Junpu Wang

Urinary microRNA-30a-5p is a potential biomarker for ovarian serous adenocarcinoma

MicroRNAs (miRNAs) can serve as biomarkers in human cancer. To determine the clinical value of urinary miRNAs for ovarian serous adenocarcinoma, we collected urine samples from 39 ovarian serous adenocarcinoma patients, 26 patients with benign gynecological disease and 30 healthy controls. The miRNA microarray data showed that only miR-30a-5p was upregulated and 37 miRNAs were downregulated in the urine samples of ovarian serous adenocarcinoma patients, when compared to healthy controls, which was confirmed after conducting quantitative PCR. The upregulation of urinary miR-30a-5p was closely associated with early stage of ovarian serous adenocarcinoma as well as lymphatic metastasis. Receiver operator characteristic (ROC) analysis demonstrated the potential use of urinary miR-30a-5p as a diagnostic marker for ovarian serous adenocarcinoma. Furthermore, a lower urine level of miR-30a-5p was found in 20 gastric cancer and 20 colon carcinoma patients when compared to ovarian serous adenocarcinoma, suggesting that the upregulation of urinary miR-30a-5p may be specific for ovarian serous adenocarcinoma. miR-30a-5p was also upregulated in ovarian serous adenocarcinoma tissues and cell lines, while urinary miR-30a-5p from ovarian cancer patients was notably reduced following the surgical removal of ovarian serous adenocarcinoma, suggesting that urinary miR-30a-5p was derived from the ovarian serous adenocarcinoma tissue. Notably, miR-30a-5p was concentrated with exosomes from the ovarian cancer cell supernatant or urine from ovarian serous adenocarcinoma patients, supporting a pathway for excretion into the urine. The results also showed that the knockdown of miR-30a-5p significantly inhibited the proliferation and migration of ovarian cancer cells. In summary, to the best of our knowledge, the present study provided the first evidence of increased miR-30a-5p in the urine of ovarian serous adeno-carcinoma patients, while the inhibition of miR-30a-5p suppressed the malignant phenotypes of ovarian cancer in vitro. Therefore, miR-30a-5p serves as a promising diagnostic and therapeutic target for ovarian serous adenocarcinoma.

The role of miR-205 in the VEGF-mediated promotion of human ovarian cancer cell invasion

OBJECTIVE Our objective was to investigate a miRNA pathway that acts downstream of VEGF-induced invasion of ovarian cancer cells. METHOD We used two paired high and low metastatic serous ovarian cancer cells to demonstrate the role of miR-205 in VEGF-induced invasion of ovarian cancer cells and to investigate the gene targets of miR-205. RESULTS Our previous comparative proteomics studies showed that VEGF decreased the expression of Ezrin and Lamin A/C, and this result was validated in the present study using qPCR and Western blotting. Then we found that VEGF enhanced the invasiveness of and inhibited apoptosis in ovarian cancer cells as assessed by transwell invasion assays and Annexin V-FITC immunostaining, respectively. VEGFR was also expressed in ovarian cancer cells, as assessed by immunocytochemical staining. Furthermore, using the dual-luciferase report assay system, we demonstrated that miR-205 targeted Ezrin and Lamin A/C. MiR-205 was up-regulated in ovarian cancer cells exposed to VEGF, as determined by miRNA microarray analysis and verified by qPCR. MiR-205 promoted the invasion and proliferation of ovarian cancer cells. CONCLUSION Our data reveal a new potential pathway in which VEGF promotes the invasion of ovarian cancer cells, partially via the down-regulation of Ezrin and Lamin A/C caused by increased expression of miR-205.

Repertaxin, an inhibitor of the chemokine receptors CXCR1 and CXCR2, inhibits malignant behavior of human gastric cancer MKN45 cells in vitro and in vivo and enhances efficacy of 5-fluorouracil.

