Xinhua Wu

Cell-Free circulating DNA: a new biomarker for the acute coronary syndrome.

Background: In recent studies, concentrations of cell-free circulating DNA (cf-DNA) have been correlated with clinical characteristics and prognosis in several diseases. The relationship between cf-DNA concentrations and the acute coronary syndrome (ACS) remains unknown. Moreover, no data are available for the detection cf-DNA in ACS by a branched DNA (bDNA)-based Alu assay. The aim of the present study was to investigate cf-DNA concentrations in ACS and their relationship with clinical features. Methods: Plasma cf-DNA concentrations of 137 ACS patients at diagnosis, of 60 healthy individuals and of 13 patients with stable angina (SA) were determined using a bDNA-based Alu assay. Results: ACS patients (median 2,285.0, interquartile range 916.4–4,857.3 ng/ml), especially in ST-segment elevation myocardial infarction patients (median 5,745.4, interquartile range 4,013.5–8,643.9 ng/ml), showed a significant increase in plasma cf-DNA concentrations compared with controls (healthy controls: median 118.3, interquartile range 81.1–221.1 ng/ml; SA patients: median 202.3, interquartile range 112.7–256.1 ng/ml) using a bDNA-based Alu assay. Moreover, we found positive correlations between cf-DNA and Gensini scoring and GRACE (Global Registry of Acute Coronary Events) scoring in ACS. Conclusion: cf-DNA may be a valuable marker for diagnosing and predicting the severity of coronary artery lesions and risk stratification in ACS.

A sensitive method to quantify human cell-free circulating DNA in blood: relevance to myocardial infarction screening.

OBJECTIVES Human cell-free circulating DNA (cf-DNA) derived mainly from cell apoptosis and necrosis can be measured by a variety of laboratory techniques, but almost all of these methods require sample preparation. We have developed a branched DNA (bDNA)-based Alu assay for quantifying cf-DNA in myocardial infarction (MI) patients. DESIGN AND METHODS A total of 82 individuals were included in the study; 22 MI and 60 normal controls. cf-DNA was quantified using a bDNA-based Alu assay. RESULTS cf-DNA was higher in serum compared to plasma and there was a difference between genders. cf-DNA was significantly higher in MI patients compared to the controls. There was no correlation between cf-DNA and creatine kinase-MB (CK-MB), troponin I (cTnI) or myoglobin (MYO). In serial specimens, cf-DNA was sensitive and peaked earlier than cTnI. CONCLUSIONS The bDNA-based Alu assay is a novel method for quantifying human cf-DNA. Increased cf-DNA in MI patients might complement cTnI, CK-MB and MYO in a multiple marker format.

Prognostic Value of Free DNA Quantification in Serum and Cerebrospinal Fluid in Glioma Patients

Unlike uniformly truncated DNA released from apoptotic nondiseased cells, free DNA released from dead tumor cells varies in size. Free DNA has been considered as a candidate biomarker for malignant tumors. We obtained serum samples from 70 patients with glioma and 22 healthy volunteers as control and cerebrospinal fluid (CSF) samples from 20 patients with glioma and eight nonneoplastic controls with hydrocephalus or arachnoid cyst and performed preoperative analysis of free DNA concentration and integrity by a quantitative polymerase chain reaction. With two primers sets amplifying short and long free DNA fragments (ALU115 and ALU247), free DNA integrity was determined by ratio of the concentration of ALU247 over ALU115 (ALU247/115). Our results indicate that free DNA integrity and the ratio of long fragments to short fragments may be a useful diagnostic assay for glioma. In summary, the CSF-free DNA concentration and integrity may serve as a new marker for the diagnosis of glioma.

miR-202 expression concentration and its clinical significance in the serum of multiple myeloma patients

Objectives To explore microRNA-202 (miR-202) expression in serum of patients with multiple myeloma (MM), and investigate correlations between serum miR-202 expression and the development and prognosis of MM. Design and methods RNA was extracted from serum by QIAGEN miRNeasy Mini kit. Reverse transcription was performed with specific stem-loop primers. SYBR Green I QF-PCR was applied to detect the relative expression of miR-202 in 40 MM patients and 30 healthy controls. The linearity, specificity and reproducibility were evaluated. In addition, correlations between the relative expression of serum miR-202 and the concentrations of lactic acid dehydrogenase (LDH), β2M, λ light chain and κ light chain were assessed. Results The relative expression of miR-202 in MM patients 1.503 (0.161–9.831) was significantly higher than that in healthy controls 1.000 (0.105–3.046) (P < 0.01) and was significantly correlated with serum β2M and κ light chain concentrations (r = 0.366, P = 0.0305; r = 0.358, P = 0.0348). Conclusions The relative expression of serum miR-202 in MM patients was significantly higher than that in healthy controls, and therefore it may prove to be useful in the auxiliary diagnosis of MM.

