Prognostic value of RNA methylation-related genes in gastric adenocarcinoma based on bioinformatics.

Background: Gastric cancer (GC) is a malignant tumor that originates from the epithelium of the gastric mucosa and has a poor prognosis. Stomach adenocarcinoma (STAD) covers 95% of total gastric cancer. This study aimed to identify the prognostic value of RNA methylation-related genes in gastric cancer. Methods: In this study, The Cancer Genome Atlas (TCGA)-STAD and GSE84426 cohorts were downloaded from public databases. Patients were classified by consistent cluster analysis based on prognosis-related differentially expressed RNA methylation genes Prognostic genes were obtained by differential expression, univariate Cox and least absolute shrinkage and selection operator (LASSO) analyses. The prognostic model was established and validated in the training set, test set and validation set respectively. Independent prognostic analysis was implemented. Finally, the expression of prognostic genes was affirmed by reverse transcription quantitative PCR (RT-qPCR). Results: In total, four prognostic genes (ACTA2, SAPCD2, PDK4 and APOD) related to RNA methylation were identified and enrolled into the risk signature. The STAD patients were divided into high- and low-risk groups based on the medium value of the risk score, and patients in the high-risk group had a poor prognosis. In addition, the RNA methylation-relevant risk signature was validated in the test and validation sets, and was authenticated as a reliable independent prognostic predictor. The nomogram was constructed based on the independent predictors to predict the 1/3/5-year survival probability of STAD patients. The gene set enrichment analysis (GSEA) result suggested that the poor prognosis in the high-risk subgroup may be related to immune-related pathways. Finally, the experimental results indicated that the expression trends of RNA methylation-relevant prognostic genes in gastric cancer cells were in agreement with the result of bioinformatics. Conclusion: Our study established a novel RNA methylation-related risk signature for STAD, which was of considerable significance for improving prognosis of STAD patients and offering theoretical support for clinical therapy.

Construction of a ceRNA network to reveal a vascular invasion associated prognostic model in hepatocellular carcinoma.

The aim of this study is to explore the prognostic value of vascular invasion (VI) in hepatocellular carcinoma (HCC) by searching for competing endogenous RNAs (ceRNA) network and constructing a new prognostic model for HCC. The differentially expressed genes (DEGs) between HCC and normal tissues were identified from GEO and TCGA. StarBase and miRanda prediction tools were applied to construct a circRNA-miRNA-mRNA network. The DEGs between HCC with and without VI were also identified. Then, the hub genes were screened to build a prognostic risk score model through the method of least absolute shrinkage and selection operator. The prognostic ability of the model was assessed using the Kaplan-Meier method and Cox regression analysis. In result, there were 221 up-regulated and 47 down-regulated differentially expressed circRNAs (DEcircRNAs) in HCC compared with normal tissue. A circRNA-related ceRNA network was established, containing 11 DEcircRNAs, 12 DEmiRNAs, and 161 DEmRNAs. Meanwhile, another DEG analysis revealed 625 up-regulated and 123 down-regulated DEGs between HCC with and without VI, and then a protein-protein interaction (PPI) network was built based on 122 VI-related DEGs. From the intersection of DEGs within the PPI and ceRNA networks, we obtained seven hub genes to build a novel prognostic risk score model. HCC patients with high-risk scores had shorter survival time and presented more advanced T/N/M stages as well as VI occurrence. In conclusion a novel prognostic model based on seven VI-associated DEGs within a circRNA-related ceRNA network was constructed in this study, with great ability to predict the outcome of HCC patients.

Plasma Methylated SEPT9 as a Novel Biomarker for Predicting Liver Metastasis in Colorectal Cancer.

