Identification and validation of DHCR7 as a diagnostic biomarker involved in the proliferation and mitochondrial function of breast cancer.

BACKGROUND: Energy metabolism has a complex intersection with pathogenesis and development of breast cancer (BC). This allows for the possibility of identifying energy-metabolism-related genes (EMRGs) as novel prognostic biomarkers for BC. 7-dehydrocholesterol reductase (DHCR7) is a key enzyme of cholesterol biosynthesis involved in many cancers, and in this paper, we investigate the effects of DHCR7 on the proliferation and mitochondrial function of BC. METHODS: EMRGs were identified from the Gene Expression Omnibus (GEO) and MSigDB databases using bioinformatics methods. Key EMRGs of BC were then identified and validated by functional enrichment analysis, interaction analysis, weighted gene co-expression network analysis (WGCNA), least absolute shrinkage and selection operator (LASSO) regression, Cox analysis, and immune infiltration. Western blot, qRT-PCR, immunohistochemistry (IHC), MTT assay, colony formation assay and flow cytometry assay were then used to analyze DHCR7 expression and its biological effects on BC cells. RESULTS: We identified 31 EMRGs in BC. These 31 EMRGs and related transcription factors (TFs), miRNAs, and drugs were enriched in glycerophospholipid metabolism, glycoprotein metabolic process, breast cancer, and cell cycle. Crucially, DHCR7 was a key EMRG in BC identified and validated by WGCNA, LASSO regression and receiver operating characteristic (ROC) curve analysis. High DHCR7 expression was significantly associated with tumor immune infiltration level, pathological M, and poor prognosis in BC. In addition, DHCR7 knockdown inhibited cell proliferation, induced apoptosis and affected mitochondrial function in BC cells. CONCLUSIONS: DHCR7 was found to be a key EMRG up-regulated in BC cells. This study is the first to our knowledge to report that DHCR7 acts as an oncogene in BC, which might become a novel therapeutic target for BC patients.

Targeted RASSF1A expression inhibits proliferation of HER2­positive breast cancer cells in vitro.

Human epidermal growth factor receptor 2-positive (HER2+) breast cancer, which accounts for 15-20% of all breast cancer, is associated with tumor recurrence and poor prognosis. RAS association domain family protein 1 subtype A (RASSF1A) is a tumor suppressor that is silenced in a variety of human cancers. The present study aimed to investigate the role of RASSF1A in HER2+ breast cancer and the therapeutic potential of RASSF1A-based targeted gene therapy for this malignancy. RASSF1A expression in human HER2+ breast cancer tissues and cell lines was evaluated by reverse transcription PCR and western blot analysis. The associations between tumorous RASSF1A level and tumor grade, TNM stage, tumor size, lymph node metastasis and five-year survival were examined. HER2+ and HER2-negative (HER2-) breast cancer cells were transfected with a lentiviral vector (LV-5HH-RASSF1A) that could express RASSF1A under the control of five copies of the hypoxia-responsive element (5HRE) and one copy of the HER2 promoter (HER2p). Cell proliferation was evaluated by the MTT and colony formation assays. It was found that tumorous RASSF1A level was negatively associated with tumor grade (P=0.014), TNM stage (P=0.0056), tumor size (P=0.014) and lymph node metastasis (P=0.029) and positively associated with five-year survival (P=0.038) in HER2+ breast cancer patients. Lentiviral transfection of HER2+ breast cancer cells resulted in increased RASSF1A expression and decreased cell proliferation, especially under hypoxic conditions. However, lentiviral transfection of HER2-breast cancer cells did not affect RASSF1A expression. In conclusion, these findings verified the clinical significance of RASSF1A as a tumor suppressor in HER2+ breast cancer and supported LV-5HH-RASSF1A as a potential targeted gene therapy for this malignancy.

Metformin and simvastatin synergistically suppress endothelin 1-induced hypoxia and angiogenesis in multiple cancer types.

