Adipocytokine alpha-2-glycoprotein 1 (AZGP1) is a 41-kDa protein which regulates insulin sensitivity and glycolipid metabolism. Recently, mounting evidence has indicated that AZGP1 plays a vital role in the progression and prognosis of many types of tumors, including hepatocellular carcinoma. Also, previous research has reported that AZGP1 levels are reduced significantly in patients with gastric carcinoma (GC). Here, we aim to assess the potential role and molecular mechanism underlying AZGP1-mediated regulation of GC progression. Both RT-PCR and Western blot methods demonstrated that AZGP1 levels were decreased in all GC cell lines tested, which included AGS, NCI-N87, MKN-28, SGC-7901 and MKN-45, relative to the normal human gastric mucosa epithelial (GES-1) cell line. Cell survival and proliferation rates were correspondingly were reduced, while cell apoptosis and caspase-3 activity were increased in NCI-N87 and SGC-7901 cells with high levels of AZGP1. Additionally, the mTOR signaling pathway was suppressed, whereas PTEN expression was elevated following transfection of NCI-N87 and SGC-7901 cells with an AZGP1 overexpressing plasmid. PTEN inhibition reversed the effects of AZGP1 on cell growth and apoptosis in SGC-7901 cells. Therefore, we conclude that AZGP1 induced apoptosis and growth inhibition in GC cells via the regulation of the mTOR/PTEN signaling pathway.

Novel diosgenin derivatives containing 1,3,4-oxadiazole/thiadiazole moieties as potential antitumor agents: Design, synthesis and cytotoxic evaluation

Diosgenin, a naturally occurring steroidal saponin, has been confirmed to possess potent anticancer properties. In the current work, two series of novel diosgenin derivatives bearing 1,3,4-oxadiazole (6a−6e and 7a−7e) or 1,3,4-thiadiazole (8a−8e and 9a−9e) moieties were designed, synthesized and evaluated for their cytotoxicities in four human cancer cell lines (HepG2, A549, MCF-7 and HCT-116) and normal human gastric epithelial cells (GES-1) using the MTT assay in vitro. The results showed that compounds 8d and 9d exhibited significant cytotoxic activities against the HepG2 and A549 cells, being more potent than their parent compound diosgenin. Furthermore, the 1,3,4-thiadiazole series of compounds generally exhibited stronger cytotoxicity compared with the 1,3,4-oxadiazole series against HepG2 and A549 cells, and the substitution of 3-pyridyl group at the C5 position of the 1,3,4-thiadiazole ring was the preferred option for these compounds to display significant cytotoxic activities. Compound 8d showed potent cytotoxic activity against A549 cell line (IC50 = 3.93 μM) and was 6.7-fold more potent than diosgenin (IC50 = 26.41 μM). Moreover, compound 8d displayed low toxicity against GES-1 cells (IC50 = 420.4 μM), showing specificity between normal and tumor cells. Further cellular mechanism studies in A549 cells indicated that compound 8d triggered the mitochondrial-mediated apoptosis by decreasing mitochondrial membrane potential, which was associated with up-regulation of Bax, down-regulation of Bcl-2 and activation levels of the caspase cascade. The above results indicated that compound 8d may be used as a promising skeleton for antitumor agents with improved efficacy.

LINC00665 induces gastric cancer progression through activating Wnt signaling pathway.

Long noncoding RNAs (lncRNAs) are recently recognized as noteworthy regulators of different tumors, counting gastric cancer (GC). Lately, long intergenic noncoding RNA (LINC) 00665 has been verified to display significant parts in several cancers. Be that as it may, its role and mechanism in GC movement still stay uninvestigated. As of now, we observed LINC00665 was obviously GC cells (MKN28, BGC-823, SGC7-901, AGS, HGC-27) in comparison to GES-1 cells, which was identified as human normal gastric epithelial cells. Then, LINC00665 was obviously downregulated in GC cells including AGS and BGC-823 cells. Loss of LINC00665 greatly repressed AGS and BGC-823 cell survival and cell expansion. Moreover, GC cell apoptosis was significantly induced by the loss of LINC00665. For another, we found that the GC cell cycle was also captured in G1 and G2 phases. The experiments on cell migration and invasion indicated that knockdown of LINC00665 restrained GC cell migration and invasion. Modifications in Wnt signaling are closely associated with the development of cancers. Here, we found that Wnt signaling was significantly inactivated by the silence of LINC00665 in GC cells. β-catenin and cyclinD1 were restrained whereas GSK-3β was induced by the inhibition of LINC00665 in GC cells. Furthermore, we confirmed the impact of LINC00665 in vivo using xenograft models. Taken these together, we indicated that LINC00665 could function as a novel biomarker in GC progression.