This study aimed to explore the role of plasma methylated SEPT9 (mSEPT9) in predicting liver metastasis (LM) in colorectal cancer (CRC) patients. The clinicopathological information of 115 consecutive CRC patients were collected. The differences of clinical characteristics and several biomarkers between CRC patients with LM and those with non-liver metastasis (NM) were analyzed. Multivariate logistic regression analysis was used to identify the risk factors for predicting LM in CRC patients. Receiver operating characteristic curve (ROC) analysis was applied to investigate the sensitivity and specificity of potential biomarkers in indicating the presence of LM in CRC. Compared with the CRC without LM, the levels of plasma mSEPT9 and carcinoembryonic antigen (CEA) were significantly increased in CRC with LM. Multivariate logistic regression analysis showed that plasma mSEPT9 was an independent risk factor for predicting LM in CRC. ROC curves showed that mSEPT9 and CEA could efficiently distinguish LM from NM in CRC. The area under the curve (AUC) of mSEPT9 was 0.850, which was slightly higher than that of CEA (0.842). The optimal cut-off value of mSEPT9 was 35.09 with a sensitivity of 81.82% and a specificity of 73.33%, both similar with that of CEA (sensitivity 87.27% and specificity 75.00%). In addition, the combination of mSEPT9 and CEA had a higher specificity than CEA alone (81.70% Vs 75.00%). Our findings suggest, for the first time, that plasma mSEPT9 might serve as a potential biomarker to predict LM in CRC, which deserves further in-depth study.

Breast cancer-derived exosomal lncRNA SNHG14 induces normal fibroblast activation to cancer-associated fibroblasts via the EBF1/FAM171A1 axis.

BACKGROUND: Exosomes released from cancer cells can activate normal fibroblasts (NFs) into cancer-associated fibroblasts (CAFs), which promotes cancer development. Our study aims to explore the role and potential mechanisms of breast cancer exosomes-delivered long non-coding RNA (lncRNA) SNHG14 in regulating CAFs transformation. METHODS: Adjacent normal tissues, cancerous and serum specimens were gathered in breast cancer patients. Exosomes and NFs were separated from breast cancer cells (SKBR-3) and normal tissues of patients, respectively. Cell viability and migration were measured with CCK-8 and Transwell assays. CAFs markers, fibroblast activation protein (FAP) and a-smooth muscle actin (α-SMA) were detected for assessing CAFs activation. The interactions between molecules were evaluated using dual luciferase reporter assay, RNA immunoprecipitation and chromatin immunoprecipitation. RESULTS: SNHG14 and FAM171A1 were upregulated in breast cancer. Exosomes secreted by SKBR-3 cells induced NFs activation in CAFs, as indicated by upregulating CAFs marker levels and facilitated cell viability and migration. Exosomal SNHG14 silencing in SKBR-3 cells inhibited CAFs activation. SNHG14 positively regulated FAM171A1 expression through EBF1. FAM171A1 overexpression eliminated the inhibition effect of exosomal SNHG14 silencing in CAFs transformation. CONCLUSION: Breast cancer-derived exosomal SNHG14 contributed to NFs transformation into CAFs by the EBF1/FAM171A1 axis.

Exploration of prognostic genes and risk signature in breast cancer patients based on RNA binding proteins associated with ferroptosis.

Background: Breast cancer (BRCA) is a life-threatening malignancy in women with an unsatisfactory prognosis. The purpose of this study was to explore the prognostic biomarkers and a risk signature based on ferroptosis-related RNA-binding proteins (FR-RBPs). Methods: FR-RBPs were identified using Spearman correlation analysis. Differentially expressed genes (DEGs) were identified by the "limma" R package. The univariate Cox and multivariate Cox analyses were executed to determine the prognostic genes. The risk signature was constructed and verified with the training set, testing set, and validation set. Mutation analysis, immune checkpoint expression analysis in high- and low-risk groups, and correlation between risk signature and chemotherapeutic agents were conducted using the "maftools" package, "ggplot2" package, and the CellMiner database respectively. The Human Protein Atlas (HPA) database was employed to confirm protein expression trends of prognostic genes in BRCA and normal tissues. The expression of prognostic genes in cell lines was verified by Real-time quantitative polymerase chain reaction (RT-qPCR). Kaplan-meier (KM) plotter database analysis was applied to predict the correlation between the expression levels of signature genes and survival statuses. Results: Five prognostic genes (GSPT2, RNASE1, TIPARP, TSEN54, and SAMD4A) to construct an FR-RBPs-related risk signature were identified and the risk signature was validated by the International Cancer Genome Consortium (ICGC) cohort. Univariate and multivariate Cox regression analysis demonstrated the risk score was a robust independent prognostic factor in overall survival prediction. The Tumor Mutational Burden (TMB) analysis implied that the high- and low-risk groups responded differently to immunotherapy. Drug sensitivity analysis suggested that the risk signature may serve as a chemosensitivity predictor. The results of GSEA suggested that five prognostic genes might be related to DNA replication and the immune-related pathways. RT-qPCR results demonstrated that the expression trends of prognostic genes in cell lines were consistent with the results from public databases. KM plotter database analysis suggested that high expression levels of GSPT2, RNASE1, and SAMD4A contributed to poor prognoses. Conclusion: In conclusion, this study identified the FR-RBPs-related prognostic genes and developed an FR-RBPs-related risk signature for the prognosis of BRCA, which will be of great significance in developing new therapeutic targets and prognostic molecular biomarkers for BRCA.

