Xiao-Zhong Wang

Exosomes: Novel Biomarkers for Clinical Diagnosis

Exosomes are 30–120 nm endocytic membrane-derived vesicles that participate in cell-to-cell communication and protein and RNA delivery. Exosomes harbor a variety of proteins, nucleic acids, and lipids and are present in many and perhaps all bodily fluids. A significant body of literature has demonstrated that molecular constituents of exosomes, especially exosomal proteins and microRNAs (miRNAs), hold great promise as novel biomarkers for clinical diagnosis. In this minireview, we summarize recent advances in the research of exosomal biomarkers and their potential application in clinical diagnostics. We also provide a brief overview of the formation, function, and isolation of exosomes.

Selective miRNA Expression Profile in Chronic Myeloid Leukemia K562 Cell-derived Exosomes

BACKGROUND Chronic myeloid leukemia (CML) is a myeloproliferative disorder of hematopoietic stem cell scarrying the Philadelphia (Ph) chromosome and an oncogenic BCR-ABL1 fusion gene. The tyrosine kinase inhibitor (TKI) of BCR-ABL1 kinase is a treatment of choice for control of CML. OBJECTIVE Recent studies have demonstrated that miRNAs within exosomes from cancer cells play crucial roles in initiation and progression. This study was performed to assess miRNAs within exosomes of K562 cells. METHODS miRNA microarray analysis of K562 cells and K562 cell-derived exosomes was conducted with the 6th generation miRCURYTM LNA Array (v.16.0). Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were also carried out. GO terms and signaling pathways were categorized into 66 classes (including homophilic cell adhesion, negative regulation of apoptotic process, cell adhesion) and 26 signaling pathways (such as Wnt). RESULTS In exosomes, 49 miRNAs were up regulated as compared to K562 cells, and two of them were further confirmed by quantitative real-time PCR. There are differentially expressed miRNAs between K562 cell derived-exosomes and K562 cells. CONCLUSION Selectively expressed miRNAs in exosomes may promote the development of CML via effects on interactions (e.g. adhesion) of CML cells with their microenvironment.

Aberrant Expression of Splicing Factors in Newly Diagnosed Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is the most common type of blood cancer in adults. Emerging evidence is establishing a connection between AML and aberrant alternative splicing of pre-mRNA, which may result from aberrant expression of splicing factors, the mediators of splicing reactions. Material and Methods: Using quantitative real-time polymerase chain reaction, we measured mRNA expression of 7 splicing factors belonging to the serine/arginine-rich (SR) protein family, SRSF1 (SF2/ASF), SRSF2 (SC35), SRSF3 (SRp20), SRSF4 (SRp75), SRSF5 (SRp40), SRSF6 (SRp55), and SRSF7 (9G8), and 1 non-SR factor, heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1), in peripheral blood mononuclear cells of 26 patients with newly diagnosed AML and 26 healthy controls. In addition, the relationship between splicing factors and the mRNA splicing patterns of the caspase-8 gene (CASP8) was investigated. Results: Compared to healthy controls, the expression of splicing factors was obviously aberrant in newly diagnosed AML patients. The expression of SRSF1, SRSF3 and SRSF4 mRNAs was significantly decreased. Moreover, a significant correlation was observed between several splicing factors and caspase-8 pre-mRNA splicing in AML patients, but not in control subjects. Conclusion: These data suggest that aberrant expression of splicing factors in AML may potentially connect with abnormal expression of oncogenes and be useful for early diagnosis, prognosis, and therapy of AML.

Long non-coding RNA NEAT1 facilitates pancreatic cancer progression through negative modulation of miR-506-3p.

Recently, long non-coding RNAs (lncRNAs) have been shown to have critical regulatory roles in tumourigenesis. Increasing evidence has suggested that lncRNA NEAT1 has been implicated in various types of human cancer. However, the potential biological roles and regulatory mechanisms of NEAT1 in pancreatic cancer (PC) remains unclear. Here, we found that the expression level of NEAT1 was higher in PC tissues compared to the corresponding non-tumor tissues. Besides, our findings indicate that high NEAT1 expression level is closely correlated with tumor progression and poor survival in PC patients. Furthermore, we also found that knockdown of NEAT1 remarkably suppressed cell proliferation by inducing cell cycle arrest and apoptosis promotion in PC cells. Moreover, bioinformatics analysis and luciferase reporter assay revealed that NEAT1 directly bound to the miR-506-3p, which has been reported to act as a tumor suppressor in diverse cancers. Additionally, our results confirmed that the tumor-promoting effects of NEAT1 in PC cells is at least partly through negative modulation of miR-506-3p. Overall, our results suggested that NEAT1 functions as an oncogenic lncRNA in PC, which could be a novel diagnostic and therapeutic target for PC.

