Identification of a Plasma Four-microRNA Panel as Potential Noninvasive Biomarker for Osteosarcoma

<div><p>Background</p><p>Circulating microRNAs (miRNAs) are emerging as promising biomarkers for human cancer. Osteosarcoma is the most common human primary malignant bone tumor in children and young adults. The objective of this study was to investigate whether circulating miRNAs in plasma could be a useful biomarker for detecting osteosarcoma and monitoring tumor removal dynamics.</p><p>Methods</p><p>Plasma samples were obtained from 90 patients before surgery, 50 patients after one month of surgery, and 90 healthy individuals. The study was divided into three steps: First, initial screening of the profiles of circulating miRNAs in pooled plasma samples from healthy controls and pre-operative osteosarcoma patients using a TaqMan low density array (TLDA). Second, evaluation of miRNA concentration in individual plasma samples from 90 pre-operative osteosarcoma patients and 90 healthy controls by a quantitative real time PCR (qRT-PCR) assay. Third, evaluation of miRNA concentration in paired plasma samples from 50 pre- and post-operative osteosarcoma patients by qRT-PCR assay.</p><p>Results</p><p>Four plasma miRNAs including miR-195-5p, miR-199a-3p, miR-320a, and miR-374a-5p were significantly increased in the osteosarcoma patients. Receiver operating characteristics curve analysis of the combined populations demonstrated that the four-miRNA signature could discriminate cases from controls with an area under the curve of 0.9608 (95% CI 0.9307-0.9912). These 4 miRNAs were markedly decreased in the plasma after operation. In addition, circulating miR-195-5p and miR-199a-3p were correlated with metastasis status, while miR-199a-3p and miR-320a were correlated with histological subtype.</p><p>Conclusions</p><p>Our data suggest that altered levels of circulating miRNAs might have great potential to serve as novel, non-invasive biomarkers for osteosarcoma.</p></div

Summary of the clinical and pathological characteristics of osteosarcoma and healthy control plasma samples.

<p>^a Student's t-test.</p><p>^b Two-sided λ² test.</p><p>Summary of the clinical and pathological characteristics of osteosarcoma and healthy control plasma samples.</p

Analysis of plasma miRNA expression profiles by TLDA.

<p>Correlation and scatter plot analysis of miRNAs expression levels in pooled plasma samples from healthy controls and pre-operative osteosarcoma patients (each pooled from 10 individuals).</p

Selected miRNAs as potential biomarkers for osteosarcoma diagnosis.

<p>The ROC curves indicate the ability of the plasma levels of the 4 miRNAs to differentiate the osteosarcoma patients from the control subjects (A-D). (E) The ROC curves for the 4-miRNA profile to distinguish the osteosarcoma samples from the control samples.</p

Relationships between the expression levels of plasma miRNAs and clinical presentations of osteosarcoma patients.

<p>(A) The correlation between the expression levels of miR-195–5p and metastasis status. (B) The correlation between the expression levels of miR-199a-3p and metastasis status. (C) The correlation between the expression levels of miR-320a and histological subtypes. (D) The correlation between the expression levels of miR-199a-3p and histological subtypes.</p

The expression levels of the 4 miRNAs in the plasma of 90 osteosarcoma patients and 90 healthy control individuals, as determined by qRT-PCR analysis.

<p>The expression levels of the 4 miRNAs in the plasma of 90 osteosarcoma patients and 90 healthy control individuals, as determined by qRT-PCR analysis.</p

The dynamic change of plasma miRNAs before and after surgery.

<p>The expression levels of the 4 miRNAs in the patients before and after surgery (A-D). Each point represents the mean of the triplicate samples. (E) The risk score of the 4 miRNAs in the osteosarcoma patients before and after surgery.</p

Hepatitis B e antigen induces the expansion of monocytic myeloid-derived suppressor cells to dampen T-cell function in chronic hepatitis B virus infection

<div><p>Chronic hepatitis B virus (HBV) infection is associated with functionally impaired virus-specific T cell responses. Although the myeloid-derived suppressor cells (MDSCs) are known to play a critical role in impairing antiviral T cell responses, viral factors responsible for the expansion of MDSCs in chronic hepatitis B (CHB) remain obscure. In order to elucidate the mechanism of monocytic MDSCs (mMDSCs) expansion and T cell function suppression during persistent HBV infection, we analyzed the circulation frequency of mMDSCs in 164 CHB patients and 70 healthy donors, and found that the proportion of mMDSCs in HBeAg (+) CHB patients was significantly increased compared to that in HBeAg (-) patients, which positively correlated with the level of HBeAg. Furthermore, exposure of peripheral blood mononuclear cells (PBMCs) isolated from healthy donors to HBeAg led to mMDSCs expansion and significant upregulation of IL-1β, IL-6 and indoleamine-2, 3-dioxygenase (IDO), and depletion of the cytokines abrogated HBeAg-induced mMDSCs expansion. Moreover, HBeAg-induced mMDSCs suppressed the autologous T-cell proliferation <i>in vitro</i>, and the purified mMDSCs from HBeAg (+) subjects markedly reduced the proliferation of CD4⁺ and CD8⁺ T cells and IFN-γ production, which could be efficiently restored by inhibiting IDO. In summary, HBeAg-induced mMDSCs expansion impairs T cell function through IDO pathway and favors the establishment of a persistent HBV infection, suggesting a mechanism behind the development of HBeAg-induced immune tolerance.</p></div