CEACAM1 Is a Prognostic Biomarker and Correlated with Immune Cell Infiltration in Clear Cell Renal Cell Carcinoma.

Background: CEACAM1 has been shown to be aberrantly expressed in a variety of tumors, and modulation of CEACAM1-related signaling pathways has been suggested as a novel approach for cancer immunotherapy in recent years. However, its role in clear cell renal cell carcinoma (ccRCC) is unclear. Methods: The relationship between CEACAM1 and ccRCC was demonstrated based on data from TCGA, GEO, and HPA databases. And the relationship between clinicopathological features and CEACAM1 expression was also assessed. Survival curve analysis was performed to analyze the prognostic relationship between CEACAM1 expression and ccRCC. Protein interaction network analysis was used to analyze the relationship between CEACAM1 and microenvironment-related proteins. In addition, the immunomodulatory role of CEACAM1 in ccRCC was assessed by analyzing CEACAM1 and immune cell infiltration. Results: The expression of CEACAM1 was lower in ccRCC tissues than in adjacent normal tissues, and its expression level was negatively correlated with tumor size status (P < 0.001), metastasis status (P = 0.009), pathological stage (P = 0.002), gender (P < 0.001), histological grade (P < 0.001), and primary therapy outcome (P = 0.045) of ccRCC. Survival curve analysis showed that ccRCC patients with lower CEACAM1 expression exhibited shorter overall survival (P < 0.001), and CEACAM1 interacted with microenvironmental molecules such as fibronectin and integrins. Furthermore, immune infiltration analysis showed that CEACAM1 expression correlated with CD8+ and CD4+ T cells, macrophage, neutrophil, and dendritic cell infiltration in ccRCC. Conclusions: CEACAM1 expression correlates with progression, prognosis, and immune cell infiltration in ccRCC patients, and it may be a promising prognostic biomarker and therapeutic target for ccRCC.

Determining the Optimal Cut-Off Values of Serum E2 and FSH for Evaluating the Menopausal Status of Breast Cancer Patients in a Southern Chinese Population.

Background: Chemotherapy-induced amenorrhea (CIA) is one of universal phenomena in breast cancer (BC) patients, and it causes difficulties in evaluating the actual menopausal status which is important for the oncologists to choose appropriate treatment. Currently, serum estradiol (E2) and follicle-stimulating hormone (FSH) levels are the most commonly used clinical parameters for the assessment of menopausal status in BC patients. However, the optimal cut-off points of serum E2 and FSH have little been explored in southern Chinese population. Objective: This study is aimed to determine the optimal cut-off values of the serum E2 and FSH levels for evaluating the menopausal status of BC patients in a southern Chinese population. Methods: A retrospective analysis was done among a total of 206 patients with BC from a southern Chinese area. The data of serum E2, FSH, and luteinizing hormone (LH) levels were collected and analyzed for the comparison purpose. The receiver-operating curve (ROC) was generated to assess the specificity and sensitivity of the three biomarkers in discriminating the menopausal status of BC patients. The optimal cut-off values were determined according to the Youden index and then compared with the recommended reference values by the Chinese Anti-cancer Association (CACA) and those recommended by the manufacturers. Results: The areas under the ROC curves (AUCs) of E2, FSH, and LH were 0.846 (95% CI: 0.790-0.903), 0.781 (95% CI: 0.714-0.847) and 0.608 (95% CI: 0.526-0.690), respectively. The optimal cut-off values were 130.0 pg/mL for E2, 23.325 IU/L for FSH, and 11.625 IU/L for LH with a maximum of the Youden index. When E2, FSH, and LH were used in combination for ROC analysis, the AUC increased to 0.847 (95% CI: 0.790-0.904), which was higher than that of any other biomarker alone. In this study, the sensitivity and specificity of E2 and FSH were 91.6% and 73.70% and 94.4% and 58.6%, respectively, in comparison with 85.0% and 75.80% and 76.6% and 65.7% according to the CACA-recommended cut-off points, or 92.5% and 68.7% and 96.3% and 53.5% according to the manufacturer recommended cut-off points. Conclusion: Considering the sensitivity and specificity of serum E2 and FSH for assessing the menopausal status, the optimal cut-off values determined in the present study were similar to the manufacturer's recommendations, but obviously superior to the cut-off points suggested by CACA. These cut-off points calculated in this study seem to be valuable in southern Chinese population and might be used by clinicians to make a correct medical decision for BC patients who would benefit from endocrine therapy of aromatase inhibitor (AI).

