Comprehensive microRNA Analysis Identifies miR-24 and miR-125a-5p as Plasma Biomarkers for Rheumatoid Arthritis

MicroRNAs (miRNAs) are present in human plasma and known as a non-invasive biomarker for cancer detection. Our study was designed to identify plasma miRNAs specific for rheumatoid arthritis (RA) by a comprehensive array approach. We performed a systematic, array-based miRNA analysis on plasma samples from three RA patients and three healthy controls (HCs). Plasma miRNAs with more than four times change or with significant (P<0.05) change in expression, or detectable only in RA plasma, were confirmed with plasma from eight RA patients and eight HCs using real-time quantitative PCR. Consistently detectable miRNAs that were significantly different between RA patients and HCs were chosen for further validation with 102 RA patients and 104 HCs. The area under curves (AUC) were calculated after plotting the receiver operating characteristic (ROC) curves. To determine if these miRNAs are specific for RA, the concentrations of these miRNAs were analyzed in 24 patients with osteoarthritis (OA), and 11 patients with systemic lupus erythematosus (SLE). The array analysis and the subsequent confirmation in larger patient cohort identified significant alterations in plasma levels of seven miRNAs. The highest AUC was found for miR-125a-5p, followed in order by miR-24 and miR-26a. Multivariable logistic regression analysis showed that miR-24, miR-30a-5p, and miR-125a-5p were crucial factors for making detection model of RA and provided a formula for Estimated Probability of RA by plasma MiRNA (ePRAM), employing miR-24, miR-30a-5p and miR-125a-5p, which showed increased diagnostic accuracy (AUC: 0.89). The level of miR-24, miR-125a-5p, and ePRAM in OA and SLE patients were lower than that in RA. There was no significant difference in detection for anti-citrullinated protein antibody (ACPA)-positive and ACPA-negative RA patients. These results suggest that the plasma concentrations of miR-24 and miR-125a-5p, and ePRAM are potential diagnostic markers of RA even if patients were ACPA-negative.     

Antileukemic Activity of hsa-miR-203a-5p by Limiting Glutathione Metabolism in Imatinib-Resistant K562 Cells

Imatinib has been the first and most successful tyrosine kinase inhibitor (TKI) for chronic myeloid leukemia (CML), but many patients develop resistance to it after a satisfactory response. Glutathione (GSH) metabolism is thought to be one of the factors causing the emergence of imatinib resistance. Since hsa-miR-203a-5p was found to downregulate Bcr-Abl1 oncogene and also a link between this oncogene and GSH metabolism is reported, the present study aimed to investigate whether hsa-miR-203a-5p could overcome imatinib resistance by targeting GSH metabolism in imatinib-resistant CML cells. After the development of imatinib-resistant K562 (IR-K562) cells by gradually exposing K562 (C) cells to increasing doses of imatinib, resistant cells were transfected with hsa-miR-203a-5p (R+203). Thereafter, cell lysates from various K562 cell sets (imatinib-sensitive, imatinib-resistant, and miR-transfected imatinib-resistant K562 cells) were used for GC-MS-based metabolic profiling. L-alanine, 5-oxoproline (also known as pyroglutamic acid), L-glutamic acid, glycine, and phosphoric acid (Pi)—five metabolites from our data, matched with the enumerated 28 metabolites of the MetaboAnalyst 5.0 for the GSH metabolism. All of these metabolites were present in higher concentrations in IR-K562 cells, but intriguingly, they were all reduced in R+203 and equated to imatinib-sensitive K562 cells (C). Concludingly, the identified metabolites associated with GSH metabolism could be used as diagnostic markers.     

