Construction of an immune-related risk score signature for gastric cancer based on multi-omics data.

Early identification of gastric cancer (GC) is associated with a superior survival rate compared to advanced GC. However, the poor specificity and sensitivity of traditional biomarkers suggest the importance of identifying more effective biomarkers. This study aimed to identify novel biomarkers for the prognosis of GC and construct a risk score (RS) signature based on these biomarkers, with to validation of its predictive performance. We used multi-omics data from The Cancer Genome Atlas to analyze the significance of differences in each omics data and combined the data using Fisher's method. Hub genes were subsequently subjected to univariate Cox and LASSO regression analyses and used to construct the RS signature. The RS of each patient was calculated, and the patients were divided into two subgroups according to the RS. The RS signature was validated in two independent datasets from the Gene Expression Omnibus and subsequent analyses were subsequently conducted. Five immune-related genes strongly linked to the prognosis of GC patients were obtained, namely CGB5, SLC10A2, THPO, PDGFRB, and APOD. The results revealed significant differences in overall survival between the two subgroups (p < 0.001) and indicated the high accuracy of the RS signature. When validated in two independent datasets, the results were consistent with those in the training dataset (p = 0.003 and p = 0.001). Subsequent analyses revealed that the RS signature is independent and has broad applicability among various GC subtypes. In conclusion, we used multi-omics data to obtain five immune-related genes comprising the RS signature, which can independently and effectively predict the prognosis of GC patients with high accuracy.

RUNX2 promotes gastric cancer progression through the transcriptional activation of MGAT5 and MMP13.

Introduction: RUNX2 is overexpressed in gastric cancer but the mechanism(s) through which it promotes tumor progression remain undefined. Here, we investigated the role of RUNX2 on gastric cancer pathogenesis at the molecular level. Methods: The qRT-PCR and western bolt were utilized to examine the mRNA and protein levels. CCK-8, Transwell and wound healing assays were used to measure cell proliferation, invasion and migration. CHIP-PCR gel electrophoresis was used to verify RUNX2 as a transcription factor for MMP13 and MGAT5. The in vivo assay was utilized to assess tumor growth. In vivo assay was used to evaluate tumor growth, aberrant expression of RUNX2 and lung metastasis of gastric cancer. Results: RUNX2 is overexpressed in MKN-45 and AGS cells. Genetic RUNX2 silencing reduced the proliferation, invasion and migration of MKN-45 and AGS cells. Analysis of the gastric cancer samples from the database revealed a significant positive correlation between MGAT5, MMP13, and RUNX2 expression. JASPAR analysis revealed that there was a potential binding site of RUNX2 in the promoter regions of MGAT5 and MMP13, and the experimental results confirmed that RUNX2 could regulate the expression of MGAT5 and MMP13 respectively. In vivo assays confirmed the aberrant expression of RUNX2 in mouse models of gastric cancer and reduced growth and lung metastasis in RUNX2 silenced xenograft tumors assessed. Conclusion: Collectively, these data reveal that RUNX2 enhances MGAT5 and MMP13 expression in gastric cancer cells and represents a biomarker and potential therapeutic target for gastric cancer therapy.

Retraction Notice to: hsa_circ_001653 Implicates in the Development of Pancreatic Ductal Adenocarcinoma by Regulating MicroRNA-377-MediatedHOXC6 Axis.

[This retracts the article DOI: 10.1016/j.omtn.2019.12.028.].

Retraction Notice to: The Potential Therapeutic Role of Exosomal MicroRNA-520b Derived from Normal Fibroblasts in Pancreatic Cancer.

[This retracts the article DOI: 10.1016/j.omtn.2019.12.029.].

MicroRNA-15a promotes prostate cancer cell ferroptosis by inhibiting GPX4 expression.