Metastasis suppressor 1 interacts with α-actinin 4 to affect its localization and regulate formation of membrane ruffling.

Membrane ruffling plays an important role in the directed cell migration and escape of tumor cells from the monolayer. Metastasis suppressor 1 (MTSS1), also known as missing in metastasis, has been implicated in cell morphology, motility, metastasis, and development. Here, the dynamic interaction proteins associated with MTSS1 and involved in membrane ruffling were determined by cross-linking and mass spectrometry analysis. We identified α-actinin 4 (ACTN4) as an interacting protein and confirmed a direct interaction between MTSS1 and ACTN4. Moreover, co-expression of MTSS1 in fibroblasts recruited cytoplasmic ACTN4 to the cell periphery, at which point ruffling became thick and rigid. In MCF-7 cells, MTSS1 knockdown did not show an obvious effect on the cell shape or the distribution of endogenous ACTN4; however, ACTN4 overexpression transformed cell morphology from an epidermal- to a fibroblast-like shape, and further MTSS1 depletion significantly increased the ratio of fibroblast cells exhibiting prominent ruffling. Furthermore, biochemical data suggested that MTSS1 cross-linking with ACTN4 induced the formation of actin fiber bundles into more organized structures in vitro. These data indicated that MTSS1 might recruit cytoplasmic ACTN4 to the cell periphery and regulate cytoskeleton dynamics to restrict its performance in membrane ruffling.

Preparation of a CaTiO3/Al3+/Pr3+/Sm3+ nanocomposite for enrichment of exosomes in human serum.

Exosomes (30-200 nm) play important roles in intercellular communication. Because their contents differ between healthy individuals and subjects diagnosed with various diseases, exosomes have been regarded as potential sources of biomarkers for clinical diagnosis. However, the accuracy of diagnosis by exosomal biomarkers is highly dependent on the extraction efficiency, yield, and the quality of exosomes. Hence, inexpensive, convenient, and fast exosome separation methods are required. In the present study, the CaTiO3/Al3+/Pr3+/Sm3+ nanocomposite was synthesized and applied in highly selective and efficient separation of exosomes. Notably, the developed material exhibited higher specificity and efficiency than commercially available TiO2. Moreover, CaTiO3/Al3+/Pr3+/Sm3+ could be reused at least three times without any significant decrease in efficiency. The synthesized material was also used for the extraction of exosomes from the serums of patients with Alzheimer's disease (AD) and healthy controls. The exosomes were subjected to two-dimensional gel electrophoresis (2-DE) separation and matrix-assisted laser desorption/ionization-time of flight (MALDI TOF/TOF) mass spectrometry analysis. It was found that five proteins in the exosomes were evidently upregulated, while one protein was downregulated. Among the detected proteins, serum amyloid P-component (SAP) has been reported to be closely related to pathogenesis of AD. The obtained results indicated that the developed method involving separation and analysis of serum exosomes could be used for disease diagnosis or postoperative clinical monitoring.

Differential mitochondrial proteomic analysis of A549 cells infected with avian influenza virus subtypes H5 and H9.