酒精性肝病合并HBV感染并发肝衰竭对NF-κB信号通路的影响

由于酗酒人数的增长,HBV感染合并酒精性肝病患者的数量在中国逐年增加,酒精性肝病与HBV感染会严重影响肝功能。目前中国已成为引起肝硬化的第二大病因。而乙型肝炎病毒感染合并酒精性肝病是最常见的,且会对肝脏造成严重的损伤。本实验的检测了NF-κB信号通路和IL-8、TNF-α和Cleaved caspase-3基因及蛋白的表达,结果表明,酒精性肝病合并HBV感染并发肝衰竭会激活NF-κB信号通路,并上调IL-8、TNF-α和Cleaved caspase-3基因和蛋白的表达,说明酒精性肝病合并HBV感染不仅对肝脏造成了极其严重的损伤,还造成肝脏组织或细胞炎症的发生和细胞凋亡及组织纤维化。通过以上的研究,本实验为揭示酒精性肝病合并HBV并发肝衰竭的分子机制,以及为后续研究酒精性肝病合并HBV并发肝衰竭的靶向治疗提供理论参考。     

hsa-miR-125a-5p通过上调Rock-1表达促进胃癌细胞侵袭

摘要：目的 探讨hsa-miR-125a-5p表达对胃癌细胞侵袭能力的影响及其作用机制。方法 在胃癌MKN45细胞株中瞬时转染hsa-miR-125a-5p-mimics使其表达明显上调,采用Transwell侵袭实验检测细胞的侵袭能力。然后,通过micRNA靶基因预测软件获得hsa-miR-125a-5p的潜在靶基因Rock-1,以抗体阻断及Western blot进行验证。结果 与对照组相比,经瞬时转染后hsa-miR-125a-5p在胃癌MKN45细胞株中表达明显增加;Western blot结果显示hsa-miR-125a-5p表达上调后Rock-1表达明显上调,Transwell实验结果表明MKN45细胞的侵袭能力明显增强（P<0.05）。而hsa-miR-125a-5p表达上调后阻断Rock-1表达,细胞的侵袭能力明显减弱（P<0.05）。结论 hsa-miR-125a-5p促进胃癌细胞的侵袭,其机制与Rock-1通路有关。     

MiR-125a-5p抑制3T3-L1前体脂肪细胞分化

MicroRNAs(miRNAs) 是一类在脂肪组织发育中发挥重要作用的小非编码RNA. 为探明miR-125a-5p在3T3-L1前体脂肪细胞中的作用,采用实时qPCR检测了miR-125a-5p在小鼠各组织及3T3-L1前体脂肪细胞分化过程中的表达|使用经化学修饰的miR-125a-5p模拟物agomir及抑制剂antagomir转染3T3-L1前体脂肪细胞,采用实时qPCR 和 Western印迹检测成脂标志基因Pparγ和aP2的表达,油红O染色观察脂肪细胞脂质积累. 结果显示,miR-125-5p在小鼠脂肪组织中高丰度表达,在3T3-L1前体脂肪细胞分化过程中表达下降.过表达miR-125a-5p,与对照组相比,成脂标志基因Pparγ和aP2在mRNA和蛋白质水平均明显下降|油红O染色及定量结果显示脂质积累减少. 抑制剂处理结果显示,Pparγ和aP2在mRNA和蛋白质水平均有不同程度上升,但油红O染色及定量结果差异不显著. 以上结果表明,miR-125a-5p在脂肪细胞分化中发挥负调控作用.     

Upregulation of hsa-miR-196a-5p is associated with MIR196A2 methylation and affects the malignant biological behaviors of glioma