Ferroptosis is a novel form of regulated cell death characterized by accumulated lipid reactive oxygen species (ROS) and inactivation of glutathione peroxidase 4 (GPX4). The present study aimed to investigate the role of microRNA (miRNA/miR)-15a in ferroptosis of prostate cancer cells. Bioinformatics analysis was performed to predict the potential interaction between miR-15a and the 3'-untranslated region (UTR) of GPX4 mRNA. The prostate cancer cell line, LNCAP was transfected with miR-15a mimics or small interfering (si)-GPX4. Reverse transcription-quantitative PCR and western blot analyses were performed to detect the mRNA and protein expression levels of GPX4, respectively. Biotin-RNA pull-down and dual-luciferase reporter assays were performed to verify the interaction between miR-15a and GPX4 mRNA. The Cell Counting Kit-8 assay was performed to assess cell proliferation, while lactate dehydrogenase (LDH) and intracellular ferrous iron levels were detected via ELISA. Lipid ROS and mitochondrial membrane potential (MMP) were assessed via flow cytometry and staining with C11-BIODIPY probes or JC-1. Furthermore, lipid peroxidation was identified by measuring malondialdehyde (MDA) levels. The results demonstrated that transfection with miR-15a mimics decreased GPX4 protein expression. Bioinformatics analysis revealed potential binding sites between miR-15a and the 3'-UTR region of GPX4, and RNA pull-down and the dual-luciferase reporter assays further confirmed the interaction between miR-15a and GPX4 mRNA. Both transfection with miR-15a mimics and si-GPX4 suppressed cell proliferation, elevated LDH release, accumulated intracellular ferrous iron and ROS, disrupted MMP and increased MDA levels. Taken together, the results of the present study suggest miR-15a induces ferroptosis by regulating GPX4 in prostate cancer cells, which provides evidence for investigating the therapeutic strategies of prostate cancer.

Orphan nuclear receptor TLX promotes immunosuppression via its transcriptional activation of PD-L1 in glioma.

BACKGROUND: High-grade gliomas are rapidly progressing tumors of the central nervous system, and are associated with poor prognosis and highly immunosuppressive microenvironments. Meanwhile, a better understanding of PD-L1, a major prognostic biomarker for checkpoint immune therapy, regulation may provide insights for developing novel immunotherapeutic strategies for treating gliomas. In the present study, we elucidate the functional significance of the orphan nuclear receptor TLX in human glioma, and its functional role in immune suppression through regulation of PD-L1/PD-1 axis. METHODS: TLX and PD-L1 expression patterns, and their association with clinicopathological parameters and immune phenotypes of glioma were analysed using CIBERSORT algorithm and single-sample gene-set enrichment analysis from The Cancer Genome Atlas (n=695) and Chinese Glioma Genome Atlas (n=1018) databases. Protein expression and cellular localization of TLX, PD-L1, and PD-1, as well as the prevalence of cytotoxic tumor-infiltrating lymphocytes (TILs), and tumor-associated macrophages (TAMs), in the glioma immune microenvironment were analyzed via tissue microarray by immunohistochemistry and multiplex immunofluorescence. Glioma allografts and xenografts with TLX manipulation (knockdown/knockout or reverse agonist) were inoculated subcutaneously, or orthotopically into the brains of immunodeficient and immunocompetent mice to assess tumor growth by imaging, and the immune microenvironment by flow cytometry. PD-L1 transcriptional regulation by TLX was analyzed by chromatin immunoprecipitation and luciferase reporter assays. RESULTS: TLX and PD-L1 expression was positively associated with macrophage-mediated immunosuppressive phenotypes in gliomas. TLX showed significant upregulation and positive correlation with PD-L1. Meanwhile, suppression of TLX significantly inhibited in vivo growth of glioma allografts and xenografts (p<0.05), rescued the antitumoral immune response, significantly decreased the PD-L1+, and glioma-associated macrophage population, and increased cytotoxic lymphocyte infiltration (p<0.05). Mechanistically, TLX binds directly to CD274 (PD-L1) gene promoter and activates CD274 transcription. CONCLUSIONS: TLX contributes to glioma malignancy and immunosuppression through transcriptional activation of PD-L1 ligands that bind to PD-1 expressed on both TILs and TAMs. Thus, targeting the druggable TLX may have potential therapeutic significance in glioma immune therapy.

