Upregulation of OTX2-AS1 is Associated With Immune Infiltration and Predicts Prognosis of Gastric Cancer.

BACKGROUND: It is unclear whether the long non-coding RNA (lncRNA) OTX2 antisense RNA 1 (OTX2-AS1) plays a pivotal role in gastric cancer (GC). An analysis of The Cancer Genome Atlas (TCGA) database data and bioinformatics was used to explore the relationship between OTX2-AS1 and GC in the current study. METHODS: We evaluated the relationship between clinical features and OTX2-AS1 expression, prognostic factors, and the significant involvement of OTX2-AS1 in function using various statistical methods, such as Kaplan-Meier method, Cox regression analysis, Gene Set Enrichment Analysis (GSEA), and immune infiltration analysis. GC cell lines were tested for OTX2-AS1 expression using qRT-PCR. RESULTS: A high level of OTX2-AS1 expression was significantly and negatively associated with Helicobacter pylori (H pylori) infection in GC patients (P = .006) and predicted a poorer overall survival (OS) (HR: 1.54; 95% CI: 1.10-2.14; P = .011), progression-free interval (PFI) (HR: 1.75; 95% CI: 1.22-2.51; P = .002) and disease-specific survival (DSS) (HR: 1.85; 95% CI: 1.21-2.85; P = .005) in GC patients. There was an independent correlation between OTX2-AS1 expression (HR: 1.771; 95% CI: 1.164-2.696; P = .008) and OS in patients with GC. There were differential enrichments for the OTX2-AS1 high expression phenotype in the olfactory transduction, G alpha (s) signaling events, keratinization, olfactory signaling pathway, and preimplantation embryo. OTX2-AS1 expression may be related to certain immune-infiltrating cells. Compared to gastric epithelial cells (GES-1), GC cell lines showed a significant increase in OTX2-AS1 expression. CONCLUSION: There was a significant association between OTX2-AS1 expression in GC patients and poor survival, suggesting that it may be a useful biomarker for prognosis and immunotherapy outcome of stomach adenocarcinoma (STAD) in GC.

LncRNA HCG11 promotes 5-FU resistance of colon cancer cells through reprogramming glucose metabolism by targeting the miR-144-3p-PDK4 axis.

BACKGROUND: Colorectal cancer (CRC), one of the most common human malignancies, is a leading cause of the cancer-related mortality. 5-FU is a first-line chemotherapeutic agent against CRC. Although CRC patients responded to 5-FU therapy initially, a part of patients succumbed to CRC due to the acquired drug resistance. Thus, investigating molecular mechanisms underlying chemoresistance will contribute to developing novel strategies against colorectal cancer. OBJECTIVE: Accumulation evidence revealed pivotal roles of long non-coding RNAs (lncRNAs) in tumorigenesis and chemoresistance of CRC. However, the precise roles and molecular mechanisms of lncRNA-HCG11 in CRC remain unclear. This study aimed to investigate the biological roles and underlying mechanisms of HCG11 as well as its molecular targets in regulating the cellular metabolism processes, which facilitate the chemoresistance of CRC. METHODS AND RESULTS: This study uncovers that HCG11 was significantly upregulated in CRC tumors tissues and cell lines. Moreover, HCG11 was elevated in 5-FU resistant CRC tumors. Silencing HCG11 inhibited colon cancer cell proliferation, migration, invasion and glucose metabolism and sensitized CRC cells to 5-FU. In addition, we detected increased HCG11 expression level and glucose metabolism in the established 5-FU resistant CRC cell line (DLD-1 5-FU Res). Furthermore, microRNA-microArray, RNA pull-down and luciferase assays demonstrated that HCG11 inhibited miR-144-3p which displays suppressive roles in colon cancer via sponging it to form a ceRNA network. We identified pyruvate dehydrogenase kinase 4 (PDK4), which is a glucose metabolism key enzyme, was directly targeted by miR-144-3p in CRC cells. Rescue studies validated that the miR-144-3p-inhibited glucose metabolism and 5-FU sensitization were through targeting PDK4. Finally, restoration of miR-144-3p in HCG11-overexpressing DLD-1 5-FU resistant cells successfully overcame the HCG11-faciliated 5-FU resistance via targeting PDK4. CONCLUSION: In summary, this study reveals critical roles and molecular mechanisms of the HCG11-mediated 5-FU resistance through modulating the miR-144-3p-PDK4-glucose metabolism pathway in CRC.