Hsa-miR-196a-5p is involved in tumorigenesis and progression. However, the driving factors for hsa-miR-196a-5p overexpression and its correlation with the clinicopathological features and prognosis of patients remain unclear in glioma. Thus, this study aimed to investigate the prognostic value of hsa-miR-196a-5p and its correlation with MIR196A2 methylation in glioma. We observed that hsa-miR-196a-5p expression was upregulated in glioma. Next, 112 patients were divided into high (n = 56) and low (n = 56) hsa-miR-196a-5p expression groups. The chi-square test showed that hsa-miR-196a-5p expression was significantly related to age, WHO grade, histopathology, IDH mutation status, and 1p/19q codeletion. Univariate and multivariate Cox regression analyses showed that hsa-miR-196a-5p expression was an independent prognostic factor. GO and KEGG enrichment analyses showed that hsa-miR-196a-5p may be involved in the MAPK signaling, focal adhesion and cancer-related pathways. Compared with the normal astrocyte cell line, glioma cell lines had an unregulated MIR196A2 methylation level, which was confirmed by TCGA data. The hypermethylated CpG sites of MIR196A2 were mainly concentrated in the gene body region, which was significantly associated with hsa-miR-196a-5p overexpression. Kaplan-Meier curves revealed that MIR196A2 hypermethylation was a poor prognostic factor. These findings suggest that hsa-miR-196a-5p overexpression may be involved in malignant biological behaviors, and MIR196A2 hypermethylation of the gene body was significantly associated with hsa-miR-196a-5p overexpression, which was a poor prognostic factor of glioma. Therefore, MIR196A2 hypermethylation may act as an early marker of prognosis of patients with glioma.     

Possible Correlation between Borna Disease Virus Infection and Japanese Patients with Chronic Fatigue Syndrome

Borna disease virus (BDV) is a neurotropic, as yet unclassified, non-segmented, negative-sense, single-strand RNA virus. Natural infection with this virus has been reported to occur in horses and sheep. In addition, antibodies to BDV in plasma or BDV RNA in peripheral blood mononuclear cells (PBMCs) were also found in patients with neuropsychiatric diseases. We describe here the possible link between the patients with chronic fatigue syndrome (CFS) and infection with BDV.     

The Role of In Vitro-Induced Lymphocyte Apoptosis in Feline Immunodeficiency Virus Infection: Correlation with Different Markers of Disease Progression

Human immunodeficiency virus infection is characterized by a progressive decline in the number of peripheral blood CD4⁺ T lymphocytes, which finally leads to AIDS. This T-cell decline correlates with the degree of in vitro-induced lymphocyte apoptosis. However, such a correlation has not yet been described in feline AIDS, caused by feline immunodeficiency virus (FIV) infection. We therefore investigated the intensity of in vitro-induced apoptosis in peripheral blood lymphocytes from cats experimentally infected with a Swiss isolate of FIV for 1 year and for 6 years and from a number of long-term FIV-infected cats which were coinfected with feline leukemia virus. Purified peripheral blood lymphocytes were either cultured overnight under nonstimulating conditions or stimulated with phytohemagglutinin and interleukin-2 for 60 h. Under stimulating conditions, the isolates from the infected cats showed significantly higher relative counts of apoptotic cells than did those from noninfected controls (1-year-infected cats, P = 0.01; 6-year-infected cats, P = 0.006). The frequency of in vitro-induced apoptosis was inversely correlated with the CD4⁺ cell count (P = 0.002), bright CD8⁺ cell count (P = 0.009), and CD4/CD8 ratio (P = 0.01) and directly correlated with the percentage of bright major histocompatibility complex class II-positive peripheral blood lymphocytes (P = 0.004). However, we found no correlation between in vitro-induced apoptosis and the viral load in serum samples. Coinfection with feline leukemia virus enhanced the degree of in vitro-induced apoptosis compared with that in FIV monoinfected cats. We concluded that the degree of in vitro-induced apoptosis was closely related to FIV-mediated T-cell depletion and lymphocyte activation and could be used as an additional marker for disease progression in FIV infection.Feline immunodeficiency virus (FIV) infection is a naturally occurring infection, and disease progression in infected cats is associated with a decline in the number of CD4⁺ cells (2, 6, 22, 23, 36), a loss of bright CD8⁺ cells in the advanced stage of the disease (22), an increased number of activated T cells (39, 41), and a changed cytokine production, i.e., decreased production of interleukin-2 (IL-2) and concomitantly increased production of tumor necrosis factor alpha (TNF-α) (25, 26). The increased production of TNF-α has been reported to induce apoptosis in chronically FIV-infected cells (38). Apoptosis, a controlled mode of cell death (34), plays an important role in the regulation of immune responses (5, 14). As described for FIV (23), the hallmark of human immunodeficiency virus (HIV)-induced disease is the loss of T-helper cells (31, 43). Theoretically, cell loss can be caused by decreased production of cells, increased destruction, or a combination of the two mechanisms. Findings of an early infection of thymocytes followed by pathologic changes in the thymus support the model of decreased T-helper cell production triggered by HIV (13) and FIV (52). The destruction model is supported by findings of an increased number of peripheral blood T cells undergoing apoptosis upon HIV (20, 32) and FIV (11, 21, 33) infection. However, increased CD4⁺-T-cell turnover may not be the main cause of the observed T-helper cell decline in HIV-1 infection, as reviewed by others (44, 51). In addition, the degree of HIV-induced apoptosis correlates with the T-helper cell decline and disease progression (19, 40). However, such a relationship has not yet been described for FIV. It has been reported that cross-linking of CD4 molecules by HIV gp160 triggers apoptosis in noninfected CD4⁺ T cells (1). Investigation of this aspect in the feline system is especially interesting since FIV does not use the feline homologue of CD4 (50).The aim of the present study was to compare the degree of in vitro-induced lymphocyte apoptosis in FIV-infected cats with normal and decreased T-helper cell counts. We used two different culture conditions to trigger apoptosis in vitro: cells were either cultured overnight under nonstimulating conditions and in the absence of growth factors or cultured for 60 h in the presence of phytohemagglutinin, IL-2, and fetal calf serum. We additionally examined cats which were coinfected with the feline leukemia virus (FeLV). This coinfection is known to accelerate the progression toward feline AIDS (23) by an unknown mechanism (8).     

