Research on Function of Exosome of miR-328-3p Secreted by Bone Marrow Mesenchymal Stem Cells (BMSCs) on Restraining the Gastric Cancer Through Being Down-Regulated with Trefoil Factor 3

Certain progress has been made in the therapeutic method against gastric cancer such as surgical operation combined with chemotherapy and radiation therapy in recent years. But the therapeutic efficacy and prognosis on gastric cancer was still not satisfactory. The function of exosome of miR-328–3p secreted by bone marrow stromal cells (BMSCs) on restraining the gastric cancer was studied in the present study. The BMSCs with highly-expressed miR-328-3p was established. The exosome in cell supernatant was collected. The exosome of BMSCs and MSCs with highlyexpressed miR-328-3p was added into SGC-7901 cells followed by analysis of miR-328-3p level by Real-time PCR and TFF3 (Trefoil Factor 3) level in exosome by Western blot, cell proliferation, expression of E-cadherin, Vimentin and Caspase-3. miR-328-39 expression was reduced and TFF3 was elevated in gastric cancer tissue (P < 0.05). miR-328-3p was upregulated and TFF3 was downregulated after addition of BMSCs exosomes along with increased cell proliferation and reduced E-cadherin and Caspase3 expression (P < 0.05). In conclusion, exosome of BMSCs could be regulated by miR-328-3p and TFF3 expression is restrained so as to regulate the biological behaviors of gastric cancer cell.

Transcriptional and Epigenetic Bioinformatic Analysis of Claudin-9 Regulation in Gastric Cancer

Gastric cancer is a heterogeneous disease that represents 5% to 10% of all new cancer cases worldwide. Advances in histological diagnosis and the discovery of new genes have admitted new genomic classifications. Nevertheless, the bioinformatic analysis of gastric cancer databases has favored the detection of specific differentially expressed genes with biological significance. Claudins, a family of proteins involved in tight junction physiology, have emerged as the key regulators of cellular processes, such as growth, proliferation, and migration, associated with cancer progression. The expression of Claudin-9 in the gastric cancer tissue has been linked to poor prognosis, however, its transcriptional and epigenetic regulations demand a more comprehensive analysis. Using the neural network promoter prediction, TransFact, Uniprot-KB, Expasy-SOPMA, protein data bank, proteomics DB, Interpro, BioGRID, String, and the FASTA protein sequence databases and software, we found the following: (1) the promoter sequence has an unconventional structure, including different transcriptional regulation elements distributed throughout it, (2) GATA 4, GATA 6, and KLF5 are the key regulators of Claudin-9 expression, (3) Oct1, NF-κB, AP-1, c-Ets-1, and HNF-3β have the higher binding affinity to the CLDN9 promoter, (4) Claudin-9 interacts with cell differentiation and development proteins, (5) CLDN9 is highly methylated, and (6) Claudin-9 expression is associated with poor survival. In conclusion, Claudin-9 is a protein that should be considered a diagnostic marker as its gene promoter region binds to the transcription factors associated with the deregulation of cell control, enhanced cell proliferation, and metastasis.

Tenascin-c knockdown suppresses vasculogenic mimicry of gastric cancer by inhibiting ERK- triggered EMT

Gastric cancer is one of the most common malignancies worldwide and vasculogenic mimicry (VM) is considered to be the leading cause for the failure of anti-angiogenesis therapy in advanced gastric cancer patients. In the present study, we investigate the role of tenascin-c (TNC) in the formation of VM in gastric cancer and found that TNC was upregulated in gastric cancer tissue than in the corresponding adjacent tissues and correlated with VM and poor prognosis of gastric cancer. Furthermore, knockdown of TNC significantly inhibited VM formation and proliferation of gastric cancer cells in vitro and in vivo, with a reduction in cell migration and invasion. Mechanistically, TNC knockdown suppressed the phosphorylation of ERK and subsequently inhibited the process of EMT, both of which play an important role in VM formation. Our results indicated that TNC plays an important role in VM formation in gastric cancer. Combining inhibition of TNC and ERK may be a potential therapeutic approach to inhibit gastric cancer growth and metastasis and decrease antiangiogenic therapeutic resistance.

