Circular RNA hsa_circ_0001306 Functions as a Competing Endogenous RNA to Regulate FBXW7 Expression by Sponging miR-527 in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common types of cancer worldwide. Circular RNAs (circRNAs) have been reported to regulate many types of cancers, including HCC. The purpose of this study was to investigate the potential roles of hsa_circ_0001306 in HCC. Firstly, the downregulation of hsa_circ_0001306 was identified by high­throughput RNA sequencing and further verified by qRT-PCR. Secondly, we evaluated the effects of hsa_circ_0001306 on HCC cell proliferation, invasion, cell cycle. Finally, we used an animal model to validate the in vitro experimental results. The expression of hsa_circ_0001306 was closely related to tumor size. Knockdown of hsa_circ_0001306 could downregulate F-box and WD repeat domain containing 7(FBXW7), a target of miR-527, thereby promoting HCC cell proliferation and invasion. Furthermore, hsa_circ_0001306 siRNA increased the multiplication rate of HCC tumors. Mechanistic studies indicated that hsa_circ_0001306 acts as a ceRNA for miR-527, which resulted in the reduction of its endogenous target, FBXW7. Hsa_circ_001306 is significantly downregulated in HCC, and the hsa_circ_0001306/miR-527/FBXW7 axis plays an important role in HCC progression.

CpG Oligodeoxynucleotide Developed to Activate Primate Immune Responses Promotes Antitumoral Effects in Combination with a Neoantigen-Based mRNA Cancer Vaccine.

PURPOSE: The purpose of our research was to identify and evaluate synthetic phosphorothioate-modified CPG oligodeoxynucleotides (CPG-ODNs) activating innate and adaptive immune responses. Furthermore, combined treatment with CpG and an mRNA cancer vaccine was evaluated in melanoma models as a therapeutic approach. METHODS: A molecular assay was used to screen new CpG molecules; mouse modeling and pathological analysis were used to confirm the antitumor effect of CpG alone or in combination with an mRNA vaccine. Finally, safety was assessed by monitoring blood biochemistry. RESULTS: We first screened and identified a new CpG-B class ODN (CpG2018B) that effectively stimulated type II interferons in both mouse plasmacytoid dendritic cells (pDCs) and human peripheral blood mononuclear cells (PBMCs). In addition, CpG2018B promoted cytokine production mainly via toll-like receptor 9 (TLR9) pathways. We further demonstrated that intratumoral (IT) injection of CpG2018B inhibited melanoma growth in syngeneic models and could turn "cold" tumors into "hot" tumors. Then, CpG2018B and an mRNA-based neoantigen cancer vaccine were encapsulated in lipid nanoparticles (LNPs) and intratumorally injected into melanoma mouse models. Interestingly, vaccination with CpG or the mRNA vaccine alone could inhibit tumor growth, while combination of CpG with the mRNA vaccine enhanced the antitumor effect. Finally, we described the long-term safety and tolerability of CpG2018B and mRNA therapy in mice model. CONCLUSION: We identified a novel CpG-B class ODN to promote the immune response, and CpG combined with mRNA cancer vaccines is an attractive candidate approach for immunostimulatory sequence (ISS)-based therapeutic strategies.

miR-137-3p Modulates the Progression of Prostate Cancer by Regulating the JNK3/EZH2 Axis.