LncRNA SNHG8 promotes proliferation and invasion of gastric cancer cells by targeting the miR-491/PDGFRA axis.

This study aimed to investigate the effects of long non-coding small nucleolar RNA host gene 8 (SNHG8) on the proliferation and invasion of gastric cancer (GC). The GC tissues and adjacent normal tissues from 30 patients were collected. Human GC cell lines, including AGS, SGC-7901, MKN-1, and BGC-803 and normal human gastric epithelial cell line GES-1 were purchased and cultured. The Cell Counting Kit-8 and Western blots were used to access cell proliferation, and platelet-derived growth factor receptor α (PDGFRA) expression, respectively. qRT-PCR was used to evaluate the expression of SNHG8 and miRNA-491. A transwell assay was applied to evaluate cell invasion. This study illustrated that SNHG8 expression was upregulated in GC tissues, as well as in cell lines. Moreover, knockdown of SNHG8 inhibited GC cell proliferation and invasion. Significantly, we determined that miRNA-491 was not only downregulated in stomach cancer but also inhibited GC cell progression induced by SNHG8. Further investigation demonstrated that SNHG8 promoted the proliferation and invasion of GC cells by targeting the miR-491/PDGFRA axis. LncRNA SNHG8 promoted the proliferation and invasion of GC cells by targeting the miR-491/PDGFRA axis, which might provide new insight for potential therapeutic strategies for GC in the future.

MicroRNA-101-3p regulates gastric cancer cell proliferation, invasion and apoptosis by targeting PIM 1 expression

To investigate the effect of microRNA-101-3p (miRNA-101-3p) on proliferation, invasion and apoptosis of gastric cancer (GC) cells, and to explore its influence mechanism. Human GC cell line (AGS) and normal human gastric epithelial cell line (GES-1) were used in this study. The cells were transfected with proto-oncogene serine/threonine-protein kinase (PIM 1) overexpression plasmid, miRNA-101-3p mimics, or miRNA-101-3p non-homologous sequence using lipofectamine 2000. Real-time quantitative polymerase chain reaction (qRT-PCR) and Western blotting were used to determine the expressions of miRNA-101-3p and PIM 1 in GC cells. Cell counting kit-8 (CCK-8) and Transwell assays were used to assess the effect of miRNA-101-3p on proliferation and invasion of GC cells. The regulatory effect of miRNA-101-3p on PIM 1 was assessed using bioinformatics analysis and luciferase reporter gene assay. The expression of miRNA-101-3p was significantly down-regulated in GC cells, relative to normal human gastric epithelial cells (p < 0.05). However, the expression of PIM 1 mRNA was significantly upregulated in GC cells, when compared with normal gastric epithelial cells (p < 0.05). The expression level of miRNA-101-3p was significantly higher in miRNA-101-3p mimic group than in miRNA-101-3p control and normal control groups (p < 0.05). Cell proliferation in miRNA-101-3p mimic group was significantly and time-dependently reduced, when compared with miRNA-101-3p control and normal control groups (p < 0.05). Transfection with miRNA-101-3p mimics significantly increased the invasiveness of AGS cells, and significantly promoted their apoptosis (p < 0.05). Results of qRT-PCR and Western blotting showed that increased miRNA-101-3p expression significantly reduced the expression of PIM 1, while decreased miRNA-101-3p expression promoted the expression of PIM 1 (p < 0.05). Results of bioinformatics showed that miRNA-101-3p had specific binding sequence in the 3'UTR region of PIM 1. Cloning of miRNA-101-3p sequence into the luciferase reporter plasmid led to significant inhibition of the expression of PIM 1 (p < 0.05), but had no inhibitory effect on mutated PIM 1 3'UTR (p > 0.05). Overexpression of PIM 1 significantly reversed the inhibition of proliferation and invasion, and promotion of apoptosis by miRNA-101-3p (p < 0.05). These results indicate that miRNA-101-3p inhibits the proliferation and invasion of GC cells, and promotes their apoptosis by regulation of the expression of PIM-1.

Inhibition of Proliferation of SGC7901 and BGC823 Human Gastric Cancer Cells by Ursolic Acid Occurs Through a Caspase-Dependent Apoptotic Pathway.

