Quantitative Proteomic Analysis of Plasma Exosomes to Identify the Candidate Biomarker of Imatinib Resistance in Chronic Myeloid Leukemia Patients.

BACKGROUND: Imatinib (IM), a tyrosine kinase inhibitor (TKI), has markedly improved the survival and life quality of chronic myeloid leukemia (CML) patients. However, the lack of specific biomarkers for IM resistance remains a serious clinical challenge. Recently, growing evidence has suggested that exosome-harbored proteins were involved in tumor drug resistance and could be novel biomarkers for the diagnosis and drug sensitivity prediction of cancer. Therefore, we aimed to investigate the proteomic profile of plasma exosomes derived from CML patients to identify ideal biomarkers for IM resistance. METHODS: We extracted exosomes from pooled plasma samples of 9 imatinib-resistant CML patients and 9 imatinib-sensitive CML patients by ultracentrifugation. Then, we identified the expression levels of exosomal proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS) based label free quantification. Bioinformatics analyses were used to analyze the proteomic data. Finally, the western blot (WB) and parallel reaction monitoring (PRM) analyses were applied to validate the candidate proteins. RESULTS: A total of 2812 proteins were identified in plasma exosomes from imatinib-resistant and imatinib-sensitive CML patients, including 279 differentially expressed proteins (DEPs) with restricted criteria (fold change≥1.5 or ≤0.667, p<0.05). Compared with imatinib-sensitive CML patients, 151 proteins were up-regulated and 128 proteins were down-regulated. Bioinformatics analyses revealed that the main function of the upregulated proteins was regulation of protein synthesis, while the downregulated proteins were mainly involved in lipid metabolism. The top 20 hub genes were obtained using STRING and Cytoscape, most of which were components of ribosomes. Moreover, we found that RPL13 and RPL14 exhibited exceptional upregulation in imatinib-resistant CML patients, which were further confirmed by PRM and WB. CONCLUSION: Proteomic analysis of plasma exosomes provides new ideas and important information for the study of IM resistance in CML. Especially the exosomal proteins (RPL13 and RPL14), which may have great potential as biomarkers of IM resistance.

Correction of Bcl-x splicing improves responses to imatinib in chronic myeloid leukaemia cells and mouse models.

Imatinib mesylate (IM) resistance has become a major clinical problem for chronic myeloid leukaemia (CML). It is known that Bcl-x splicing is deregulated and is involved in multiple malignant cancer initiation and chemotherapy resistance, including CML. The aim of the present study was to correct the abnormal splicing of Bcl-x in CML and investigate the subsequent malignant phenotype changes, especially response to IM. The aberrant Bcl-x splicing in CML cells was effectively restored using vivo-Morpholino Antisense Oligomer (vMO). CCK-8 cell viability assay and flow cytometry showed that restoring of Bcl-x splicing increases IM-induced growth inhibition and apoptosis of K562 cells. Moreover, a more significant similar phenomenon was observed in imatinib-resistant CML cell lines K562/G01. Finally, establishment of CML xenograft model had also proved that correcting Bcl-x splicing in vivo can also enhance the anti-tumor effect of IM. Our findings suggest that vMO co-operating with IM can effectively increase the sensitivity of CML cells to IM both in vitro and in vivo, and Bcl-x splicing could become good candidates for chemotherapy-sensitized target in IM-resistant CML.

K562 cell-derived exosomes suppress the adhesive function of bone marrow mesenchymal stem cells via delivery of miR-711.

A failure of bone marrow mesenchymal stem cells (BM-MSCs) to adhere to hematopoietic cells is an essential cause of the progression of chronic myelogenous leukemia and is also a cause of failure of bone marrow (BM) transplantation, but the exact mechanisms of this have not been fully elucidated. Recent studies have indicated that microRNAs (miRNAs) are contained in leukemia-derived exosomes and are involved in modulating the BM microenvironment. In this study, we found that K562 cell-derived exosomes transfer miR-711 to BM-MSCs and suppress the adhesive function of BM-MSCs. Using qRT-PCR, we also confirmed a significantly higher level of miR-711 in exosomes derived from K562 cells than in exosomes derived from parental cells. The BM-MSCs co-cultured with exosomes derived from K562 cells showed a lower adhesion rate than did controls. We further demonstrated that exosomal transfer of miR-711 induced decreased adhesive abilities by inhibiting expression of adhesion molecule CD44 in BM-MSCs. In conclusion, our study reveals that K562 cell-derived exosomal miR-711 can be transferred to BM-MSCs and weaken adhesive abilities by silencing the expression of the adhesion molecule CD44.

Preoperative fibrinogen to prealbumin ratio as a novel predictor for clinical outcome of hepatocellular carcinoma.