BACKGROUND: Prostate cancer (PCa) is one of the most common cancers in men worldwide. Early detection of prostate cancer by prostate-specific antigen (PSA) screening still has limitations. The discovery of new candidates is urgent and can provide insights into the mechanism involved in prostate cancer tumorigenesis. METHODS: We conducted a cross-sectional study involving prostate cancer cell lines and clinical samples. qPCR and IHC were used to evaluate the expression of miR-137-3p/JNK3/EZH2. Furthermore, cell growth, migration, invasion, cell cycle and apoptosis were analyzed to describe the function of this axis. Moreover, xenograft models, pathology platforms and TCGA data were generated to confirm the role of the miR-137-3p/JNK3/EZH2 axis. RESULTS: In this study, we determined that miR-137-3p was significantly reduced in prostate cancer, and low expression of miR-137-3p was correlated with tumor stage . The overexpression of miR-137-3p suppressed cell proliferation, migration and invasion in prostate cancer by enhancing cell apoptosis. We also validated JNK3 (MAPK10) as a direct target gene of miR-137-3p. Down-regulation of JNK3 in prostate cancer also inhibited cell proliferation and invasion and promoted apoptosis. Moreover, JNK3 expression was up-regulated and negatively correlated with miR-137-3p in prostate cancer tissues. Furthermore, JNK3 modulated EZH2 expression, which is a key oncogene in prostate cancer. Survival data indicated that patients with high levels of JNK3 and EZH2 had a worse prognosis. CONCLUSION: Collectively, the identification of miR-137-3p and the JNK3/EZH2 pathway might facilitate the development of biomarkers and therapeutic targets for prostate cancer.

Dysregulation of SPRR3/miR-876-3p Axis Contributes to Tumorigenesis in Non-Small-Cell Lung Cancer.

BACKGROUND: SPRR3, also known as esophagin, has been shown to be involved in the initiation and progression of numerous types of tumor. However, the biological function of SPRR3 that contributes to non-small-cell lung cancer (NSCLC) growth and migration is largely unknown. METHODS: The expression of SPRR3 and its association with EZH2 and miR-876-3p in NSCLC cells were determined by real-time PCR. Protein levels were measured by immunohistochemistry (IHC) and Western blot. Cell functions were studied by CCK-8, transwell assay, flow cytometry and dual-luciferase reporter assay. The effect of SPRR3 on tumor growth in vivo was evaluated in patient-derived xenograft (PDX) models. RESULTS: SPRR3 was up-regulated in most NSCLC cell lines and clinical tissues. Also, the correlation between SPRR3 expression and clinical features was significant. Functional studies confirmed that SPRR3 modulates cell proliferation, invasion and cell apoptosis in NSCLC via regulating EZH2, which is a well-known oncogene in NSCLC. Furthermore, SPRR3 was found to be a direct target of miR-876-3p that also plays a suppressor role in NSCLC. CONCLUSION: These findings indicated that miR-876-3p/SPRR3/EZH2 signaling cascade exerts important roles in the regulation of NSCLC, suggesting that this pathway can serve as a potential therapeutic target in NSCLC.

Subtype-specific secretomic characterization of pulmonary neuroendocrine tumor cells.

Pulmonary neuroendocrine (NE) cancer, including small cell lung cancer (SCLC), is a particularly aggressive malignancy. The lineage-specific transcription factors Achaete-scute homolog 1 (ASCL1), NEUROD1 and POU2F3 have been reported to identify the different subtypes of pulmonary NE cancers. Using a large-scale mass spectrometric approach, here we perform quantitative secretome analysis in 13 cell lines that signify the different NE lung cancer subtypes. We quantify 1,626 proteins and identify IGFBP5 as a secreted marker for ASCL1High SCLC. ASCL1 binds to the E-box elements in IGFBP5 and directly regulates its transcription. Knockdown of ASCL1 decreases IGFBP5 expression, which, in turn, leads to hyperactivation of IGF-1R signaling. Pharmacological co-targeting of ASCL1 and IGF-1R results in markedly synergistic effects in ASCL1High SCLC in vitro and in mouse models. We expect that this secretome resource will provide the foundation for future mechanistic and biomarker discovery studies, helping to delineate the molecular underpinnings of pulmonary NE tumors.

miR-140-3p functions as a tumor suppressor in squamous cell lung cancer by regulating BRD9.