Neutrophil-lymphocyte ratio and platelet-lymphocyte ratio are 2 new inflammatory markers associated with pulmonary involvement and disease activity in patients with dermatomyositis

BACKGROUND The neutrophil-lymphocyte ratio (NLR) and platelet-lymphocyte ratio (PLR) have emerged as useful biomarkers to predict systemic inflammation. However, there is no study to investigate the relationship between the biomarkers and dermatomyositis (DM). METHODS Seventy-three newly diagnosed patients with DM and 147 healthy subjects were selected in this retrospective study. We divided the 73 DM patients into 2 groups: 55 without interstitial lung disease (ILD) and 18 with ILD. Complete clinical characteristics were extracted from the medical records of DM patients. The correlations between NLR, PLR, the clinical characteristics and the disease activity were analyzed. RESULTS For DM patients without ILD, the NLR and PLR were significantly higher than those in the control group (both P<0.001). For DM patients with ILD, the NLR and PLR were higher than in DM patients without ILD (P=0.004 and P=0.026, respectively). The NLR was positively correlated with C-reactive protein (CRP) (r=0.543, P<0.001) and the erythrocyte sedimentation rate (ESR) (r=0.513, P=0.001). The global activity scores correlated positively and significantly with NLR, PLR, and CRP (r=0.486, P<0.001; r=0.240, P=0.041; and r=0.343, P=0.003, respectively). Based on the ROC curve, to predict DM patients with ILD, the best cut-off value of the NLR was 3.98 (sensitivity 88.9%, specificity 52.7%, AUC=0.727), and the best cutoff value of PLR was 221.69 (sensitivity 77.8%, specificity 69.1%, AUC=0.722). CONCLUSIONS Both NLR and PLR exhibit favorable diagnostic performance in predicting pulmonary involvement and disease activity in patients with DM. We provide the optimal cut-off values for DM patients with ILD that would maximize the diagnostic efficiency.

Identification of differential expressed PE exosomal miRNA in lung adenocarcinoma, tuberculosis, and other benign lesions.

Abstract Pleural effusion (PE) is a common clinical complication of many pulmonary and systemic diseases, including lung cancer and tuberculosis. Nevertheless, there is no clinical effective biomarker to identify the cause of PE. We attempted to investigate differential expressed exosomal miRNAs in PEs of lung adenocarcinoma (APE), tuberculous (TPE), and other benign lesions (NPE) by using deep sequencing and quantitative polymerase chain reaction (qRT-PCR). As a result, 171 differentiated miRNAs were observed in 3 groups of PEs, and 11 significantly differentiated exosomal miRNAs were validated by qRT-PCR. We identified 9 miRNAs, including miR-205-5p, miR-483-5p, miR-375, miR-200c-3p, miR-429, miR-200b-3p, miR-200a-3p, miR-203a-3p, and miR-141-3p which were preferentially represented in exosomes derived from APE when compared with TPE or NPE, while 3 miRNAs, including miR-148a-3p, miR-451a, and miR-150-5p, were differentially expressed between TPE and NPE. These different miRNAs profiles may hold promise as biomarkers for differential diagnosis of PEs with more validation based on larger cohorts.

Alternative splicing of apoptosis-related genes in imatinib-treated K562 cells identified by exon array analysis

Imatinib is the therapeutic standard for newly diagnosed patients with chronic myeloid leukemia (CML). In these patients, imatinib has been shown to induce an apoptotic response specifically in cells expressing the oncogenic fusion protein BCR-ABL. Previous studies in our lab revealed that imatinib-induced apoptosis in K562 cells involves a shift in production of Bcl-x splice isoforms towards the pro-apoptotic Bcl-xS splice variant. Here, we report the findings from our subsequent study to identify other apoptosis-related genes that are differentially spliced in response to imatinib treatment. Gene expression profiling of imatinib-treated K562 cells was performed by the Affymetrix GeneChip® Human Exon 1.0 ST array, and differences in exon-level expression and alternative splicing were analyzed using the easyExon software. Detailed analysis by reverse transcription-PCR (RT-PCR) and sequencing of key genes confirmed the experimental results of the exon array. Our results suggest that imatinib treatment of K562 cells causes a transcriptional shift towards alternative splicing in a large number of apoptotic genes. The present study provides insight into the molecular character of apoptotic leukemia cells and may help to improve the mechanism of imatinib therapy in patients with CML.