AIM: To investigate prognostic value of preoperative inflammatory biomarkers in hepatocellular carcinoma (HCC). PATIENTS & METHODS: Preoperative circulating fibrinogen, prealbumin, fibrinogen to prealbumin ratio (FPR), neutrophil to lymphocyte ratio, derived neutrophil to lymphocyte ratio, lymphocyte to monocyte ratio, platelet to lymphocyte ratio were detected and calculated in 230 HCC patients. X-tile software, Kaplan-Meier curve, Cox regression, time-dependent receiver-operating characteristic were used to explored prognostic roles of them in HCC. RESULTS: Multivariate Cox regression showed that high FPR was significantly associated with decreased recurrence-free survival (p = 0.034) and overall survival (p < 0.001) within HCC patients. FPR generated the largest area under curve of time-dependent receiver-operating characteristic comparing to the other biomarkers. Overall survival of HCC patients receiving chemotherapy was superior to the cases without receiving chemotherapy only in high FPR subgroup (p = 0.028). CONCLUSION: Preoperative FPR was superior to other biomarkers to independently predict survival of HCC patients, and it could identify the patients who could benefit from adjuvant chemotherapy.

The predictive and prognostic role of a novel ADS score in esophageal squamous cell carcinoma patients undergoing esophagectomy.

BACKGROUND: Chronic inflammation is deemed to play a significant effect on initiation and progression of esophageal squamous cell carcinoma (ESCC). In current study, we investigated the prognostic and predictive role of albumin (Alb) to fibrinogen (Fib) ratio (AFR) and a novel AFR-Alb-derived neutrophil/lymphocyte ratio (dNLR) score (ADS) in ESCC patients undergoing esophagectomy and compared them with Fib, Alb, neutrophil to lymphocyte ratio (NLR), dNLR, platelet to lymphocyte ratio (PLR) and lymphocyte to monocyte ratio (LMR). MATERIALS AND METHODS: A total of 153 clinical confirmed ESCC patients undergoing esophagectomy between January 2011 and December 2013 were included in present study. We detected preoperative Alb, Fib and neutrophil, monocyte, lymphocyte and platelet count, and obtained overall survival (OS) by 3 years' follow-up in the cases. X-tile software, Kaplan-Meier curve, Cox regression and predicted nomogram were used to evaluate the predictive and prognostic role of them in ESCC patients. RESULTS: The optimal cut-off values of Fib, Alb, AFR, NLR, dNLR, PLR and LMR were 3.2 mg/dL, 38.2 g/L, 9.3, 2.1, 4.3, 145.9 and 2.3, respectively. High levels of Fib [(adjusted hazard ratio (HR) = 2.148, 95% confidential interval (CI) (1.229-3.753)], dNLR (adjusted HR = 2.338, 95% CI 1.626-5.308) and PLR (adjusted HR = 1.964, 95% CI 1.129-3.415) as well as low AFR (adjusted HR = 2.381, 95% CI 1.152-4.926) and Alb (adjusted HR = 2.398, 95% CI 1.342-4.273) were significantly associated with decreased OS in ESCC patients. The survival predictive areas under the time-dependent receiver operating characteristics curve of AFR, dNLR and Alb were higher than Fib and PLR, respectively. High ADS score was significantly associated with short 3 years' OS of ESCC patients (adjusted HR = 2.94, 95% CI 1.70-5.08). Moreover, OS of ESCC patients receiving adjuvant radio-chemotherapy was longer than those without the treatment in high ADS score subgroup (p = 0.001), however, no significant survival difference was observed in the patients with or without treatment radio-chemotherapy (p = 0.297). Additionally, a significant difference was observed in c-index values of the nomograms including or without ADS (0.720 vs. 0.670, p < 0.05). CONCLUSIONS: Preoperative ADS was a prospective biomarker to predict clinical efficacy of adjuvant radio-chemotherapy and clinical prognosis of ESCC patients undergoing esophagectomy, and the score could apparently improve predicted efficacy of the nomogram.

Two new inflammatory markers associated with disease activity score-28 in patients with rheumatoid arthritis: Albumin to fibrinogen ratio and C-reactive protein to albumin ratio.

BACKGROUND: The albumin to fibrinogen ratio (AFR) and C-reactive protein to albumin ratio (CAR) have emerged as useful biomarkers to predict systemic inflammation. The aim here is to investigate the relation between AFR/CAR and Disease Activity Score of 28 joints (DAS 28) in rheumatoid arthritis (RA). METHODS: This retrospective study included 160 patients with RA and 159 healthy controls. We divided the RA patients into two groups according to the DAS 28-ESR score. Group 1 included 40 patients with a score of lower than 2.6 (patients in remission) and Group 2 included 120 patients with a score of 2.6 or higher (patients with active disease). The correlations between AFR, CAR and the disease activity were analyzed. RESULTS: For RA patients, the AFR was lower than those in the control group (P < 0.001). Patients in group 2 had higher CAR than those in group 1 (P < 0.001). The AFR was lower in group 2 than that in group 1. A positively correlation was observed between DAS 28-ESR score and CAR (r = 0.645, P < 0.001), while the correlation between DAS 28-ESR and AFR (r = -0.836, P < 0.001) was negative. AFR was related with decreased risk of RA disease activity (EXP (B) = 0.33, 95% CI (0.21-0.53), P < 0.001). CONCLUSIONS: AFR and CAR are two novel inflammatory markers for monitoring disease activity in patients with RA.

Differential expression of urinary exosomal microRNAs in IgA nephropathy.