Multiple cancers have been reported to be associated with angiogenesis and are sensitive to anti-angiogenic therapies. Vascular normalization, by restoring proper tumor perfusion and oxygenation, could limit tumor cell invasiveness and improve the effectiveness of anticancer treatments. However, the underlying anticancer mechanisms of antiangiogenic drugs are still unknown. Metformin (MET) and simvastatin (SVA), two metabolic-related drugs, have been shown to play important roles in modulating the hypoxic tumor microenvironment and angiogenesis. Whether the combination of MET and SVA could exert a more effective antitumor effect than individual treatments has not been examined. The antitumor effect of the synergism of SVA and MET was detected in mouse models, breast cancer patient-derived organoids, and multiple tumor cell lines compared with untreated, SVA, or MET alone. RNA sequencing revealed that the combination of MET and SVA (but not MET or SVA alone) inhibited the expression of endothelin 1 (ET-1), an important regulator of angiogenesis and the hypoxia-related pathway. We demonstrate that the MET and SVA combination showed synergistic effects on inhibiting tumor cell proliferation, promoting apoptosis, alleviating hypoxia, decreasing angiogenesis, and increasing vessel normalization compared with the use of a single agent alone. The MET and SVA combination suppressed ET-1-induced hypoxia-inducible factor 1α expression by increasing prolyl hydroxylase 2 (PHD2) expression. Furthermore, the MET and SVA combination showed a more potent anticancer effect compared with bosentan. Together, our findings suggest the potential application of the MET and SVA combination in antitumor therapy.

Establishment and verification of a nomogram for predicting survival in patients with triple-positive breast cancer.

Background: Triple-positive breast cancer (TPBC) is a specific type of breast cancer characterized by the positive expression of estrogen receptor (ER)/progesterone receptor (PR)/human epidermal growth factor receptor 2 (HER-2). In recent years, the research on breast cancer has been increasing year by year, but there are few studies on TPBC, especially the lack of analysis with large sample size. In this study, sufficient samples were provided through the SEER database, explore the factors affecting the prognosis of TPBC, and construct a prediction model, in order to assess the individual survival of patients, and help clinicians accurately identify high-risk patients and develop personalized treatment plans. Methods: Patients pathologically diagnosed with TPBC were recruited from Surveillance, Epidemiology, and End Results (SEER) database and randomly divided into training and validation groups (7:3 ratio). Univariate analysis was used to analyze the related factors affecting the prognosis of TPBC patients in the modeling group, and then multivariate Cox proportional hazards model was used to analyze the significant factors to screen out the independent risk factors affecting the 3- and 5-year overall survival (OS) rate and construct the prediction model. Using the concordance index (C-index) and calibration curve were performed to evaluate the predictive ability of the model. Results: The results of the Cox risk-scale model showed that race, age, marital status, tumor grade, tumor, node, metastasis stage, surgical treatment, chemotherapy, and radiotherapy affected the prognosis of TPBC patients (P<0.05) in the training group, and the factors were used to construct a nomogram. The internal and external validation of the nomogram chart indicated that the C-index of the training group was 0.85 [95% confidence interval (CI): 0.836, 0.863] and that of the verification group was 0.833 (95% CI: 0.807, 0.858). The calibration curves of the 2 groups showed that the OS predicted by the model was consistent with the actual survival of the patients. Conclusions: The prediction model accurately predicted the prognosis of and identified high-risk TPBC patients.

Development and Validation of a 6-Gene Hypoxia-Related Prognostic Signature For Cholangiocarcinoma.

Cholangiocarcinoma (CHOL) is highly malignant and has a poor prognosis. This study is committed to creating a new prognostic model based on hypoxia related genes. Here, we established a novel tumor hypoxia-related prognostic model consisting of 6 hypoxia-related genes by univariate Cox regression and the least absolute shrinkage and selection operator (LASSO) algorithm to predict CHOL prognosis and then the risk score for each patient was calculated. The results showed that the patients with high-risk scores had poor prognosis compared with those with low-risk scores, which was verified as an independent predictor by multivariate analysis. The hypoxia-related prognostic model was validated in both TCGA and GEO cohorts and exhibited excellent performance in predicting overall survival in CHOL. The PPI results suggested that hypoxia-related genes involved in the model may play a central role in regulating the hypoxic state. In addition, the presence of IDH1 mutations in the high-risk group was high, and GSEA results showed that some metabolic pathways were upregulated, but immune response processes were generally downregulated. These factors may be potential reasons for the high-risk group with worse prognosis. The analysis of different immune regulation-related processes in the high- and low-risk groups revealed that the expression of genes related to immune checkpoints would show differences between these two groups. We further verified the expression of the oncogene PPFIA4 in the model, and found that compared with normal samples, CHOL patients were generally highly expressed, and the patients with high-expression of PPFIA4 had a poor prognosis. In summary, the present study may provide a valid prognostic model for bile duct cancer to inform better clinical management of patients.