BACKGROUND: Both the highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic avian influenza (LPAI) H9N2 viruses have been reported to cross species barriers to infect humans. H5N1 viruses can cause severe damage and are associated with a high mortality rate, but H9N2 viruses do not cause such outcomes. Our purpose was to use proteomics technology to study the differential expression of mitochondrial-related proteins related to H5N1 and H9N2 virus infections. METHODS: According to the determined viral infection titer, A549 cells were infected with 1 multiplicity of infection virus, and the mitochondria were extracted after 24 h of incubation. The protein from lysed mitochondria was analyzed by the BCA method to determine the protein concentration, as well as SDS-PAGE (preliminary analysis), two-dimensional gel electrophoresis, and mass spectrometry. Differential protein spots were selected, and Western blotting was performed to verify the proteomics results. The identified proteins were subjected to GO analysis for subcellular localization, KEGG analysis for functional classification and signaling pathways assessment, and STRING analysis for functional protein association network construction. RESULTS: In the 2-D gel electrophoresis analysis, 227 protein spots were detected in the H5N1-infected group, and 169 protein spots were detected in the H9N2-infected group. Protein spots were further subjected to mass spectrometry identification and removal of redundancy, and 32 differentially expressed proteins were identified. Compared with the H9N2 group, the H5N1-infected group had 16 upregulated mitochondrial proteins and 16 downregulated proteins. The differential expression of 70-kDa heat shock protein analogs, short-chain enoyl-CoA hydratase, malate dehydrogenase, and ATP synthase was verified by Western blot, and the results were consistent with the proteomics findings. Functional analysis indicated that these differentially expressed proteins were primarily involved in apoptosis and metabolism. CONCLUSIONS: Compared with their expression in the H9N2 group, the differential expression of eight mitochondrial proteins in the H5N1 group led to host T cell activation, antigen presentation, stress response, ATP synthesis and cell apoptosis reduction, leading to higher pathogenicity of H5N1 than H9N2.

Increased SPHK1 and HAS2 Expressions Correlate to Poor Prognosis in Pancreatic Cancer.

OBJECTIVE: SPHK1 and HAS2 have been reported to play important roles in tumorigenesis and development. However, their expression and prognostic value in pancreatic cancer (PC) remain unclear. This study is aimed at investigating the expression of SPHK1 and HAS2 on the prognosis of pancreatic cancer. MATERIALS AND METHODS: The expression of SPHK1 and HAS2 in pancreatic cancer tissues was analyzed through TCGA and GTEx databases and validated by qRT-PCR and Western blot in pancreatic cancer cell lines. χ 2 test was used to explore the correlation of the SPHK1 and HAS2 expressions with clinical characteristics. Kaplan-Meier survival analysis and ROC curve were used to evaluate the prognostic and diagnostic roles of SPHK1 and HAS2 in pancreatic cancer. Additionally, Spearman correlation analysis was applied to assess the correlation between the SPHK1 and HAS2 in pancreatic cancer. GO analysis and KEGG analysis were applied to explore the possible signaling pathway that SPHK1 and HAS2 coregulated genes mediated. RESULTS: The expression of SPHK1 and HAS2 was markedly upregulated in pancreatic cancer tissue and cell lines, respectively. Furthermore, there was a significant positive correlation between SPHK1 and HAS2 expressions. ROC curves showed that SPHK1 combine with HAS2 has good diagnostic value in pancreatic cancer patients with 85% sensitivity and 99.4% specificity. Kaplan-Meier analysis showed that increased expression of SPHK1 and HAS2 was significantly associated with short overall survival (OS) of pancreatic cancer patients. GO and KEGG results revealed that SPHK1 and HAS2 mainly involved cell proliferation and invasion mediated by extracellular matrix- (ECM-) receptor interaction, focal adhesion, and PI3K-AKT signaling pathways. CONCLUSIONS: Overexpression of SPHK1 and HAS2 could be important markers for the prognosis of pancreatic cancer.

Serum ProGRP as a novel biomarker of bone metastasis in prostate cancer.