E2F7, regulated by miR­30c, inhibits apoptosis and promotes cell cycle of prostate cancer cells.

Prostate cancer (PCa) remains a leading cause of mortality among men in the United States and Western Europe. The molecular mechanism of PCa pathogenesis has not been fully elucidated. In the present study, the expression profile of E2F transcription factor 7 (E2F7) in PCa was examined using immunohistochemistry and reverse transcription­quantitative PCR, whilst cell cycle progression and apoptosis were determined using fluorescent cell activated sorting techniques. Cell viability was measured using Cell Counting Kit­8 in loss­ and gain­of­function studies. Dual­luciferase reporter assay was used to verify if E2F7 was one of the potential targets of miR­30c. The staining score of E2F7 of PCa tissues was found to be notably higher compared with that of adjacent normal tissues. Suppression of E2F7 expression in PCa cell lines led to significantly reduced proliferation rates, increased proportion of cells in the G1 phase of the cell cycle and higher apoptotic rates compared with those in negative control groups. Dual­luciferase reporter assay revealed E2F7 to be one of the binding targets of microRNA (miR)­30c. In addition, transfection of miR­30c mimics into PCa cells resulted in reduced cell viability, increased proportion of cells in the G1 phase and higher apoptotic rates. By contrast, transfection with the miR­30c inhibitor led to lower apoptosis rates of PCa cells compared with negative control groups, whilst E2F7 siRNA co­transfection reversed stimulatory effects of miR­30c inhibitors on cell viability. In addition, the expression of cyclin­dependent kinase inhibitor p21 were found to be upregulated by transfection with either E2F7 siRNA or miR­30c mimics into PCa cells. In conclusion, the present study suggested that E2F7 may be positively associated with PCa cell proliferation by inhibiting p21, whereas E2F7 is in turn under regulation by miR­30c. These observations suggest the miR­30c/E2F7/p21 axis to be a viable therapeutic target for PCa.

The Potential Therapeutic Role of Exosomal MicroRNA-520b Derived from Normal Fibroblasts in Pancreatic Cancer.

Pancreatic cancer (PC) remains a major health concern, with conventional cancer treatments exerting little influence on the disease course. MicroRNA-520b (miR-520b) functions as a tumor suppressor in several types of human cancers, whereas its anti-tumor property in the context of PC is still fundamental. The aim of this study is to identify the potential therapeutic role of miR-520b, transferred by exosomes, derived from normal fibroblasts (NFs) in PC progression. A gain-of-function study was performed to examine the roles of miR-520b in PC cell line SW1990, which suggested that miR-520b served as a tumor suppressor in PC. In order to confirm the role of exosomal miR-520b, exosomes were isolated from NF culture medium and cocultured with SW1990 cells. During the coculture experiments, we disrupted exosome secretion and upregulated exosomal miR-520b. The in vitro coculture studies revealed that miR-520b was transferred from NF-derived exosomes to PC cells and thereby suppressed PC cell proliferation, invasion, migration, and stimulated apoptosis. Furthermore, inhibited tumor growth and live metastasis upon elevated miR-520b in exosomes were observed in vivo. Conjointly, our study demonstrates that NF-derived exosomal miR-520b impedes the progression of PC, which contributes to a novel, therapeutic role of exosomal miR-520b for treating PC.

hsa_circ_001653 Implicates in the Development of Pancreatic Ductal Adenocarcinoma by Regulating MicroRNA-377-Mediated HOXC6 Axis.