Exosomes derived from imatinib-resistant chronic myeloid leukemia cells mediate a horizontal transfer of drug-resistant trait by delivering miR-365

&NA; Chronic myeloid leukemia (CML) is a malignant disorder of hematopoietic stem/progenitor cells. Majority of patients can be effectively treated with tyrosine kinase inhibitors (TKIs) such as imatinib, but a portion of patients will develop drug resistance. Accumulated evidences have identified exosomes in cancer as promoters of tumor progression. Herein, we found that exosomes derived from imatinib resistant CML cells can be internalized into sensitive CML cells and confer drug‐resistance traits. We also demonstrated a significant higher level of miR‐365 in exosomes derived from drug‐resistant CML cells compared with those from sensitive ones using microarray and qRT‐PCR. The imatinib sensitive CML cells transfected with pre‐miR‐365 displayed lower chemosensitivity and apoptosis rate compared with controls. We further confirmed that exosomal transfer of miR‐365 induced drug resistance by inhibiting expression of pro‐apoptosis protein in sensitive CML cells. In conclusion, our study reveals that exosomes mediate a horizontal transfer of drug‐resistant trait in chronic myeloid leukemia cell by delivering miR‐365.

Prognostic value of a novel FPR biomarker in patients with surgical stage II and III gastric cancer

Background Inflammation and nutrition are two main causes contributing to progression of gastric cancer (GC), and inflammatory biomarker may be presented as its valuable prognostic factor. Thus, this study was carried out to investigate the prognostic significance of preoperative circulating albumin/fibrinogen ratio (AFR), fibrinogen/pre-Albumin ratio (FPR), fibrinogen (Fib), albumin (Alb) and pre-Albumin (pAlb) in surgical GC. Materials and Methods Three hundred and sixty surgical stage II and III GC patients from June 2011 to December 2013 were enrolled in this retrospective study. X-tile software, Kaplan–Meier curve and Cox regression model were used to evaluate the prognostic role of them. A predictive nomogram was established to predict prognosis of overall survival (OS), and its accuracy was assessed by concordance index (c-index). Results Decreased Alb, pAlb, AFR and elevated FPR were significantly associated with shorter OS. FPR was identified as the most effective prognostic factor to predict 3-year’s OS by time-dependent ROC analysis. A long survival was observed in patients with low level of FPR and the prognosis of stage III FPR-low GC patients undergoing chemotherapy was significantly superior to the patients without the treatment (P=0.002). However, no difference of survival was examined in stage II subgroups stratified by FPR and high FRP of stage III patients with or not the treatment of chemotherapy. C-index of nomogram containing FPR (c-index=0.756) was high in comparison with the nomogram without FPR (c-index =0.748). Conclusion Preoperative FPR might be a feasible prognostic biomarker in surgical stage II and III GC and it could precisely distinguish stage III patients who appeared to obviously benefit from adjuvant chemotherapy. Meanwhile established nomogram based on clinical parameters and FPR could improve its predictive efficacy.

Differential expression and alternative splicing of cell cycle genes in imatinib-treated K562 cells

Cancer progression often involves the disorder of the cell cycle, and a number of effective chemotherapeutic drugs have been shown to induce cell cycle arrest. The purpose of this study was to comprehensively investigate the effects of imatinib on the expression profile of cell cycle genes in the chronic myeloid leukemia (CML) K562 cell line. In addition, we also investigated alternative splicing of the cell cycle genes affected by imatinib, since an important relationship has been shown to exist between RNA splicing and cell cycle progression. Exon array analysis was performed using total RNA purified from normal and imatinib-treated K562 cells. We identified 185 differentially expressed genes and 277 alternative splicing events between the two cell groups. A detailed analysis by reverse transcription-PCR (RT-PCR) of key genes confirmed the experimental results of the exon array. These results suggested that treatment of K562 cells with imatinib shifts the expression and alternative splicing profiles of several cell cycle-related genes. Importantly, these findings may help improve imatinib treatment strategies in patients with CML and may be useful for imatinib resistance research and CML drug development.