BACKGROUND: The prognosis of prostate cancer (PCa) is related to tumor metastasis, among which 80% were bone metastasis. In this study, we investigated the correlation between diverse clinical factors and bone metastasis in PCa patients and identified potential biomarkers of bone metastasis in PCa patients. METHODS: The clinical data of 150 PCa patients were reviewed consecutively from January 2015 to March 2020 in this study. The relationships between clinical characteristics, serum biomarkers and bone metastasis in PCa patients were analyzed, respectively. Multivariate logistic regression analysis was applied to identify potential markers of bone metastasis in prostate cancer. Receiver operating characteristic (ROC) curve was used to explore the diagnostic values of potential biomarkers. RESULTS: Compared with the PCa patients without bone metastasis, the serum levels of CA-125, T-PSA, F-PSA, CYFRA21-1 and ProGRP were significantly elevated in PCa patients with bone metastasis. Multivariate logistic regression analysis showed that T-PSA (OR 1.014, P = 0.021), F-PSA (OR 1.124, P = 0.016) and Pro-gastrin-releasing peptide (ProGRP) (OR 1.057, P = 0.026) were significantly associated with the bone metastasis of PCa patients. ROC curves indicated that T-PSA, F-PSA and ProGRP could effectively discriminate bone metastasis from non-bone metastasis PCa patients, and the AUCs (area under the curves) were 0.885, 0.919 and 0.752, respectively. According to the Youden index, the cut-off values of T-PSA, F-PSA and ProGRP were defined as 56.50 ng/ml, 6.96 ng/ml and 31.60 pg/ml, respectively. T-PSA, F-PSA and ProGRP produced a sensitivity of 78.30%, 81.70% and 61.70%, a specificity of 93.30%, 88.90% and 82.20%, respectively. The AUC for the combination of T-PSA, F-PSA with ProGRP was 0.941 with 90.00% sensitivity, much better than that of any single biomarker or two biomarkers combinate. CONCLUSIONS: Serum ProGRP might be a potential tumor marker of bone metastasis in prostate cancer, which may contribute to the early diagnosis of bone metastasis when used alone or in combination with other commonly used biomarkers.

Bioinformatical Analysis of Gene Expression Omnibus Database Associates TAF7/CCNB1, TAF7/CCNA2, and GTF2E2/CDC20 Pathways with Glioblastoma Development and Prognosis.

OBJECTIVE: This study bioinformatically analyzed aberrant genes and pathways for associations with glioblastoma development and prognosis. METHODS: The Gene Expression Omnibus (GEO) database was searched and 4 GEO datasets (GSE4290, GSE50161, GSE116520, and GSE90598) were retrieved for limma and RobustRankAggreg package analyses of differentially expressed genes (DEGs) between glioblastoma and normal brain tissues. Functional enrichment analysis was conducted for the main biological functions of these DEGs, whereas the hub genes were identified using the protein-protein interaction network and confirmed for transcriptional and translational levels using the Cancer Genome Atlas, the Genotype-Tissue Expression, and the Human Protein Atlas data. The prognostic values of these hub genes were analyzed using the Chinese Glioma Genome Atlas. Their transcriptional factor regulation network was constructed to assess the roles in glioblastoma development and progression. RESULTS: A total of 473 DEGs (182 upregulated and 291 downregulated) were identified and the hub genes (including CCNB1, CDC20, CCNB2, BUB1, and CCNA2) were shown in module 1 and enriched in the cell cycle or p53 signaling pathway. The highly expressed CCNB1, CDC20, BUB1, and CCNA2 in patients with glioblastoma were associated with poor overall survival, whereas TAF7 could upregulate expression of CCNB1 and CCNA2 and GTF2E2 could upregulate CDC20 expression in glioblastoma. CONCLUSIONS: This study showed several DEGs in glioblastoma, and aberrant expression of their hub genes was associated with glioblastoma pathogenesis and poor prognosis, especially the signaling axes of TAF7/CCNB1, TAF7/CCNA2, and GTF2E2/CDC20.

Long noncoding RNA OR3A4 promotes the proliferation and invasion of osteosarcoma cells by sponging miR-1227-5p.

BACKGROUND: Long noncoding RNAs (lncRNAs) have been identified as key players in promoting tumourigenesis in osteosarcoma. LncRNA OR3A4 (OR3A4) has been reported as an oncogene in a number of tumours. However, the clinical value of OR3A4 in osteosarcoma and the role of OR3A4 in osteosarcoma progression are still unknown. METHODS: The expression levels of OR3A4 in the tumour tissue of osteosarcoma patients and osteosarcoma cell lines were detected by RT-PCR. Kaplan-Meier analysis and log-rank test were performed to evaluate the relationship between the level of OR3A4 expression and the prognosis of osteosarcoma patients. We investigated the association between the tissue expression levels of OR3A4 and different clinicopathological characteristics of osteosarcoma patients by χ2 tests. Bioinformatic databases and luciferase reporter assays were used to predict and validate the target microRNA of OR3A4. Finally, the role of OR3A4 in the proliferation and invasion of osteosarcoma cells was tested by MTT and Transwell assays, respectively. RESULTS: We observed that the expression level of OR3A4 was upregulated in the tumour tissue of osteosarcoma patients (p < 0.001) and osteosarcoma cell lines (p < 0.01) compared with the normal adjacent tissue and a normal human foetal osteoblastic cell line, respectively. The survival curve revealed that patients with high expression levels of OR3A4 had lower overall survival. Increased OR3A4 expression in osteosarcoma patients was associated with distant metastasis (p = 0.02) and advanced clinical stage (p < 0.001). In addition, bioinformatics analysis and luciferase reporter assays verified the complementary binding between OR3A4 and miR-1227-5p. Furthermore, we found that OR3A4 acted as a miR-1227-5p "sponge" to modulate osteosarcoma cell proliferation and invasion via downregulation of miR-1227-5p. CONCLUSION: OR3A4 promotes osteosarcoma cell proliferation and invasion by sponging miR-1227-5p, which might be related to the metastasis of osteosarcoma and could be used as a potential prognostic biomarker and therapeutic target in osteosarcoma.