BACKGROUND: Immunoglobulin A nephropathy (IgAN) is the most common type of primary glomerulonephritis in the world. Reliable biomarkers are required for the non-invasive diagnosis and monitoring of IgAN. This study aims to investigate the difference in urinary exosomal microRNA (miRNA) expression profiles between patients with IgA nephropathy (IgAN) and healthy controls, which may provide clues to identify novel potential non-invasive miRNA biomarkers for renal diseases. METHODS: Urine samples were collected from eighteen healthy controls and eighteen patients with IgAN. Differential centrifugation was performed to isolate exosomes from urine samples. High-throughput sequencing and real-time quantitative polymerase chain reaction (RT-qPCR) were sequentially used to screen and further validate miRNA expression profiles in urinary exosomes of patients with IgAN in two independent cohorts. RESULTS: Urinary exosomes were successfully isolated to obtain exosomal miRNAs. MiR-215-5p and miR-378i were significantly upregulated in urinary exosomes of patients with IgAN compared with healthy controls (P<.01), while miR-29c and miR-205-5p were significantly downregulated (P<.05). MiR-215-5p, miR-378i, miR-365b-3p and miR-135b-5p were found to have altered expression in patients with IgAN from validation cohorts, which was consistent with the high-throughput sequencing analysis. CONCLUSION: This study suggests that there is a significant difference in urinary exosomal miRNA profiles between patients with IgAN and healthy controls. These exosomal miRNAs, such as miR-29c, miR-146a and miR-205 may potentially serve as novel non-invasive biomarkers for IgAN.

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

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.

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

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.

Association Between TNF-α -308G/A Polymorphism and Risk of Immune Thrombocytopenia: A Meta-Analysis.

OBJECTIVE: Previous studies have investigated the association between tumor necrosis factor-alpha (TNF-α) -308G/A polymorphism and risk of immune thrombocytopenia (ITP), but the reported results have been inconsistent. Thus, a systematic meta-analysis was performed to resolve this discrepancy. METHODS: Electronic databases and the cited references of the obtained published articles were manually searched. Quality assessment of each study was conducted using the Newcastle-Ottawa Scale (NOS). All case-control studies were used to assess the strength of the association. Statistical analysis was performed using Stata version 12.0. RESULTS: Eight high-quality studies, including 947 patients and 1911 controls, were selected for the final meta-analysis. There was no significant association between TNF-α -308G/A polymorphism and ITP in overall and Asian populations. However, a significant positive association was observed between them in the dominant genetic model (AA+AG versus GG) in the Caucasian population (OR = 1.35, 95% confidence interval [CI]: 1.07-1.71, PH = 0.173). CONCLUSIONS: Our finding suggested that TNF-α -308G/A might be involved in development of ITP in the Caucasian population, but not in the Asian population. Among Caucasians the A allele (AA+AG) was associated with ITP. However, larger-scale studies are required to confirm our findings.

Differential miRNA expression in pleural effusions derived from extracellular vesicles of patients with lung cancer, pulmonary tuberculosis, or pneumonia.

MicroRNAs (miRNAs) have been found to play important regulatory roles in various physiological and pathological processes. MiRNAs also exhibit high stability and are present at high concentrations in human bodily fluids. Consequently, miRNAs may represent attractive and novel diagnostic biomarkers for certain clinical conditions. Recently, the capacity for extracellular vesicles, including microvesicles and exosomes, to carry miRNAs that participate in cell-to-cell communication has been described. In the present study, the miRNA expression patterns for three kinds of pleural effusions that were obtained from patients with pneumonia (group A), pulmonary tuberculosis (group B), and lung cancer (group C) were detected with high-throughput sequencing. When the expression levels of these miRNAs were compared among the three groups, three differentially expressed miRNAs were detected between groups A and B, while 27 differentially expressed miRNAs were detected between groups A and C. Notably, miR-378i was significantly elevated only in group B, while miR-205-5p and miR-200b were markedly increased only in group C (p < 0.01). Further studies are needed to confirm whether these differentially expressed miRNAs may serve as prospective diagnostic markers for pulmonary diseases.

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.

Mechanism of alternative splicing and its regulation.

Alternative splicing of precursor mRNA is an essential mechanism to increase the complexity of gene expression, and it plays an important role in cellular differentiation and organism development. Regulation of alternative splicing is a complicated process in which numerous interacting components are at work, including cis-acting elements and trans-acting factors, and is further guided by the functional coupling between transcription and splicing. Additional molecular features, such as chromatin structure, RNA structure and alternative transcription initiation or alternative transcription termination, collaborate with these basic components to generate the protein diversity due to alternative splicing. All these factors contributing to this one fundamental biological process add up to a mechanism that is critical to the proper functioning of cells. Any corruption of the process may lead to disruption of normal cellular function and the eventuality of disease. Cancer is one of those diseases, where alternative splicing may be the basis for the identification of novel diagnostic and prognostic biomarkers, as well as new strategies for therapy. Thus, an in-depth understanding of alternative splicing regulation has the potential not only to elucidate fundamental biological principles, but to provide solutions for various diseases.