Squamous cell lung cancer (SqCLC) is among the most malignant lung cancers worldwide, lacking biomarkers for diagnostic and targets for treatment. In this study, we observed that miR-140-3p was expressed at low levels both in SqCLC cell lines and patient samples, while overexpression of miR-140-3p dramatically reduced the cell proliferation and invasion in SqCLC cells and Patient derived xenograft (PDX) models. Our further investigation indicated miR-140-3p negatively affected the tumorigenesis of SqCLC by down-regulating the expression of BRD9, an oncogene in SqCLC. Inhibition of BRD9 repressed SqCLC tumorigenesis by regulating c-myc expression. Meanwhile, BRD9 expression is up-regulated and negatively correlated with miR-140-3p in clinical samples; a meta-analysis of survival data indicates that SqCLC patients with high levels of BRD9 in their tumors have a worse prognosis. Collectively, our study suggests the prognostic and therapeutic roles of miR-140-3p and BRD9 axis in squamous cell lung cancer.

Development and validation of serum exosomal microRNAs as diagnostic and prognostic biomarkers for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide. China accounts for over half of the new cases and deaths. Diagnostic imprecision and a lack of complimentary molecular biomarkers are partially responsible for this lack of progress. Herein, serum-derived exosomal microRNA (miRNA) profiling was performed on 80 patients which histologically confirmed HCC and 30 normal controls. A classification of 8 exosomal miRNAs had biologically and statistically significant differences between HCC and normal serum samples, including miR-122, miR-125b, miR-145, miR-192, miR-194, miR-29a, miR-17-5p, and miR-106a. Online algorithm showed strong independent classification accuracy (area under the curve) reached 0.535 to 0.850, separately. The significant correlation between serum exosomal miRNAs and tumor size was observed. In addition, the survival difference of HCC patients with high or low exosomal miR-106a was statistically significant using Kaplan-Meier analysis. Besides, we also measured the proliferation and invasion ability of HCC cells following exosomal miR-106a mimics or inhibitor treatment. After prediction with algorithms, mitogen-activated protein kinase and c-Jun N-terminal kinase pathways were identified associated with miR-106a's function. In summary, differentially expressed serum exosomal miRNAs can be helpful for diagnostic and prognostic of HCC.

Circulating HER-2 mRNA in the peripheral blood as a potential diagnostic and prognostic biomarker in females with breast cancer.

Breast cancer is a prevalent malignant cancer worldwide, and a lack of defined biomarkers for early prognostication contributes to its high associated mortality rate, especially in human epidermal growth factor receptor 2 (HER-2)-positive breast cancer. In the present study, HER-2 mRNA levels in patients were detected prior to surgery and during neoadjuvant chemotherapy to explore its potential diagnostic and prognostic value. Blood samples were collected from 70 patients with breast cancer, including 50 HER-2-negative and 20 HER-2-positive patients, prior to and following surgery (postoperative, n=13; neoadjuvant chemotherapy, n=5); the control group included 35 samples from healthy individuals. The relative mRNA level of HER-2 in blood was determined by one-step reverse transcription-quantitative polymerase chain reaction. HER-2 expression curves of measurements taken during neoadjuvant chemotherapy were compared with the tumor size. A significant difference in the blood HER-2 mRNA level was observed between healthy women and patients with breast cancer (P<0.0001). A cutoff value of 1.512 was established for the circulating HER-2 level in healthy subjects based on the upper 95% confidence interval value of samples from the control group. The level of HER-2 mRNA in blood was associated with the HER-2 status, Ki-67 expression, and lymphovascular invasion in primary tumor tissue samples; however, there was no association with the lymph node status, tumor stage, tumor grade, tumor size, patient age, estrogen or progesterone receptor status of the primary tumor. HER-2 mRNA levels were associated with the response rate, as determined by primary tumor size, in patients who received neoadjuvant chemotherapy. In conclusion, baseline and early changes in peripheral blood HER-2 mRNA indicated that HER-2 mRNA may be a potential diagnostic biomarker for breast cancer and a prognostic marker for predicting the efficacy of neoadjuvant therapy.

Surgical excision and oncoplastic breast surgery in 32 patients with benign phyllodes tumors.

