Proteomic analysis of plasma exosomes to differentiate malignant from benign pulmonary nodules.

BACKGROUND: It is difficult to distinguish benign pulmonary nodules (PNs) from malignant PNs by conventional examination. Therefore, novel biomarkers that can identify the nature of PNs are needed. Exosomes have recently been identified as an attractive alternative approach since tumor-specific molecules can be found in exosomes isolated from biological fluids. METHODS: Plasma exosomes were extracted via the exoEasy reagent method. The major proteins from plasma exosomes in patients with PNs were identified via labelfree analysis and screened for differentially expressed proteins. A GO classification analysis and KEGG pathway analysis were performed on plasma exosomal protein from patients with benign and malignant PNs. RESULTS: Western blot confirmed that protein expression of CD63 and CD9 could be detected in the exosome extract. Via a search of the human Uniprot database, 736 plasma exosome proteins from patients with PNs were detected using high-confidence peptides. There were 33 differentially expressed proteins in the benign and malignant PNs. Of these, 12 proteins were only expressed in the benign PNs group, while 9 proteins were only expressed in the malignant PNs group. We further obtained important information on signaling pathways and nodal proteins related to differential benign and malignant PNs via bioinformatic analysis methods such as GO, KEGG, and String. CONCLUSIONS: This study provides a new perspective on the identification of novel detection strategies for benign and malignant PNs. We hope our findings can provide clues for the identification of benign and malignant PNs.

LncRNA H19 overexpression induces bortezomib resistance in multiple myeloma by targeting MCL-1 via miR-29b-3p.

Radiotherapy, chemotherapy, autologous/allogeneic stem cell transplantation, and targeted drug therapy are currently available therapeutic options for multiple myeloma (MM), but the clinical outcome remains unsatisfactory owing to frequent occurrence of drug resistance. Anti apoptosis is one of the main mechanisms to mediate drug resistance. Studies have shown that MCL-1 plays a key role in the growth of cancer cells "escaping" drug attacks, but the underlying mechanism remains unclear. Our previous study demonstrated that lncRNA H19 was highly expressed in the serum of MM patients. Bioinformatics predicts that miR-29b-3p is the downstream target gene, and MCL-1 is the downstream target protein of miR-29b-3p. Therefore, we speculated that MCL-1 may be involved in the occurrence of drug resistance through epigenetics. On the basis of these previous findings, the present study was intended to explore the biological function of H19, interactions between the downstream target genes, and the effect of H19 on BTZ resistance of myeloma cells. In addition, in vivo experiments we have also confirmed that H19 promoted tumor growth and may develop resistance to bortezomib partly. It was found that H19 reduced cell sensitivity to the chemotherapeutic drug BTZ by working as a miRNA sponge to inhibit the expression of miR-29b-3p, enhance MCL-1 transcriptional translation and inhibit apoptosis. These findings may help gain insights into the molecular mechanism of acquired BTZ resistance and develop new drug targets for the clinical treatment of MM.

Serum miR-30d as a novel biomarker for multiple myeloma and its antitumor role in U266 cells through the targeting of the MTDH/PI3K/Akt signaling pathway.

Multiple myeloma (MM) is a hematological tumor and is characterized by the infiltration of malignant clonal plasma cells (PCs) in bone marrow. MicroRNAs (miRNAs or miRs) have been reported to play an important role in the genesis and progression of MM. However, little is known about the clinical diagnostic value and biological functions of miR-30d in MM. In this study, to investigate the role of miR-30d in MM, we used reverse transcription-quantitative polymerase chain reaction quantitative (RT-qPCR) to detect the relative expression level of miR-30d in the serum of 81 patients with primary MM and 78 healthy donors (HDs). The biological functions of miR-30d were then assessed by CCK-8 assay, flow cytometric analysis of apoptosis and western blot (WB) analysis in U266 cells. Moreover, the confirmation of the target gene of miR-30d was conducted by luciferase reporter assay. Our results indicated that miR-30d expression was significantly downregulated in the serum of patients with primary MM compared with that of the HDs and that it was significantly associated with several clinical indicators of MM. Further cell functional analyses using the U266 cells revealed that miR-30d functions as a tumor suppressor gene in MM by inhibiting cell viability and promoting cell apoptosis. Moreover, miR-30d was confirmed to directly bind to the 3'UTR of its target gene, metadherin (MTDH) and inhibit the activation of the downstream PI3K/Akt signaling pathway. On the whole, the findings of this study indicate that the serum expression level of miR-30d is of great significance to the diagnosis and treatment monitoring of patients with MM. Moreover, miR-30d carries out its antitumor role in U266 cells through the inhibition of the activation of the PI3K/Akt signaling pathway by negatively regulating MTDH, which reveals its potential for use as a therapeutic target for MM.

Serum level of long noncoding RNA H19 as a diagnostic biomarker of multiple myeloma.

Circulating long noncoding RNA (lncRNA) H19 has been reported to be a biomarker for cancer monitoring. The purpose of this study was to determine whether serum lncRNA could serve as a novel biomarker for the diagnosis of multiple myeloma (MM) and evaluate its value of clinical application. In our study, the expression of lncRNA H19 was up-regulated in 80 patients with MM and MM cell lines by RT-PCR analysis. Clinicopathological analysis showed the expression level of H19 could assist clinical staging, and the severity of the disease could be roughly determined according to the amount of H19 expressed in the patient serum. This is the first report to show that H19 was expressed in the serum of MM patients, suggesting that upregulation of serum lncRNA H19 may prove to be a novel biomarker for early diagnosis and clinical treatment of MM.