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive pancreatic cancer with poor survival rate. Circular RNAs (circRNAs) signatures have been identified in some human cancers, but there are little data concerning their presence in PDAC. We investigated the role of hsa_circ_001653, a newly identified circRNA, in the development of PDAC. hsa-circ-001653 expression was measured in 83 paired normal and tumor tissues surgically resected from PDAC patients. Phenotypic changes of PDAC cells were evaluated by assays for cell viability, cell cycle, invasion, and apoptosis. Tube-like structure formation of human umbilical vein endothelial cells (HUVECs) was examined in the presence of PDAC cells. Cross-talk between hsa_circ_001653 and microRNA-377 (miR-377)/human homeobox C6 (HOXC6) was assessed using dual-luciferase reporter assay, Ago2 immunoprecipitation, and northern blot analysis. Nude mice were inoculated with human PDAC cells for in vivo analysis. hsa_circ_001653 was an upregulated circRNA in PDAC. Silencing of hsa_circ_001653 in PDAC cells via RNA interference inhibited cell viability, cell-cycle progression, in vitro angiogenesis, and invasive properties, showing a pro-apoptotic effect. hsa_circ_001653 was found to bind to miR-377, which in turn repressed HOXC6 expression. Inhibition of miR-377 by its specific inhibitor restored cell viability, cell-cycle progression, in vitro angiogenesis, and invasive properties in PDAC cells lacking endogenous hsa_circ_001653. When nude mice were inoculated with human PDAC cells, inhibition of hsa_circ_001653 had a therapeutic effect. Collectively, the present study provides an enhanced understanding of hsa_circ_001653 as a therapeutic target for PDAC.

DNA methylation of a non-CpG island promoter represses NQO1 expression in rat arsenic-transformed lung epithelial cells.

NAD(P)H:quinone oxidoreductase 1 (NQO1), a phase II flavoenzyme that catalyzes reduction reactions to protect cells against electrophiles and oxidants, is involved in tumorigenesis. Altered methylation of the NQO1 gene has been observed and is speculated to result in aberrant NQO1 expression in rat cells undergoing chemical carcinogenesis, although this has not been proven experimentally. In this study, we first investigated the potential epigenetic mechanisms underlying the phenomenon of NQO1 differential expression in individual subclones of rat arsenic-transformed lung epithelial cells (TLECs). NQO1 expression of TLEC subclones with or without 5-aza-2'-deoxycytidine (5-Aza-CdR) treatment was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR), western blot analysis, and real-time PCR. Methylation status of the NQO1 promoter in TLEC subclones was analyzed by bisulfite sequencing. Transcriptional activity of NQO1 promoter in vitro methylated was determined by luciferase assay using a CpG-free luciferase reporter driven by the NQO1 promoter region (-435 to +229). We found that non-CpG island (non-CpGI) within the NQO1 promoter was hyper- or hypo-methylated in TLEC subclones and corresponded to low and high gene expressions, respectively. Following the treatment with 5-Aza-CdR, transcription of the NQO1 gene in the hypermethylated subclones was restored, accompanied by demethylation of the NQO1 promoter. In vitro promoter methylation almost completely silenced reporter activity in TLECs. These results indicate that DNA methylation of the non-CpGI promoter contributes to epigenetic silencing of NQO1 in rat TLECs.

Oncogenic function of TUSC3 in non-small cell lung cancer is associated with Hedgehog signalling pathway.

Non-small cell lung cancer (NSCLC) represents 75-80% of all lung carcinomas, which is the most common cause of death from cancer. Tumour suppressor candidate 3 (TUSC3) is pivotal in many biochemical functions and cytological processes. Dis-regulation of TUSC3 is frequently observed in epithelial cancers. In this study, we observed up-regulated TUSC3 expression at the mRNA and protein levels in clinical NSCLC samples compared with adjacent non-tumorous lung tissues. The expression level of TUSC3 is significantly correlated with tumour metastasis and patient survival. Overexpression of TUSC3 in NSCLC cells led to increased proliferation, migration, and invasion in vitro and accelerated xenograft tumour growth in vivo, while the opposite effects were achieved in TUSC3-silenced cells. Increased GLI1, SMO, PTCH1, and PTCH2 abundance were observed in TUSC3 overexpressed cells using western blotting. Co-immunoprecipitation and immunofluorescence analyses further revealed interaction between TUSC3 and GLI1. In conclusion, our study demonstrated an oncogenic role of TUSC3 in NSCLC and showed that dis-regulation of TUSC3 may affect tumour cell invasion and migration through possible involvement in the Hedgehog (Hh) signalling pathway.