BACKGROUND: The purpose of this study was to assess the effectiveness and safety in patients with benign phyllodes after performing local excision and following with intra-operative breast flap reconstruction. METHODS: Patients (n = 32) with eligible breast cystosarcoma phyllodes underwent wide local excision followed by intra-operative breast flap reconstruction. Primary outcome measures included average operative time, length of in-hospital stay, postoperative recurrence, and intra-operative and postoperative complications. RESULTS: Thirty-two patients who underwent surgical excision and oncoplastic breast surgery were evaluated using the BCCT.core software. A satisfactory symmetrical breast shape was achieved. The average operative time was 56.3 ± 8.2 min. The average postoperative duration of hospitalization was 3.7 ± 1.2 days. While there was no breast disease recurred during the 1 to 8-year follow-up period. CONCLUSIONS: Wide local excision accompanied by intra-operative breast flap reconstruction could be adopted for removing benign phyllodes tumors while retaining the basic shape of the breast.

Exosomal miR-93 promotes proliferation and invasion in hepatocellular carcinoma by directly inhibiting TIMP2/TP53INP1/CDKN1A.

Hepatocellular carcinoma (HCC) is a malignant cancer worldwide; lacking biomarkers for early prognostication contributes to its high lethality. Herein, we report a novel biomarker, exosome delivered miR-93, is up-regulated in HCC cell line media and serum samples of HCC patients. We measured the proliferation and invasion ability of HCC cell lines following exosomal miR-93 treatment. After prediction with online algorithms, we further confirmed that TP53INP1, TIMP2 and CDKN1A are direct targets of miR-93 by dual-luciferase reporter assay. In addition, the diagnostic value of exosomal miR-93 was evaluated by qPCR and ROC analysis. The significant correlation between serum exosomal miR-93 and clinical information including stage, tumor size were observed. Furthermore, the survival differences of HCC patients with high or low miR-93 were statistically significant using Kaplan-Meier analysis. In summary, our work identified exosomal miR-93 as a novel biomarker for both diagnosis and prognosis in HCC.

miR-342-3p suppresses cell proliferation and migration by targeting AGR2 in non-small cell lung cancer.

AGR2 is a well-studied secreted protein that is involved in multiple biological processes including cell proliferation and migration. The mechanism by which AGR2 increases the growth and migration of non-small cell lung cancer cells (NSCLC) is still unknown. In this study, we report that AGR2 is directly targeted by miR-342-3p. Functional studies suggest that overexpression of miR-342-3p inhibits the proliferation and migration of non-small cell lung cancer cells. Overexpression of AGR2 counteracts the phenotypes induced by miR-342-3p. Moreover, AGR2 expression is up-regulated and negatively correlated with miR-342-3p levels in NSCLC cells and tissues. A meta-analysis of survival data indicates that NSCLC patients with high levels of AGR2 in their tumors have a worse prognosis. Collectively, the identification of miR-342-3p and AGR2 might facilitate the development of biomarkers and therapeutic targets for this devastating disease.

Modulation of glucose metabolism by a natural compound from Chloranthus japonicus via activation of AMP-activated protein kinase.

AMP-activated protein kinase (AMPK) is a key sensor and regulator of glucose metabolism. Here, we demonstrated that shizukaol F, a natural compound isolated from Chloranthus japonicus, can activate AMPK and modulate glucose metabolism both in vitro and in vivo. Shizukaol F increased glucose uptake in differentiated C2C12 myotubes by stimulating glucose transporter-4 (GLUT-4) membraned translocation. Treatment of primary mouse hepatocytes with shizukaol F decreased the expression of phosphoenolpyruvate carboxykinase 2 (PEPCK), glucose-6-phosphatase (G6Pase) and suppressed hepatic gluconeogenesis. Meanwhile, a single oral dose of shizukaol F reduced gluconeogenesis in C57BL/6 J mice. Further studies indicated that shizukaol F modulates glucose metabolism mainly by AMPKa phosphorylation activity. In addition, we also found that shizukaol F depolarizes the mitochondrial membrane and inhibits respiratory complex I, which may result in AMPK activation. Our results highlight the potential value of shizukaol F as a possible treatment of metabolic syndrome.

Quantitative Secretomic Analysis Identifies Extracellular Protein Factors That Modulate the Metastatic Phenotype of Non-Small Cell Lung Cancer.