Alu-based cell-free DNA: a potential complementary biomarker for diagnosis of colorectal cancer.

OBJECTIVES Many patients with colorectal cancer (CRC) present with regional or widespread metastasis, partially reflecting limitations of the current screening programs. This study was aimed to find a complementary marker that can improve the diagnostic accuracy. DESIGN AND METHODS Concentrations of cell-free DNA based on Alu (Alu-based CFD) in 31 unselected CRC patients, 30 intestinal polyp patients and 92 healthy individuals were detected by branch DNA (bDNA). Concentrations of carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) were detected by ARCHITECT assay. RESULTS There was significant difference in concentrations of CFD between CRC and intestinal polyp patients or healthy individuals (P<0.0001). There was no statistically significant difference in CFD in different subgroups of CRC patients with respect to gender, age, tumor site and pathologic stage, suggesting that CFD might be an independent marker relative to CEA and CA19-9. There was a significant correlation between pathologic stage and CEA or CA19-9. Although no significant correlation was observed between pathologic stage and CFD, CFD (the area under the receiver operating characteristic curve (AUC)=0.904) seemed to be a better indicator to distinguish CRC patients from intestinal polyp patients as compared with CEA (AUC=0.681) or CA19-9 (AUC=0.651). CFD was more accurate than CEA or CA19-9 in diagnosing CRC. CONCLUSIONS Combination of CFD, CEA and CA19-9 may be a better option for the diagnosis of CRC than any of them used alone. Discrimination CRC from intestinal polyp patients with CFD and staging with CEA and CA19-9 may substantially improve the accuracy CRC diagnosis.

Upregulated lncRNA-PCAT1 is closely related to clinical diagnosis of multiple myeloma as a predictive biomarker in serum.

OBJECTIVE The purpose of this study was to explore serum PCAT-1 expression in multiple myeloma (MM) and examine the potential usefulness of this molecule as a biomarker for diagnosis in MM. METHODS Serum samples were collected from 60 newly diagnosed untreated MM patients, and 48 healthy controls. Serum PCAT-1 expression levels were detected by RT-qPCR. In addition, correlations between the relative expression of serum PCAT-1 and the concentrations of lactic acid dehydrogenase (LDH), β2M, λ light chain and κ light chain were assessed. RESULTS It was found that the relative expression of serum PCAT-1 in MM patients (81.02 ± 136.9) was higher than that in healthy controls (3.17 ± 5.75) (U= 307.0, P< 0.0001) and was significantly correlated with β2M concentration (r= 0.461, P= 0.0002), but not with LDH, κ light and λ light chain concentration (r= 0.061, P= 0.641; r= 0.007, P= 0.956; r=-0.090, P= 0.499 respectively). Additionally, it was significantly correlated with different isotype of MM (H= 7.464, P= 0.024). The AUC of the ROC curve of serum PCAT-1 was 0.892 (95% CI 0.833-0.950), which was higher than other markers. Combining PCAT-1 and β2M together, the sensitivity was highest compared with other markers alone, or combined. CONCLUSION The expression levels of serum PCAT-1 in MM patients were significantly higher than that in healthy controls, suggesting that it may be useful in the auxiliary diagnosis of MM.

miRNA-202 in bone marrow stromal cells affects the growth and adhesion of multiple myeloma cells by regulating B cell-activating factor

Abstract Bone marrow stromal cells (BMSCs) up-regulate B cell-activating factor (BAFF) in multiple myeloma. Increasing experimental evidence has shown that microRNAs play a causal role in hematology tumorigenesis. In this study, we characterized the role of miR-202 in regulating the expression of BAFF in BMSCs. It was found that expressions of BAFF mRNA and protein were increased in BMSCs treated with miR-202 inhibitor. The growth rate of miR-202 mimics transfection cells was significantly lower than that of non-transfected cells. The expression of Bcl-2 protein was down-regulated, and Bax protein was up-regulated after miR-202 mimics transfection. Over-expression of miR-202 in BMSCs rendered MM cells more sensitive to bortezomib. More significantly, the regulatory effect of miR-202 could inhibit the activation of NF-κB pathway in BMSCs. These results suggest that miR-202 functions as a modulator that can negatively regulate BAFF by inhibiting MM cell survival, growth, and adhesion in the bone marrow microenvironment.