BackgroundWorldwide, gastric cancer is one of the most common malignant tumors. Ursolic acid is a plant metabolite and pentacyclic triterpenoid used in traditional Chinese medicine. This study aimed to investigate the effects of ursolic acid the growth and apoptosis of SGC7901 and BGC823 human gastric cancer cells in vitro.Material/MethodsSGC7901 and BGC823 human gastric cancer cells and normal GES-1 gastric epithelial cells were cultured with increasing doses of ursolic acid at 50, 60, and 100 μM. Cell viability and proliferation were assessed using an MTT assay. Flow cytometry was used to assess cell apoptosis. Western blot was used to measure procaspase-8, procaspase-9, procaspase-3, and cleaved poly (ADP-ribose) polymerase (PARP) expression. The expression of receptor interaction protein 3 (RIP3) was examined by Western blot and reverse transcription polymerase chain reaction (RT-PCR). Morphological changes in the gastric cancer cells were determined using Hoechst 33342 staining following ursolic acid treatment.ResultsUrsolic acid inhibited the viability of SGC7901 and BGC823 cells but not GES-1 cells. Ursolic acid treatment significantly induced apoptosis in SGC7901 and BGC823 cells when compared with GES-1 cells (P<0.05), and significantly increased the activation of caspase-3, caspase-8, caspase-9, poly ADPribose polymerase (PARP), and the production of reactive oxygen species (ROS). Treatment of SGC7901 and BGC823 cells with ursolic acid for 72 h did not induce necroptosis.ConclusionsUrsolic acid inhibited the proliferation of SGC7901 and BGC823 human gastric cancer cells in vitro through a caspase-dependent apoptotic pathway.

MiR-135b-5p promotes viability, proliferation, migration and invasion of gastric cancer cells by targeting Krüppel-like factor 4 (KLF4).

IntroductionThe expression of MiR-135b-5p was up-regulated while Krüppel-like factor 4 (KLF4) expression was extremely low in human gastric carcinoma (GC) tissues. This study aimed to explore the role of miR-135b-5p in GC cells and its influence on various cell capacity and viability by targeting KLF4.Material and methodsThe dual-luciferase reporter assay was first performed and the target relationship between miR-135b-5p and KLF4 was confirmed. Then three GC cell lines and the human normal gastric epithelial cell line (GES1) were analyzed for the expression level of miR-135b-5p and KLF4 mRNA by RT-qPCR. The BGC-823 GC cell line was chosen for subsequent assays.ResultsThe expression of miR-135b-5p and KLF4 was manipulated via transfection. The changes of proliferation, invasion, migration, viability, cycle and apoptosis of GC cells were evaluated by MTS, colony formation assay, transwell assay, wound healing assay and flow cytometry assay, respectively. Overexpression of MiR-135b-5p enhanced viability, proliferation, invasion and migration of GC cells, increased cell viability and reduced cell apoptosis. Replenishing of KLF4 functioned oppositely.ConclusionsThe inhibitory effects of ectopic KLF4 could be attenuated by co-transfection of miR-135b-5p. Collective data suggested that miR-135b-5p has a tumor-promoting role in GC cells via downregulating KLF4. Hence, inhibition of miR-135b-5p could be valuable for treatment of gastric cancer.

The activation of high mobility group Box1 and toll-like receptor 4 is involved in clopidogrel-induced gastric injury through p38 MAPK.

Clopidogrel-induced gastric injury is an important clinical problem. However, the exact mechanism was still unclarified. This study aimed to investigate the role of high mobility group box protein 1 (HMGB1) and the toll-like receptor 4 (TLR4) pathway in normal human gastric epithelial (GES-1) cells under clopidogrel exposure. Morphological alterations of the gastric epithelial cells were observed under a microscope. A laser scanning confocal microscope was used to determine the distribution of HMGB1 and TLR4 in clopidogrel-induced injury. MTT was used to compare the viability of GES-1 cell among the pretreated Cli-095 (TLR4 inhibitor) group, pretreated ethyl pyruvate (HMGB1 inhibitor) group, clopidogrel group, and control group. Moreover, expression of the HMGB1, TLR4, tight junction (TJ) proteins occludin and the phosphorylation of the mitogen-activated protein kinases (MAPK) p38 were examined by western blot. We found the expression of HMGB1 and TLR4 in the cytoplasm increased after clopidogrel administration. Besides, inhibited TLR4, which is a receptor of HMGB1, prevented the injury and occludin reducing caused by clopidogrel challenge. Furthermore, blocking HMGB1 or TLR4 activity by ethyl pyruvate (HMGB1 inhibitor) or cli-095(TLR4 inhibitor) can partially diminish the activation of p38MAPK. Gastric mucosal damages observed by clopidogrel challenge are associated with HMGB1, TLR4, through p38MAPK signal pathway.