A Crucial Angiogenesis-Associated Gene MEOX2 Could Be a Promising Biomarker Candidate for Breast Cancer.

Background: Angiogenesis plays a critical role in the growth and metastasis of breast cancer and angiogenesis inhibition has become an effective strategy for cancer therapy. Our study aimed to clarify the key candidate genes and pathways related to breast cancer angiogenesis. Methods: Differentially expressed genes (DEGs) in the raw breast cancer (BRCA) gene dataset from the Cancer Genome Atlas (TCGA) database were identified and gene ontology analysis of the DEGs was performed. Hub genes were subsequently determined using the Gene Expression Omnibus database. The expression of the mesenchyme homeobox 2 (MEOX2) in breast cancer cells and tissues was assessed by quantification real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC), respectively. The prognostic value of the MEOX2 gene in breast cancer tissue was evaluated with the Kaplan-Meier plotter. Results: A total of 61 angiogenesis-related DEGs were identified in the TCGA dataset, among which the gene MEOX2 was significantly down-regulated. GO functional annotation and pathway enrichment analyses showed that MEOX2 was significantly enriched in the regulation of vasculature development. The IHC results confirmed that MEOX2 expression was repressed in breast cancer tissues and the relatively low level indicated the tissue was densely vascularized. Moreover, MEOX2 expression was significantly elevated in breast cancer cells after treatment with cisplatin (DDP) and epirubicin (EPI). Finally, the Kaplan-Meier plotter confirmed that higher expression levels of MEOX2 were related to better overall survival. Conclusion: Our study revealed that the angiogenesis-associated gene MEOX2 can be used as a novel biomarker for breast cancer diagnosis and clinical therapy.

Low expression of MEOX2 is associated with poor survival in patients with breast cancer.

Aim: To investigate associations of MEOX2 expression with clinicopathological features and survival of breast cancer patients. Materials & methods: We used a breast cancer tissue microarray for immunohistochemistry. Associations between MEOX2 expression and clinicopathological features were analyzed using the χ-square test. Survival analysis was determined using a Kaplan-Meier curve. Multivariate Cox regression was used to determine associations of MEOX2 expression with overall survival. Results: We found that 74.1% of patients (100/135) had expression of MEOX2 at varying levels. MEOX2 was associated with histological grade and negatively correlated with Ki67 expression. Lower MEOX2 expression was significantly associated with decreased overall survival (p = 0.0011). Conclusion: MEOX2 expression could be a novel diagnostic and prognostic biomarker of breast cancer.

In this study we found that lower expression of the protein MEOX2 was associated with poor overall survival in breast cancer. MEOX2 is an independent prognostic factor for breast cancer patients. It would be a new diagnostic and prognostic biomarker for breast cancer.

MicroRNA-1269 promotes cell proliferation via the AKT signaling pathway by targeting RASSF9 in human gastric cancer.

BACKGROUND: MicroRNAs (miRNAs) play key roles in tumorigenesis and progression of gastric cancer (GC). miR-1269 has been reported to be upregulated in several cancers and plays a crucial role in carcinogenesis and cancer progression. However, the biological function of miR-1269 in human GC and its mechanism remain unclear and need to be further elucidated. METHODS: The expression of miR-1269 in GC tissues and cell lines was detected by quantitative real-time PCR (qRT-PCR). Target prediction programs (TargetScanHuman 7.2 and miRBase) and a dual-luciferase reporter assay were used to confirm that Ras-association domain family 9 (RASSF9) is a target gene of miR-1269. The expression of RASSF9 was measured by qRT-PCR and Western blotting in GC tissues. MTT and cell counting assays were used to explore the effect of miR-1269 on GC cell proliferation. The cell cycle and apoptosis were measured by flow cytometry. RASSF9 knockdown and overexpression were used to further verify the function of the target gene. RESULTS: We found that miR-1269 expression was upregulated in human GC tissues and cell lines. The overexpression of miR-1269 promoted GC cell proliferation and cell cycle G1-S transition and suppressed apoptosis. The inhibition of miR-1269 inhibited cell growth and G1-S transition and induced apoptosis. miR-1269 expression was inversely correlated with RASSF9 expression in GC tissues. RASSF9 was verified to be a direct target of miR-1269 by using a luciferase reporter assay. The overexpression of miR-1269 decreased RASSF9 expression at both the mRNA and protein levels, and the inhibition of miR-1269 increased RASSF9 expression. Importantly, silencing RASSF9 resulted in the same biological effects in GC cells as those induced by overexpression of miR-1269. Overexpression of RASSF9 reversed the effects of miR-1269 overexpression on GC cells. Both miR-1269 overexpression and RASSF9 silencing activated the AKT signaling pathway, which modulated cell cycle regulators (Cyclin D1 and CDK2). In contrast, inhibition of miR-1269 and RASSF9 overexpression inhibited the AKT signaling pathway. Moreover, miR-1269 and RASSF9 also regulated the Bax/Bcl-2 signaling pathway. CONCLUSIONS: Our results demonstrate that miR-1269 promotes GC cell proliferation and cell cycle G1-S transition by activating the AKT signaling pathway and inhibiting cell apoptosis via regulation of the Bax/Bcl-2 signaling pathway by targeting RASSF9. Our findings indicate an oncogenic role of miR-1269 in GC pathogenesis and the potential use of miR-1269 in GC therapy.