Leucine-rich alpha-2-glycoprotein-1, relevant with microvessel density, is an independent survival prognostic factor for stage III colorectal cancer patients: a retrospective analysis.

BACKGROUND: Leucine-rich alpha-2-glycoprotein-1 (encoded by LRG1) has been shown to be involved in multiple cancer progression and angiogenesis. LRG1 has been shown to be one of the five plasma proteins that can be used for colorectal cancer (CRC) diagnosis. The objective of the current study was to explore relationship between LRG1 protein expression and microvessel density (MVD) in stage III CRC. METHODS: A single-center retrospective analysis of all stage III CRC who underwent surgery and adjuvant chemotherapy was carried out. LRG1 and CD34 were tested in tumor tissues by immunohistochemistry (IHC). RESULTS: LRG1 protein expression was significantly associated with MVD (P <0.001) and other clinicopathological parameters, including T stage (P=0.028), differentiation (P=0.035) and vascular invasion (P=0.007). Cox multivariate regression analysis showed that LRG1 protein expression was an independent poor predictive factor for both disease-free and overall survival. CONCLUSION: LRG1 protein expression can be used as a prognostic marker for stage III CRC along with its use as a diagnostic marker for CRC in general.

Oral attenuated Salmonella typhimurium vaccine against MG7-Ag mimotope of gastric cancer.

AIM: To develop an oral attenuated Salmonella typhimurium vaccine against gastric cancer and to evaluate its efficacy in mice. METHODS: A complementary sequence of Nco I site and a sequence coding for MG7-Ag mimotope were designed at the 5' terminus of forward primer. Using p1.2 II-HBCAg plasmid as template, PCR was performed to get a fusion gene of the mimotope and a HBcAg gene. The fusion gene was then subcloned into the plasmid pYA3341 complementary to Salmonella typhimurium X4550, and the recombinant plasmid was then transformed into attenuated Salmonella typhimurium X4550. Balb/c mice were orally immunized with the recombinant Salmonella typhimurium X4550. The mice were immunized every 2 wk to reinforce the immunity. At the 6th wk, serum titer of antibody was detected by ELISA, and at the 8th wk, cellular immunity was detected by (51)Cr release test. Ehrlich ascites carcinoma cells expressing MG7-Ag were used in tumor challenge assay as a model to evaluate the protective effect of the vaccine. RESULTS: Serum titer of antibody against MG7-Ag was significantly higher in mice immunized with the vaccine than in control groups (0.9538+/-0.043 vs 0.6531+/-0.018, P<0.01; 0.9538+/-0.043 vs 0.6915+/-0.012, P<0.01), while in vitro (51)Cr release assay of the splenocytes showed no statistical difference in the three groups. Two weeks after tumor challenge, 1 in 5 immunized mice was tumor free, while all the mice in the control group presented tumor. CONCLUSION: Oral attenuated Salmonella typhimurium vaccine against the MG7-Ag mimotope of gastric cancer is immunogenic. It can induce significant humoral immunity against tumors in mice, and has some protective effects.

Screening and identification of mimotope of gastric cancer associated antigen MGb1-Ag.