Prognostic Value of Highly Expressed Type VII Collagen (COL7A1) in Patients With Gastric Cancer

Collagen is a major component in the tumor microenvironment. This study reveals a novel biomarker candidate, type VII collagen (COL7A1), in patients with gastric cancer. To identify genes differentially expressed in gastric cancer tissue, we analyzed cancerous (n = 20) and noncancerous tissues (n = 13) using a DNA microarray. To perform immunohistochemistry and validate the upregulation of COL7A1 expression, we collected 200 more gastric cancer tissues and 100 normal gastric tissues from 200 randomly selected patients who underwent gastrectomy for gastric cancer between January 2010 and December 2013. The correlations between COL7A1 expression and clinicopathological parameters and patients’ overall survival (OS) were analyzed. In the microarray, COL7A1 was upregulated in gastric cancer tissue compared with normal tissue. In the immunohistochemistry study, COL7A1 was more highly expressed in cancer tissue than in normal tissue (p = 0.001). Patients with intracellular COL7A1 expression had significantly poorer five-year OS than those with only extracellular expression (41.5 versus 69.7%, p = 0.001), and the site of expression was an independent prognostic factor of OS (hazard ratio 2.00, 95% CI 1.26–3.16, p = 0.003). Also, we found a significant association between the COL7A1 immunohistochemistry score and distant metastasis (high versus low, odds ratio 4.45, 95% CI 1.40–14.16, p = 0.011). The site and total immunohistochemistry score of COL7A1 expression in gastric cancer showed prognostic significance for OS and distant metastasis, respectively. COL7A1 could be a novel biomarker with diagnostic and therapeutic value.

Norepinephrine Enhances Aerobic Glycolysis and May Act as a Predictive Factor for Immunotherapy in Gastric Cancer

Objective/Background and Aims. The gastrointestinal tract is rich in neurotransmitters, which play an essential role in the occurrence and development of gastrointestinal tumors. We aimed to explore the function of neurotransmitters in gastric cancer and identify a suitable target to treat gastric cancer patients in the future. Methods. Monoamine neurotransmitters were detected in gastric cancer tissue and paired normal tissue, and The Cancer Genome Atlas was used to identify differentially expressed norepinephrine-degrading and synthetic enzymes. Quantitative real-time PCR and the Seahorse assay were used to determine the effect of norepinephrine on gastric cancer cell glycolysis. MAOA expression in cancer tissues was analyzed by immunohistochemistry and was compared with the patient SUVmax value of PET-CT and other clinicopathological characteristics. Results. The norepinephrine levels were markedly high in gastric cancer tissue, while the norepinephrine-degrading enzymes MAOA and MAOB showed low expression. High norepinephrine levels were associated with activated glycolysis. The MAOA or MAOB expression levels in tumor tissue were closely correlated with the patient SUV max values of PET-CT and immunotherapy evaluation indices, such as PD-L1 and the microsatellite status. Conclusions. Norepinephrine shows relatively higher expression in gastric cancer tissue than in normal tissue, and its expression level is associated with the glycolysis levels in patients. The norepinephrine-degrading enzymes MAOA and MAOB have significant expression differences in cancer and normal tissue, and their missing or low expression may predict immune therapy outcomes for gastric cancer patients. High norepinephrine levels with metabolic abnormalities may be more suitable for metabolic targeted therapy or immunotherapy.