Single nucleotide polymorphism-based microarray analysis for the diagnosis of hydatidiform moles.

In clinical diagnostics, single nucleotide polymorphism (SNP)-based microarray analysis enables the detection of copy number variations (CNVs), as well as copy number neutral regions, that are absent of heterozygosity throughout the genome. The aim of the present study was to evaluate the effectiveness and sensitivity of SNP­based microarray analysis in the diagnosis of hydatidiform mole (HM). By using whole­genome SNP microarray analysis, villous genotypes were detected, and the ploidy of villous tissue was determined to identify HMs. A total of 66 villous tissues and two twin tissues were assessed in the present study. Among these samples, 11 were triploid, one was tetraploid, 23 were abnormal aneuploidy, three were complete genome homozygosity, and the remaining ones were normal ploidy. The most noteworthy finding of the present study was the identification of six partial HMs and three complete HMs from those samples that were not identified as being HMs on the basis of the initial diagnosis of experienced obstetricians. This study has demonstrated that the application of an SNP­based microarray analysis was able to increase the sensitivity of diagnosis for HMs with partial and complete HMs, which makes the identification of these diseases at an early gestational age possible.

Lung adenocarcinoma harboring L858R and T790M mutations in epidermal growth factor receptor, with poor response to gefitinib: A case report.

Lung cancer is the leading cause of mortality among malignant diseases in humans worldwide. During the last decade, molecular targeted therapies for non-small cell lung cancer using first-generation, reversible epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), including gefitinib, have been shown to be a promising approach for patients harboring activating mutations in EGFR. The current study reports a 77-year-old patient diagnosed with adenocarcinoma harboring L858R and T790M point mutations in the EGFR gene. The patient was treated with gefitinib as the second-line therapy, but no clinical benefit was observed. As the majority of patients with lung cancer receiving EGFR-TKI therapy acquire resistance, repeated biopsies and detection of the EGFR mutation state are beneficial for selecting appropriate treatments.

Increased expression of macrophage migration inhibitory factor and DJ-1 contribute to cell invasion and metastasis of nasopharyngeal carcinoma.

BACKGROUND AND AIM: Both macrophage migration inhibitory factor (MIF) and DJ-1 protein have been shown to relate with cell invasion and metastasis in tumors. However, the role of DJ-1 in invasion and metastasis of nasopharyngeal carcinoma (NPC) and its relation to MIF expression in NPC are not fully understood. The aim of present study is to determine whether or not MIF and DJ-1 are correlated with tumor invasion and influence a worse outcome in NPC, as well as its related mechanism. METHODS: 125 cases of NPC and 45 normal tissues of nasopharynx were collected. The expression of MIF and DJ-1 in tissue microarray was evaluated by immunohistochemical staining. Correlation between immunostainings and clinicopathological parameters, as well as the follow-up data of patients, was analyzed statistically. The association of MIF and DJ-1 with cell invasion and migration in NPC cell line were evaluated by small interfering RNA (siRNA) transfection, invasion assay and Western blotting. RESULTS: MIF and DJ-1 staining was diffused and strong in tumor cells, whereas they were generally weaker and less common in normal lining epithelia of nasopharynx. High MIF expression in tumor cells (71.2%, 89/125 cases) were significantly associated with advanced clinical stage, lymph node metastasis, and worse prognosis of NPC patients. High expression of DJ-1 (75.2%, 94/125 cases) were closely correlated to lymph node metastasis and MIF high-expression. Only MIF high expression (P = 0.010) and lymph node metastasis (P = 0.004) emerged as strong independent prognostic factors for overall survival of NPC patients. In vitro, down-regulated expression of DJ-1 in NPC cell lines by siRNA was observed to reduce cell migration and invasion potential, however, exogenous MIF promoted cells invasion. CONCLUSIONS: The data provided evidence that increased expression of MIF and DJ-1 induced cell invasion and metastasis of NPC, supporting the idea that MIF and DJ-1 may play important roles as regulators in the progression of NPC.

