Development and characterization of a novel specific monoclonal antibody against mink enteritis virus and its antigen epitope analysis.

This study prepared a novel monoclonal antibody (MAb) against mink enteritis parvovirus (MEV) and identified its antigen epitope. The antibody subclass is identified as IgG1, the titers of the MAb is up to 1:1 × 106 and keeps stably after low-temperature storage for 9 months or 11 passages of the MAb cells. The MAb can specifically recognize MEV in the cells in IFA, but not Aleutian disease virus (ADV) or canine distemper virus (CDV). Its antigen epitope was identified as a polypeptide containing 5 key amino acids (378YAFGR382) and the homology in 20 MEV strains, 4 canine parvovirus strains, and 4 feline panleukopenia virus strains was 100%. This study supplies a biological material for developing new methods to detect MEV.

Long Non-Coding RNA LOC339059 Attenuates IL-6/STAT3-Signaling-Mediated PDL1 Expression and Macrophage M2 Polarization by Interacting with c-Myc in Gastric Cancer.

Background: Long non-coding RNA (lncRNA) was identified as a novel diagnostic biomarker in gastric cancer (GC). However, the functions of lncRNAs in immuno-microenvironments have not been comprehensively explored. In this study, we explored a critical lncRNA, LOC339059, that can predict the clinical prognosis in GC related to the modulation of PD-L1 and determined its influence upon macrophage polarization via the IL-6/STAT3 pathway. Methods: To date, accumulating evidence has demonstrated that the dysregulation of LOC339059 plays an important role in the pathological processes of GC. It acts as a tumor suppressor, regulating GC cell proliferation, migration, invasion, tumorigenesis, and metastasis. A flow cytometry assay showed that the loss of LOC339059 enhanced PDL1 expression and M2 macrophage polarization. RNA sequencing, RNA pull-down, RNA immunoprecipitation, Chip-PCR, and a luciferase reporter assay revealed the pivotal role of signaling alternation between LOC339059 and c-Myc. Results: A lower level of LOC339059 RNA was found in primary GC tissues compared to adjacent tissues, and such a lower level is associated with a poorer survival period (2.5 years) after surgery in patient cohorts. Moreover, we determined important immunological molecular biomarkers. We found that LOC339059 expression was correlated with PD-L1, CTLA4, CD206, and CD204, but not with TIM3, FOXP3, CD3, C33, CD64, or CD80, in a total of 146 GC RNA samples. The gain of LOC339059 in SGC7901 and AGS inhibited biological characteristics of malignancy, such as proliferation, migration, invasion, tumorigenesis, and metastasis. Furthermore, our data gathered following the co-culture of THP-1 and U937 with genomic GC cells indicate that LOC339059 led to a reduction in the macrophage cell ratio, in terms of CD68+/CD206+, to 1/6, whereas the selective knockdown of LOC339059 promoted the abovementioned malignant cell phenotypes, suggesting that it has a tumor-suppressing role in GC. RNA-Seq analyses showed that the gain of LOC339059 repressed the expression of the interleukin family, especially IL-6/STAT3 signaling. The rescue of IL-6 in LOC339059-overexpressing cells reverted the inhibitory effects of the gain of LOC339059 on malignant cell phenotypes. Our experiments verified that the interaction between LOC339059 and c-Myc resulted in less c-Myc binding to the IL-6 promoter, leading to the inactivation of IL-6 transcription. Conclusions: Our results establish that LOC339059 acts as a tumor suppressor in GC by competitively inhibiting c-Myc, resulting in diminished IL-6/STAT3-signaling-mediated PDL1 expression and macrophage M2 polarization.

Long non-coding RNA SNHG1 suppresses cell migration and invasion and upregulates SOCS2 in human gastric carcinoma.