Exosome-transmitted miR-567 reverses trastuzumab resistance by inhibiting ATG5 in breast cancer.

Trastuzumab is commonly used in the treatment of human epidermal growth factor receptor-2 positive (HER-2+) breast cancer, but its efficacy is often limited by the emergence of chemoresistance. Recent studies indicate that exosomes act as vehicles for exchange of genetic cargo between heterogeneous populations of tumor cells, engendering a transmitted drug resistance for cancer development and progression. However, the specific contribution of breast cancer-derived exosomes is poorly understood. In this study, publicly available expression profiling data from breast cancer and bioinformatics analyses were used to screen potential miRNAs in trastuzumab resistance. A series of gain- or loss-functional assays were performed to define the function of miR-567 and ATG5 in trastuzumab resistance and autophagy, both in vitro and in vivo. Our results showed that miR-567 was significantly decreased in trastuzumab-resistant patients compared with responding patients. Moreover, miR-567 was also downregulated in trastuzumab-resistant cells compared with parental cells. Overexpression of miR-567 reversed chemoresistance, whereas silence of miR-567 induced trastuzumab resistance, both in vitro and in vivo. In addition, enhanced miR-567 could be packaged into exosomes, incorporated into receipt cells, suppressing autophagy and reversed chemoresistance by targeting ATG5. To conclude, exosomal miR-567 plays a key role in reversing trastuzumab resistance via regulating autophagy, indicating it may be a promising therapeutic target and prognostic indicator for breast cancer patients.

Analysis of cyclin E co-expression genes reveals nuclear transcription factor Y subunit alpha is an oncogene in gastric cancer.

OBJECTIVE: To explore genes potentially co-expressed with cyclin E in gastric cancer and discover possible targets for gastric cancer treatment. METHODS: The Cancer Genome Atlas (TCGA) stomach adenocarcinoma sequencing data were used to predict genes co-expressed with cyclin E. Co-expression genes predicted by cBioPortal online analysis with Pearson correlation coefficient ≥0.4 were analyzed by gene ontology (GO) enrichment annotation using the PANTHER online platform (Ver. 7). Interactions between proteins encoded by these genes were analyzed using the STRING online platform (Ver. 10.5) and Cytoscape software (Ver. 3.5.1). Genes displaying a high degree of connection were analyzed by transcription factor enrichment prediction using FunRich software (Ver. 3). The significant transcription factor and cyclin E expression levels and their impact on gastric cancer progression were analyzed by Western blotting and Kaplan-Meier survival curve analysis. RESULTS: After filtering the co-expression gene prediction results, 78 predicted genes that included 73 protein coding genes and 5 non-coding genes with Pearson correlation coefficient ≥0.4 were selected. The expressions of the genes were considered to be correlated with cyclin E expression. Among the 78 genes co-expressed with cyclin E, 19 genes at the central of the regulatory network associated with cyclin E were discovered. Nuclear transcription factor Y subunit alpha (NF-YA) was identified as a significant transcription factor associated with cyclin E co-expressing genes. Analysis of specimen donors' clinical records revealed that high expression of NF-YA tended to be associated with increased cyclin E expression. The expression of both was associated with progression of gastric cancer. Western blotting results showed that compared with normal tissues, NF-YA and cyclin E were highly expressed in tumor tissues (P < 0.001). Survival curve analysis clearly demonstrated relatively poor overall survival of gastric cancer patients with high cyclin E or high NF-YA expression level, compared to patients with low cyclin E or NF-YA expression (P < 0.05). CONCLUSIONS: NF-YA may promote gastric cancer progression by increasing the transcription of cyclin E and other cell cycle regulatory genes. NF-YA might be a potential therapeutically useful prognostic factor for gastric cancer.