BLyS expression and JNK activation may form a feedback loop to promote survival and proliferation of multiple myeloma cells.

B-Lymphocyte stimulator (BLyS), a member of tumor necrosis factor superfamily, is a potent co-activator of B cells in vitro, and in vivo induces B cell proliferation and immunoglobulin secretion. Multiple myeloma (MM) is an incurable malignancy of terminally differentiated B cells (plasma cells). Previous studies have well ascertained that BLyS plays an important contributory role in the pathogenesis and propagation of multiple myeloma by virtue of its ability to promote B cell survival, expansion, and differentiation. However, the intracellular signaling of BLyS in human MM cells remains undefined. This study was designed to see whether there was interaction between MAPK signaling pathway and BLyS expression. It was found that the active protein p-JNK was expressed in KM3, U266 and PBMCs of MM patients, and that the expression of BLyS could be changed by JNK pathway activator and inhibitor. In addition, recombinant BLyS activated JNK pathway, while BLyS siRNA treatment inhibited the activation of JNK pathway. The level of BLyS expression and the activation of JNK pathway were positively correlated. These findings suggest that JNK activation and BLyS expression in MM cells may form a positive feedback loop that promotes the survival and proliferation of MM cells, and these may shed some light on the pathogenesis and treatment of MM.

Study on the Association Between miRNA-202 Expression and Drug Sensitivity in Multiple Myeloma Cells.

An increasing amount of experimental evidence has shown that miRNAs play a causal role in hematologic tumorigenesis. In this study, we characterized the role of miR-202 in multiple myeloma (MM) drug sensitivity. The potential binding site of miR-202 and B cell-activating factor (BAFF) was confirmed by luciferase reporter assay. MM cells were transfected with miR-202 mimics and inhibitor. Cells growth was measured by WST-1 cell proliferation assay and Annexin V-FLUOS apoptosis assay. BAFF and miR-202 mRNA levels were measured by real-time PCR. Meanwhile, BAFF, Bcl-2 family survival proteins and MAPK pathway proteins were measured by Western blot. It was found that miR-202 was functioned as a modulator of BAFF expression. miR-202 over-expression sensitized MM cells to bortezomib (Bort) but less to Thalidomide (Thal) and dexamethasone (Dex). miR-202 mimics in combination with Bort inhibited MM cell survival more effectively as compared with Bort treatment alone. Our study also provided experimental evidence that JNK/SAPK signaling pathway was involved in the regulatory effect of miR-202 on drug resistance of MM cells. These results suggest that the regulatory mechanism of miR-202 expression may be a promising target for fine-tuning anti-myeloma therapy.

Binding of B-cell maturation antigen to B-cell activating factor induces survival of multiple myeloma cells by activating Akt and JNK signaling pathways

B‐cell maturation antigen (BCMA) is expressed on normal and malignant plasma cells and represents a potential target for therapeutic intervention. In this study, we characterized the mechanism underlying the protein kinase B (Akt) and c‐Jun N‐terminal kinase (JNK) pathways and BCMA interactions in regulating multiple myeloma (MM) cell survival. It was found that the expression levels of B cell‐activating factor (BAFF) and BCMA were increased in MM cells as compared with those in normal controls. The proliferation of U266 cells was induced by recombinant human BAFF (rhBAFF) and could also be decreased by BCMA siRNA. The expression of Bcl‐2 protein was up‐regulated, and Bax protein was down‐regulated after rhBAFF treatment, which could be reversed by BCMA siRNA. Similarly, the protein p‐JNK and p‐Akt were activated by rhBAFF and could be changed by BCMA siRNA. In addition, the BCMA mRNA and protein expression levels were decreased after treatment with Akt and JNK pathway inhibitors. These results suggest that Akt and JNK pathways are involved in the regulation of BCMA. A novel BAFF/BCMA signalling pathway in MM may be a new therapeutic target for MM. Copyright