Chemokine-mediated activation of G protein-coupled receptors CXCR1/2 promotes tumor growth, invasion, inflammation and metastasis. Repertaxin, a CXCR1/2 small-molecule inhibitor, has been shown to attenuate many of these tumor-associated processes. The present study aimed to investigate the effects of repertaxin alone and in combination with 5-fluorouracil (5-FU) on the malignant behavior of gastric cancer and the potential mechanisms. Gastric cancer MKN45 cells were treated in vitro with repertaxin and 5-FU, either alone or in combination. MTT and colony formation assay were performed to assess proliferation. Cell cycle progression and apoptosis was completed by flow cytometry. Migration and invasion were also assessed by Transwell and wound-healing assay. Western blot analysis and quantitative RT-PCR were performed to determine expression of signaling molecules. MKN45 cells were also grown as xenografts in nude mice. Mice were treated with repertaxin and 5-FU, and tumor volume and weight, angiogenesis, proliferation and apoptosis were monitored. Combination of repertaxin and 5-FU inhibited MKN45 cell proliferation and increased apoptosis better than either agent alone. Similarly, enhanced effect of the combination was also observed in migration and invasion assays. The improved effect of repertaxin and 5-FU was also observed in vivo, as xenograft models treated with both compounds exhibited significantly decreased tumor volume and increased apoptosis. In conclusion, repertaxin inhibited malignant behavior of human gastric cancer MKN45 cells in vitro and in vivo and enhances efficacy of 5-fluorouracil. These data provide rationale that targeting CXCR1/2 with small molecule inhibitors may enhance chemotherapeutic efficacy for the treatment of gastric cancer.

Increases urinary HMGA1 in serous epithelial ovarian cancer patients.

BACKGROUND Serous epithelial ovarian cancer is the most common variety of ovarian cancer and is currently diagnosed using serum CA-125 levels. HMGA1 a small 10.6-12 kDa protein, has been implicated as a potentially important tumor biomarker and may enter the urinary trace, thus potentially able to serve as a disease biomarker. OBJECTIVE To determine if urine HMGA1 can be detected and potentially serve as a clinical diagnostic biomarkers. METHOD Urine was collected from 20 healthy normal control patients, 20 patients with benign gynecological disease and 55 epithelial ovarian specimens of which 20 exhibited G1/2 ovarian cancer and 35 G3 ovarian cancers. Serum was also collected from 20 healthy normal control patients and 55 serous epithelial ovarian cancers patients. HMGA1 levels were examined via enzyme-linked immunosorbent assay (ELISA) and were reported independently and normalized to urine creatinine levels. Serum CA-125 levels were examined via enzyme assay and the data was analyzed via box and ROC analysis. RESULTS Urine HMGA1 was significantly elevated in serous epithelial ovarian cancer specimen relative to healthy control specimens with G3 specimens exhibiting higher levels than G1-G2 specimens. ROC analysis revealed a high degree of sensitivity and specificity for urine HMGA1 detection in ovarian cancer, with a higher AUC value noted for urine HMGA1 than serum CA-125. Furthermore, urine HMGA1 and serum CA-125 combined AUC indicated that urine HMGA1 is an excellent diagnostic biomarker for serous epithelial ovarian cancer. CONCLUSION Our data indicates that measuring urine HMGA1 may serve as a useful diagnostic tool.

CXCR1 promotes malignant behavior of gastric cancer cells in vitro and in vivo in AKT and ERK1/2 phosphorylation.

CXCR1 is a member of the chemokine receptor family, which was reported to play an important role in several cancers. The present study investigated the influence of CXCR1 stable knockdown or overexpression on the malignant behavior of gastric cancer cells in vitro and in vivo and the potential mechanisms. MKN45 and BGC823 cells were stably transfected with plasmid pYr-1.1-CXCR1-shRNA (knockdown) and pIRES2-ZsGreen1-CXCR1 (overexpression), respectively. Malignant behavior was evaluated in vitro for changes in proliferation by MTT and colony forming assays; cell cycle and apoptosis by flow cytometry; and migration and invasion using transwell and wound-healing assays. Proliferation, cell cycle, apoptosis, migration and invasion-related signaling molecule expression were measured by real-time RT-PCR and western blot analysis. CXCR1 knockdown and overexpressing xenografts were monitored for in vivo tumor growth. Stable knockdown of CXCR1 inhibited MKN45 cell proliferation, migration and invasion, but were reversed in BGC823 cells stably overexpressing CXCR1. In addition, MKN45 cells stably transfected with CXCR1 shRNA inhibited AKT and ERK1/2 phosphorylation, protein expression of cyclin D1, EGFR, VEGF, MMP-9, MMP-2 and Bcl-2, and increased protein expression of Bax and E-cadherin (all P<0.05). In vivo CXCR1-shRNA-MKN45 cells transplanted into nude mice formed smaller tumors than non-transfected or scrambled-shRNA cells (both P<0.05). In contrast BGC823 cells overexpressing CXCR1 formed larger tumors in mice than cells carrying an empty expression plasmid or non-transfected cells (both P<0.05). CXCR1 promoted gastric cancer cell proliferation, migration and invasion. The present study provides preclinical data to support CXCR1 as a novel therapeutic target for gastric cancer.