Changes in the mitochondrial proteome in human hepatocytes in response to alpha-amanitin hepatotoxicity.

Amanitin-induced apoptosis is proposed to have a significant effect on the pathogenesis of liver damage. However, few reports have focused on proteome changes induced by α-amanitin (α-AMA). Here, we evaluated changes in mitochondrial proteins of hepatocytes in response to 2 µM α-AMA, a concentration at which α-AMA-induced cell damage could be rescued at cellular level by common clinical drugs. We found 56 proteins were differentially expressed in an α-AMA-treated group. Among them, 38 proteins were downregulated and 18 were upregulated. Downregulated functional proteins included importer TOMM40, respiratory chain component cytochrome C, and metabolic enzymes of citrate acid cycle such as malate dehydrogenase, which localize on the mitochondrial outer membrane, inner membrane and matrix respectively. Immunoblot analysis showed that α-AMA decreased mitochondrial import receptor subunit TOMM40 and cytochrome c accompanied by an increase in the cytosol although their total protein levels were not affected significantly. The mitochondrial membrane potential was also destroyed by α-AMA and was restored by the clinical drug silibinin. Immunofluorescence suggested that mitochondrial morphology did not change. Taken together, our results provide further insights into the toxic mechanism of α-AMA on hepatocytes.

Inhibition of cell invasion and migration by CEACAM1-4S in breast cancer.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), a cell-cell adhesion molecule, has been revealed to perform an important role in tumor progression. Although there are a number of studies on CEACAM1 in patients with breast cancer, there is limited information on the roles of CEACAM1 in breast cancer metastasis. The present study aimed to identify whether CEACAM1 is involved in breast cancer development and to investigate the underlying mechanisms. First, the expression of CEACAM1 was observed in patients with breast cancer, and the association between CEACAM1 expression levels and migration and invasion of breast cancer cells was analyzed. As there are 12 isoforms of CEACAM1, of which CEACAM1-4S dominates in the human breast epithelium, subsequent study focused on CEACAM1-4S as a representative of all the isoforms. Results of the present study demonstrated that CEACAM1-4S suppresses breast cancer cell invasion and migration in a manner that is dependent on the balance between matrix metalloproteinase 2/tissue inhibitor of metalloproteinase 2 and E-/N-cadherin expression. In addition, CEACAM1-4S was likely to cause reversal of epithelial-mesenchymal transition of breast cancer cells through repressing Smad2 and signal transducer and phosphorylation of activator of transcription 3. In conclusion, the present study demonstrated that CEACAM1-4S performs an inhibitory role in breast cancer metastasis, and restoring CEACAM1-4S expression may provide a novel strategy for therapy of patients with metastatic breast cancer.

Assay of serum CEACAM1 as a potential biomarker for breast cancer.

BACKGROUND: Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a widely expressed multi-functional adhesion molecule reported to serve as a serum biomarker in several types of cancer. However, the serum CEACAM1 expression in breast cancer is unclear. We investigated the serum concentrations of CEACAM1 in patients with breast cancer and determine the potential of serum CEACAM1 as a breast cancer biomarker. METHODS: Serum specimens were obtained from 33 patients with breast cancer, 30 patients with benign breast diseases and 34 healthy donors. The serum CEACAM1 concentrations were examined by an enzyme-linked immunosorbent assay (ELISA). RESULTS: The serum CEACAM1 concentrations in the malignant group (532 ng/ml) were significantly higher than those of the benign group (423 ng/ml) and healthy control group (386 ng/ml) (both p<0.001). Based on univariable logistic regression, serum CEACAM1 concentrations significantly predicted breast cancer versus normal controls or benign breast diseases. Area under receiver operating characteristic curve (ROC) for serum CEACAM1 was 0.925(95% CI: 0.866-0.984). The optimal cut-off concentration of CEACAM1 was 475.82 ng/ml for discriminating breast cancer from normal controls. CONCLUSION: Serum concentrations of CEACAM1 may serve as a useful indicator for the presence of breast cancer.