YAP Suppresses Lung Squamous Cell Carcinoma Progression via Deregulation of the DNp63-GPX2 Axis and ROS Accumulation.

Lung squamous cell carcinoma (SCC), accounting for approximately 30% of non-small cell lung cancer, is often refractory to therapy. Screening a small-molecule library, we identified digitoxin as a high potency compound for suppressing human lung SCC growth in vitro and in vivo Mechanistic investigations revealed that digitoxin attenuated YAP phosphorylation and promoted YAP nuclear sequestration. YAP activation led to excessive accumulation of reactive oxygen species (ROS) by downregulating the antioxidant enzyme GPX2 in a manner related to p63 blockade. In patient-derived xenograft models, digitoxin treatment efficiently inhibited lung SCC progression in correlation with reduced expression of YAP. Collectively, our results highlight a novel tumor-suppressor function of YAP via downregulation of GPX2 and ROS accumulation, with potential implications to improve precision medicine of human lung SCC. Cancer Res; 77(21); 5769-81. ©2017 AACR.

Aspirin-induced inhibition of adipogenesis was p53-dependent and associated with inactivation of pentose phosphate pathway.

Obesity has become a major public health problem of global significance. Today, aspirin remains the most commonly used medication for the treatment of pyrexia, pain, inflammation and antiplatelet. The present study aims at evaluating the possible existence of a putative p53-dependent pathway underlying the aspirin-induced inhibition of adipogenesis. Cell migration assay was identified by the ability to migrate through Transwell insert. Oil Red O staining was employed to quantify adipose accumulation. The concentration of glucose and triglyceride were measured by using assay kits. The expression levels of several master regulatory molecules controlling various signal pathways were monitored using the immunoblotting techniques. Aspirin significantly inhibited preadipocyte migration and adipose accumulation. The p53-p21 signaling and the expression of differentiation marker glycerol-3-phosphate dehydrogenase were increased in a dose-dependent manner. It indicated that aspirin induced adipocyte differentiation through p53-p21 pathway. The oncogenic ERK 1/2 MAPK signaling was induced, whereas, the expression of adipogenic markers peroxisome proliferator-activated receptor γ (PPARγ), adipocyte fatty acid-binding protein (A-FABP) and inflammatory factors cyclooxygenase-2 (Cox-2), tumor necrosis factor α (TNFα) and inducible nitric oxide synthase (iNOS) were inhibited. Aspirin negatively regulated the pentose phosphate pathway (PPP) by inhibiting the expression of rate-limiting enzyme glucose-6-phosphate dehydrogenase. Knockdown the expression of oncogenic ERK 1/2 MAPK by using 10 µM PD98059 significantly increased triglyceride synthesis, adipose accumulation and activated PPP, however, decreased glucose uptake. Diverted the glucose flux to PPP, rather than increased glucose uptake, was associated with adipogenesis. Down-regulated the expression of tumor suppressor p53 by 10 µM pifithrin-α (PFTα) alone had no effect on adipose accumulation. However, administration of aspirin accompanied with PFTα abolished aspirin-induced inhibition of adipogenesis. We demonstrated that aspirin-induced inhibition of adipogenesis was p53-dependent and associated with inactivation of PPP. Blockade PPP may be a novel strategy for obesity prevention and therapy. Moreover, when use aspirin in therapeutic strategy, the p53 status should be considered.

The novel p53-dependent metastatic and apoptotic pathway induced by vitexin in human oral cancer OC2 cells.

Vitexin, identified as apigenin-8-C-D-glucopyranoside, a natural flavonoid compound found in certain herbs such as hawthorn herb, has been reported to exhibit anti-oxidative, anti-inflammatory, anti-metastatic and antitumor properties. The aim of this study was to investigate the possible existence of p53-dependent pathway underlying vitexin-induced metastasis and apoptosis in human oral cancer cells, OC2 cells. Vitexin decreased cell viability significantly. Meanwhile, the expression of tumor suppressor p53 and a small group of its downstream genes, p21(WAF1) and Bax, were upregulated. The p53 inhibitor pifithrin-α (PFT-α) knockdown of the signaling of p53 led vitexin to lose its antitumor effect and inhibited the expression of p53 downstream genes, p2(WAF1) and Bax. Vitexin had anti-metastatic potential accompanied with increasing plasminogen activator inhibitor 1 (PAI-1) accumulation and decreasing matrix metalloproteinase-2 expression. Our present study evidenced, by using p53 inhibitor PFT-α, PAI-1 and peroxisome proliferator-activated receptor γ are downstream genes of p53 in vitexin-induced signaling. MAPK inhibitor PD98059 decreased the OC2 cells viability significantly. The expression of p53 and its downstream genes p21(WAF1) and Bax were enhanced by blocking the activation of p42/p44 MAPK in response to treatment with vitexin. Moreover, p42/p44 MAPK played a negative role in p53-dependent metastasis and apoptosis. We give evidence for the first time that the novel p53-dependent metastatic and apoptotic pathway induced by vitexin in human oral cancer OC2 cells.