Knockdown of Microtubule Associated Serine/threonine Kinase Like Expression Inhibits Gastric Cancer Cell Growth and Induces Apoptosis by Activation of ERK1/2 and Inactivation of NF-κB Signaling

SummaryMicrotubule-associated serine/threonine kinase (MASTL) functions to regulate chromosome condensation and mitotic progression. Therefore, aberrant MASTL expression is commonly implicated in various human cancers. This study analyzed MASTL expression in gastric cancer vs. adjacent normal tissue for elucidating the association with clinicopathological data from patients. This work was then extended to investigate the effects of MASTL knockdown on tumor cells in vitro. The level of MASTL expression in gastric cancer tissue was assessed from the UALCAN, GEPIA, and Oncomine online databases. Lentivirus carrying MASTL or negative control shRNA was infected into gastric cancer cells. RT-qPCR, Western blotting, cell viability, cell counting, flow cytometric apoptosis and cell cycle, and colony formation assays were performed. MASTL was upregulated in gastric cancer tissue compared to the adjacent normal tissue, and the MASTL expression was associated with advanced tumor stage, Helicobacter pylori infection and histological subtypes. On the other hand, knockdown of MASTL expression significantly reduced tumor cell viability and proliferation, and arrested cell cycle at G2/M stage but promoted tumor cells to undergo apoptosis. At protein level, knockdown of MASTL expression enhanced levels of cleaved PARP1, cleaved caspase-3, Bax and p-ERK1/2 expression, but downregulated expression levels of BCL-2 and p-NF-κB-p65 protein in AGS and MGC-803 cells. MASTL overexpression in gastric cancer tissue may be associated with gastric cancer development and progression, whereas knockdown of MASTL expression reduces tumor cell proliferation and induces apoptosis. Further study will evaluate MASTL as a potential target of gastric cancer therapeutic strategy.

Polymorphisms in Pepsinogen C and miRNA Genes Associate with High Serum Pepsinogen II in Gastric Cancer Patients

Background: Pepsinogen (PG) II (PGII) is a serological marker used to estimate the risk of gastric cancer but how PGII expression is regulated is largely unknown. It has been suggested that PGII expression, from the PGC (Progastricsin) gene, is regulated by microRNAs (miRNA), but how PGII levels vary with Helicobacter pylori (H. pylori) infection and miRNAs genotype remains unclear. Methods: Serum levels of PGI and PGII were determined in 80 patients with gastric cancer and persons at risk for gastric cancer (74 first-degree relatives of patients, 62 patients with autoimmune chronic atrophic gastritis, and 2 patients with dysplasia), with and without H. pylori infection. As control from the general population, 52 blood donors were added to the analyses. Associations between PGII levels and genetic variants in PGC and miRNA genes in these groups were explored based on H. pylori seropositivity and the risk for gastric cancer. The two-dimensional difference in gel electrophoresis (2D-DIGE) and the NanoString analysis of messenger RNA (mRNAs) from gastric cancer tissue were used to determine the pathways associated with increased PGII levels. Results: PGII levels were significantly higher in patients with gastric cancer, and in those with H. pylori infection, than in other patients or controls. A PGI/PGII ratio ≤ 3 was found better than PGI < 25 ng/mL to identify patients with gastric cancer (15.0% vs. 8.8%). For two genetic variants, namely rs8111742 in miR-Let-7e and rs121224 in miR-365b, there were significant differences in PGII levels between genotype groups among patients with gastric cancer (p = 0.02 and p = 0.01, respectively), but not among other study subjects. Moreover, a strict relation between rs9471643 C-allele with H. pylori infection and gastric cancer was underlined. Fold change in gene expression of mRNA isolated from gastric cancer tissue correlated well with polymorphism, H. pylori infection, increased PGII level, and pathway for bacteria cell entry into the host. Conclusions: Serum PGII levels depend in part on an interaction between H. pylori and host miRNA genotypes, which may interfere with the cut-off of PGI/PGII ratio used to identify persons at risk of gastric cancer. Results reported new findings regarding the relation among H. pylori, PGII-related host polymorphism, and genes involved in this interaction in the gastric cancer setting.