Gastric carcinoma (GC) is one of the most common malignancies and the third leading cause of cancer-related deaths worldwide. Long noncoding RNAs (lncRNAs) may be an important class of functional regulators involved in human gastric cancers development. In this study, we investigated the clinical significance and function of lncRNA SNHG1 in GC. SNHG1 was significantly downregulated in GC tumor tissues compared with adjacent noncancerous tissues. Overexpression of SNHG1 in BGC-823 cells remarkably inhibited not only cell proliferation, migration, invasion in vitro, but also tumorigenesis and lung metastasis in the chick embryo chorioallantoic membrane (CAM) assay in vivo. Conversely, inhibition of SNHG1 by transfection of siRNA in AGS cells resulted in opposite phenotype changes. Mechanically, SNHG1 was found interacted with ILF3, NONO and SFPQ. RNA-seq combined with bioinformatic analysis identified a serial of downstream genes of SNHG1, including SOCS2, LOXL2, LTBP3, LTBP4. Overexpression of SNHG1 induced SOCS2 expression whereas knockdown of SNHG1 decreased SOCS2 expression. In addition, knockdown of SNHG1 promoted the activation of JAK2/STAT signaling pathway. Taken together, our data suggested that SNHG1 suppressed aggressive phenotype of GC cells and regulated SOCS2/JAK2/STAT pathway.

Monoclonal antibody 3H11 chimeric antigen receptors enhance T cell effector function and exhibit efficacy against gastric cancer.

Although chimeric antigen receptor T cell (CAR-T) therapies for certain types of solid tumors have been used in clinical trials, novel CARs that are able to target gastric cancer (GC) are still required. In our previous study, monoclonal antibody 3H11 (mAb 3H11), generated from immunization with five human GC cell lines, was demonstrated to have a 93.5% positive reaction with a clear membrane location and more than 5% cancer cell staining in GC tissues in our previous study. In the present study, 3H11-CARs were designed for modified T cell therapy. To begin with, it was confirmed that the single-chain variable fragment (scFV) of the mAb 3H11, known as scFV-3H11, exhibited similar activity with the natural antibody. In addition, scFV-3H11 CAR-T cells are able to kill tumor cells accompanied with increased interleukin-2 and interferon-γ secretion in vitro, and reduced the tumor burden in GC cell lines and patient-derived GC cells in vivo. In conclusion, scFV-3H11 CARs may have the potential to treat mAb 3H11-positive GC.

MicroRNA-130a-3p suppresses cell migration and invasion by inhibition of TBL1XR1-mediated EMT in human gastric carcinoma.

MiR-130a-3p was found to play tumor suppressor role in most human cancers, except for gastric cancer. However, in this study, we demonstrated that miR-130a-3p was significantly down-regulated in gastric carcinoma (GC) tissues compared with adjacent non-neoplastic tissues, and decreased miR-130a-3p expression was associated with shorter overall survival (OS) and was an independent prognostic factor for OS in GC patients. Over-expression of miR-130a-3p remarkably inhibited not only GC cell migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro, but also tumorigenesis and lung metastasis in the chick embryo chorioallantoic membrane (CAM) assay in vivo. Conversely, inhibition of miR-130a-3p resulted in opposite phenotype changes in GC cells. Furthermore, TBL1XR1 was identified as a direct target of miR-130a-3p, and reintroduction of TBL1XR1 into miR-130a-3p-transfected MGC-803 cells reversed the inhibitory effects of miR-130a-3p on GC cell migration, invasion and EMT. Taken together, our data suggested that miR-130a-3p suppressed aggressive phenotype of GC cells partially by direct targeting and decreasing TBL1XR1 and subsequent EMT process.

SLC3A2, antigen of mAb 3G9, promotes migration and invasion by upregulating of mucins in gastric cancer.

Solute carrier family 3 member 2 (SLC3A2) has been reported to be highly expressed in a variety of carcinomas. However, the function of SLC3A2 in gastric carcinoma (GC) has not been well explored. Monoclonal antibody (mAb) 3G9, generated from immunogen of various human GC cell lines, has been shown to bind to GC tissues specifically. In this study, we identified the target antigen of mAb 3G9 as SLC3A2, and detected the expression profile of SLC3A2 in a panel of gastric cancer cell lines and GC tumor tissues. We found that the increased expression of SLC3A2 was associated with serosal invasion in GC patients. Knockout of SLC3A2 suppressed the migration and invasion of BGC-823 cells in vitro and in vivo, whereas overexpression of SLC3A2 in NCI-N87 cells promoted the migration and invasion in vitro and in vivo. Mechanistic investigations suggested that MUC1, MUC16 and MUC5B were the downstream genes of SLC3A2 in GC cells. Taken together, our data suggested that SLC3A2 promoted the aggressive phenotype of GC by upregulating several mucin genes expression and may serve as a potential biomarker for diagnosis and target therapy.