Secondary cutaneous Epstein-Barr virus-associated diffuse large B-cell lymphoma in a patient with angioimmunoblastic T-cell lymphoma: a case report and review of literature.

Only a few cases of extranodal Epstein-Barr virus (EBV)-associated B-cell lymphomas arising from patients with angioimmunoblastic T-cell lymphoma (AITL) have been described. We report a case of AITL of which secondary cutaneous EBV-associated diffuse large B-cell lymphoma (DLBCL) developed after the initial diagnosis of AITL. A 65-year-old Chinese male patient was diagnosed as AITL based on typical histological and immunohistochemical characteristics in biopsy of the enlarged right inguinal lymph nodes. The patient initially received 6 cycles of chemotherapy with CHOP regimen (cyclophosphamide, vincristine, adriamycin, prednisone), but his symptoms did not disappear. Nineteen months after initial diagnosis of AITL, the patient was hospitalized again because of multiple plaques and nodules on the skin. The skin biopsy was performed, but this time the tumor was composed of large, polymorphous population of lymphocytes with CD20 and CD79a positive on immunohistochemical staining. The tumor cells were strong positive for EBER by in situ hybridization. The findings of skin biopsy were compatible with EBV-associated DLBCL. CHOP-R chemotherapy (cyclophosphamide, doxorubicin, vincristine, prednisone and rituximab) was then administered, resulting in partial response of the disease with pancytopenia and suppression of cellular immunity. To our knowledge, this is the first case of cutaneous EBV-associated DLBCL originated from AITL in Chinese pepole. We suggest the patients with AITL should perform lymph node and skin biopsies regularly in the course of the disease to detect the progression of secondary lymphomas.

[Expression of angiogenesis-related factors in invasive breast cancer and its clinical significance].

OBJECTIVE: To investigate the relation between expression of angiogenesis-related factors, namely vascular endothelial growth factor (VEGF) and transforming growth factor-beta1 (TGFbeta(1)), and microvessel count (MVC) in invasive breast cancer and analyze its clinical implications. METHODS: VEGF, TGFbeta (1) and CD34 expressions in 62 surgical specimens of invasive breast cancer and 12 normal breast specimens were examined by immunohistochemistry and HE staining. MVC was calculated according to the quantification of positive CD34 expression. Clinicopathological characteristics of the patients including age, tumor size, histological type and auxiliary lymph node metastasis were recorded and compared with the results of MVC VEGF and TGFbeta1 expression and detection. RESULTS: MVC and of VEGF and expressions TGFbeta (1) in invasive breast cancer group (55.62-/+11.07, 51.61%, 56.45%, respectively) were greater than those in the normal control group (12.65-/+5.73, 16.67%, 16.67%, respectively, P<0.05). MVC and the positivity rates of VEGF and TGFbeta (1) expressions were 65.53-/+20.36, 68.75% and 78.13%, respectively, in invasive breast cancer patients with axillary lymph node metastasis, significantly higher than those without metastasis (P<0.05). MVC was correlated with VEGF and TGFbeta (1) expressions in that MVC was significantly higher in patients positive for VEGF and TGFbeta (1) (62.82-/+16.31 and 59.35-/+12.76) than in those negative for their expressions (51.16-/+12.53 and 50.80-/+15.62, P<0.05). Significant correlation was also found between VEGF and TGFbeta (1) expressions (P<0.05). CONCLUSION: The interaction between VEGF and TGFbeta (1) mediates angiogenesis, and MVC and VEGF and TGFbeta (1) expressions are correlated to lymph node metastasis, which may provide reference for prognostic evaluation of invasive breast cancer.