Exosomal MiR-744 Inhibits Proliferation and Sorafenib Chemoresistance in Hepatocellular Carcinoma by Targeting PAX2.

BACKGROUND Hepatocellular carcinoma (HCC) is a commonly occurring liver malignancy. Its prognosis remains unsatisfactory. Accumulating evidence has revealed that exosomal microRNAs (miRNAs) act as biomarkers and play crucial roles in the advancement of HCC. The current study explored the biological role and fundamental mechanism of exosomal miR-744 in HCC. MATERIAL AND METHODS The serum exosomes of HCC patients were isolated by differential ultracentrifugation. MiR-744 expression in HCC tissues, cell lines and serum exosomes were detected by quantitative real-time polymerase chain reaction (qRT-PCR). EdU (5-ethynyl-2'-deoxyuridine) assay and Cell Counting Kit-8 (CCK-8) assay were conducted to show the impacts of miR-744 or exosomal miR-744 on proliferation and sorafenib resistance in HepG2 cells. The target of miR-744 was ascertained by regulating the level of miR-744 in HepG2 cells. RESULTS MiR-744 is downregulated in HCC tissues and cell lines as well as in exosomes derived from patient serum and HepG2 cells. Additionally, downregulated miR-744 promotes HepG2 cell proliferation and inhibits the chemosensitivity of HepG2 cells to sorafenib. PAX2 was identified as the functional target of miR-744. Interestingly, miR-744 is decreased in exosomes derived from sorafenib-resistant HepG2 cells. Furthermore, when treated with the miR-744-enriched exosomes, the proliferation of HepG2 cells was significantly suppressed, and the sorafenib resistance was reduced. CONCLUSIONS MiR-744 has an imperative role in the propagation and chemoresistance of HCC. Serum exosomal miR-744 might act as a biomarker of HCC, and exosomal miR-744 might offer an innovative strategy for HCC treatment.

Overexpression of Contactin 1 promotes growth, migration and invasion in Hs578T breast cancer cells.

BACKGROUND: Contactin1 (CNTN1) has been shown to play an important role in the invasion and metastasis of several tumors; however, the role of CNTN1 in breast cancer has not been fully studied. The purpose of this study is to investigate the role of CNTN1 in regulating tumor growth, migration and invasion in breast cancer. RESULTS: To investigate its function, CNTN1 was expressed in Hs578T cells. CNTN1 expression was confirmed by western blot, immunohistochemistry and real-time RT-PCR. The effect of CNTN1 overexpression on proliferation, migration and invasion of Hs578T breast cancer cells was assessed in vitro and in vivo. Our results showed that CNTN1 overexpression promoted Hs578T cell proliferation, cell cycle progression, colony formation, invasion and migration. Notably, overexpression of CNTN1 in Hs578T cells enhanced the growth of mouse xenograft tumors. CONCLUSIONS: CNTN1 promotes growth, metastasis and invasion of Hs578T breast cancer cell line. Thus, therapies targeting CNTN1 may prove efficacious for breast cancer. However, further investigation is required to understand the mechanism by which CNTN1 influences proliferation, metastasis and invasion in breast cancer.