Expression of miR-224, miR-145, and their putative target ADAM17 in hepatocellular carcinoma

Yuwu Liu1,2,3, Chang Wu4, Ying Wang1,2, Sailan Wen1,2, Junpu Wang1,2, Zhihong Chen1,2, Qiongqiong He1,2, and Deyun Feng1,2* Department of Pathology, Xiangya Hospital, Central South University, Changsha 410008, China Department of Pathology, School of Basic Medical Sciences, Central South University, Changsha 410013, China Department of Morphology, The Institute of Advanced Occupation Technology, Xinjiang Medical University, Urumqi 830011, China Department of Pathology, Shenzhen Sixth People’s Hospital (Nanshan Hospital), Shenzhen 518052, China *Correspondence address: Tel/Fax: þ86-731-82650410; E-mail: DeyunFeng2014@126.com

A highly efficient method for isolating urinary exosomes

In the present study, a highly efficient method, referred to as optimized ultrafiltration (OUF), was developed. This method is effective for exosome purification and also facilitates clinical work involving substantial urinary exosome isolation. In the OUF method, 0.22-µm filters along with a dialysis membrane with a molecular weight cut-off of 10,000 kDa were introduced, in order to remove extracellular microvesicles that were >200 nm and concentrate the supernatant up to 1/50 of the initial volume. The existence, purity and production of the exosomes isolated by OUF and conventional ultracentrifugation (UC) were systematically compared by transmission electron microscopy, western blotting and nanoparticle tracking analysis. In addition, colloidal Coomassie-stained gel and reverse transcription-quantitative polymerase chain reaction were used to investigate the stability and integrity of exosomes isolated by these two protocols. The time required and cost of these two methods in the process of isolating urinary exosomes were also estimated. The results indicated that OUF clearly outperforms UC in quantity, quality and biological stability, and this improved method may have extensive applications in the growing fields of clinical biomarker discovery and exosome research.

Loss of plexin-B3 in hepatocellular carcinoma.

Plexins are the primary receptors of semaphorins, and participate in the majority of intracellular pathways triggered by semaphorins, including the regulation of cell adhesion and the motility of numerous cell types. Recently, several studies have reported that plexins can significantly affect different aspects of cancer cell biology, and the aberrant expression of plexins has been observed in a wide variety of tumor types. However, the expression and role of plexin-B3 in hepatocellular carcinoma (HCC) is yet to be investigated. In the present study, plexin-B3 expression was measured in 14 paired HCC samples and the corresponding adjacent non-cancerous tissue by quantitative polymerase chain reaction and western blot analysis. The results indicated that the mRNA and protein expression levels of plexin-B3 were downregulated in HCC samples when compared with the corresponding adjacent non-cancerous tissue. In order to elucidate the correlation between clinicopathological data and the expression of plexin-B3 in patients with HCC, 84 HCC archived specimens were analyzed by immunohistochemistry (IHC). The IHC results revealed that the protein expression level of plexin-B3 was lower in the HCC samples compared with the corresponding adjacent non-cancerous tissue, and plexin-B3 underexpression was correlated with the patient gender and tumor size. In conclusion, these results indicated that loss of plexin-B3 in HCC may be of predictive value for the occurrence and progression of HCC. Thus, plexin-B3 may be a promising biomarker for the diagnosis and treatment of tumors in the future.