Lung cancer is the leading cause of cancer-related deaths for men and women in the United States, with non-small cell lung cancer (NSCLC) representing 85% of all diagnoses. Late stage detection, metastatic disease and lack of actionable biomarkers contribute to the high mortality rate. Proteins in the extracellular space are known to be critically involved in regulating every stage of the pathogenesis of lung cancer. To investigate the mechanism by which secreted proteins contribute to the pathogenesis of NSCLC, we performed quantitative secretomic analysis of two isogenic NSCLC cell lines (NCI-H1993 and NCI-H2073) and an immortalized human bronchial epithelial cell line (HBEC3-KT) as control. H1993 was derived from a chemo-naïve metastatic tumor, while H2073 was derived from the primary tumor after etoposide/cisplatin therapy. From the conditioned media of these three cell lines, we identified and quantified 2713 proteins, including a series of proteins involved in regulating inflammatory response, programmed cell death and cell motion. Gene Ontology (GO) analysis indicates that a number of proteins overexpressed in H1993 media are involved in biological processes related to cancer metastasis, including cell motion, cell-cell adhesion and cell migration. RNA interference (RNAi)-mediated knock down of a number of these proteins, including SULT2B1, CEACAM5, SPRR3, AGR2, S100P, and S100A14, leads to dramatically reduced migration of these cells. In addition, meta-analysis of survival data indicates NSCLC patients whose tumors express higher levels of several of these secreted proteins, including SULT2B1, CEACAM5, SPRR3, S100P, and S100A14, have a worse prognosis. Collectively, our results provide a potential molecular link between deregulated secretome and NSCLC cell migration/metastasis. In addition, the identification of these aberrantly secreted proteins might facilitate the development of biomarkers for early detection of this devastating disease.

Shizukaol D isolated from Chloranthus japonicas inhibits AMPK-dependent lipid content in hepatic cells by inducing mitochondrial dysfunction.

This study is the first to demonstrate that shizukaol D, a natural compound isolated from Chloranthusjaponicus, can activate AMP- activated protein kinase (AMPK), a key sensor and regulator of intracellular energy metabolism, leading to a decrease in triglyceride and cholesterol levels in HepG2 cells. Furthermore, we found that shizukaol D induces mitochondrial dysfunction by depolarizing the mitochondrial membrane and suppressing energy production, which may result in AMPK activation. Our results provide a possible link between mitochondrial dysfunction and AMPK activation and suggest that shizukaol D might be used to treat metabolic syndrome.

Preparation and characterization of paclitaxel-loaded DSPE-PEG-liquid crystalline nanoparticles (LCNPs) for improved bioavailability.

Lipid-based liquid crystalline nanoparticles (LCNPs) have attracted growing interest as a new drug nanocarrier system for improving bioavailability for both hydrophilic and hydrophobic drugs. In this study, self-assembled LCNPs based on soy phosphatidyl choline and glycerol dioleate, which was known possessing low toxicity and negligible hemolysis, were prepared using poly(ethylene glycol)-grafted 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE-PEG) as the dispersing agent. Paclitaxel (PTX) was used as a model hydrophobic drug. The particle size of the optimized DSPE-PEG-LCNPs and PTX-loaded DSPE-PEG-LCNPs were around 70nm. Crossed polarized light microscopy was used to characterize the phase behavior of liquid crystalline (LC) matrices, which showed a fan-like birefringent texture in dark background indicating the coexistence of reversed cubic and hexagonal phase in the optimized LC matrix. Transmission electron microscopy and cryo-field emission scanning electron microscopy revealed its internal water channel and "twig-like" surface morphology. PTX-loaded DSPE-PEG-LCNPs exhibited a biphasic drug sustained release pattern with a relatively fast release at the initial stage and a sustained release afterwards. PTX-loaded DSPE-PEG-LCNPs presented higher AUC (410.942±72.522µg/Lh) when compared with commercial product Taxol (212.670±41.396µg/Lh). These results indicated that DSPE-PEG-LCNPs might serve as a potential sustained release system for poorly water-soluble agents.