AIM: Using a monoclonal antibody against gastric cancer antigen named MGb1 to screen a phage-displayed random peptide library fused with coat protein pIII in order to get some information on mimotopes. METHODS: Through affinity enrichment and ELISA screening, positive clones of phages were amplified. 10 phage clones were selected after three rounds of biopanning and the ability of specific binding of the positive phage clones to MGb1-Ab were detected by ELISA assay (DNA sequencing was performed and the amino acid sequences were deduced) By blocking test, specificity of the mimic phage epitopes was identified. RESULTS: There were approximately 200 times of enrichment about the titer of bound phages after three rounds of biopanning procedures. DNA of 10 phage clones after the third biopanning was assayed and the result showed that the positive clones had a specific binding activity to MGb1-Ab and a weak ability of binding to control mAb or to mouse IgG. DNA sequencing of 10 phage clones was performed and the amino acid sequences were deduced. According to the homology of the amino acid sequences of the displayed peptides, most of the phage clones had motifs of H(x)Q or L(x)S. And these 10 phage clones could also partly inhibit the binding of MGb1-Ab to gastric cancer cell KATO-III. The percentage of blocking was from (21.0+/-1.6) % to (39.0+/-2.7) %. CONCLUSION: Motifs of H(x)Q and L(x)S selected and identified show a high homology in the mimic epitopes of gastric cancer associated antigen. There may be one or more clones which can act as candidates of tumor vaccines.

Development of an oral DNA vaccine against MG7-Ag of gastric cancer using attenuated salmonella typhimurium as carrier.

AIM: To develop an oral DNA vaccine against gastric cancer and evaluate its efficacy in mice. METHODS: The genes of the MG7-Ag mimotope and a universal Th epitope (Pan-DR epitope, PADRE) were included in the PCR primers. By PCR, the fusion gene of the two epitopes was amplified. The fusion gene was confirmed by sequencing and was then cloned into pcDNA3.1(+) plasmid. The pcDNA3.1 (+)-MG7/PADRE was used to transfect an attenuated Salmonella typhimurium. C57BL/6 mice were orally immunized with 1X10(8) cfu Salmonella transfectants. Salmonella harboring the empty pcDNA3.1(+) plasmid and phosphate buffer saline (PBS) were used as negative controls. At the 6th week, serum titer of MG7-Ag specific antibody was detected by ELISA. At the 8th week cellular immunity was detected by an unprimed proliferation test of the spleenocytes by using a ((3)H)-thymidine incorporation assay. Ehrlich ascites carcinoma cells expressing MG7-Ag were used as a model in tumor challenge assay to evaluate the protective effect of the vaccine. RESULTS: Serum titer of antibody against MG7-Ag was significantly higher in mice immunized with the vaccine than that in control groups (0.841 vs 0.347, P<0.01; 0.841 vs 0.298, P<0.01), while in vitro unprimed proliferation assay of the spleenocytes showed no statistical difference between those three groups. Two weeks after tumor challenge, 2 in 7 immunized mice were tumor free, while all the mice in the control groups showed tumor formation. CONCLUSION: Oral DNA vaccine against the MG7-Ag momitope of gastric cancer is immunogenic. It can induce significant humoral immunity against tumor in mice, and the vaccine has partially protective effects.

[In-vivo screening and characterization of peptides specific for vasculature of gastric cancer].

AIM: To screen in vivo from a phage-displayed peptide library polypeptide fragments specific binding to vascular endothelial cells of gastric cancer xenografts, so as to provide for anti-angiogenesis therapy of tumor. METHODS: Immunosupressed mice models for human gastric cancer xeno-grafts were established by subrenal capsular assay (SR-CA). The 12-peptide library was panned through 4 rounds. Phages were recovered and titrated from tumor xenografts and control tissue (brain). The distribution of phage were detected in transplanted tumor tissues by immunohistochemical staining. RESULTS: Phage homing to gastric cancer xenografts were enriched through four rounds of panning,being 3.4-fold of that recovered from brain tissue. Peptide sequences were characterized for randomly picked-upclones and the peptide sequence YESIRIGVAPSQ appeared most frequently. Immunohistochemical staining for the homing phage revealed a specific vascular endothelial cell localization in gastric cancer xenografts 5 min after injection of the enriched phages. When the specific phage individually test-ed, the phage recovered from gastric cancer xenografts were as 4. 2 times as those from control tissue ( brain) , as 4.9 times as those from lung, as 5.4 times as those from heart. CONCLUSION: The tumor-specific homing peptides may provide a effective tool for targeting tumor vasculature in anti-angiogenesis therapy of cancer. The in vivo selection technique in this study was feasible and applicable to screening peptides homing to vascular endothelial cells.