慢性肝炎中乙型和丙型病毒混合感染的重症化研究

在慢性肝炎中,乙、丙型病毒性肝炎混合感染相当多见,可使肝炎慢性化、重症化,肝组织损伤加重,肝硬化(LC)和肝癌(HCC)发生率增加[1]。本文应用血清学和分子生物学方法对196例肝病患者的血清进行检测,初步探讨了乙型肝炎病毒(Hepatitis B virus,HBV)、丙型肝类病毒(Hepatitis C virus,HCV)的复制状况以及两者间的相互作用与预后的关系。1材料和方法1.1病例受检的196例病例均为2004年1月至2005年7月我院住院及门诊病人,男149例,女47例,年龄15~82岁,其中慢性肝炎(CH)患者139例,肝硬化(LC)患者42例,肝癌(HCC)患者15例。所有病例诊断符合…     

慢性肝炎中乙型和丙型病毒混合感染的重症化研究

在慢性肝炎中,乙、丙型病毒性肝炎混合感染相当多见,可使肝炎慢性化、重症化,肝组织损伤加重,肝硬化(LC)和肝癌(HCC)发生率增加[1].本文应用血清学和分子生物学方法对196例肝病患者的血清进行检测,初步探讨了乙型肝炎病毒(Hepatitis B virus,HBV)、丙型肝类病毒(Hepatitis C viruS,HCV)的复制状况以及两者间的相互作用与预后的关系.     

Association of T-Cell Immunoglobulin and Mucin Domain-Containing Molecule 3 (Tim-3) Polymorphisms with Susceptibility and Disease Progression of HBV Infection

Purpose

T-cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3) plays an important role in regulating T cells in hepatitis B virus (HBV) infection and hepatocellular carcinoma (HCC). However, few researches have reported the association of Tim-3 genetic variants with susceptibility and progression of HBV infection. In this study, we focused on the association of Tim-3 polymorphisms with HBV infection, HBsAg seroclearance and hepatocellular carcinoma.

Methods

A total of 800 subjects were involved in this study. Four groups were studied here, including HBV, HBsAg seroclearance, HBV-associated HCC and healthy controls. Three single-nucleotide polymorphisms (SNPs) of Tim-3, rs246871, rs25855 and rs31223 were genotyped to analyze the association of Tim-3 polymorphisms with susceptibility and disease progression of HBV infection.

Results

Our study found that rs31223 and rs246871 were associated with disease progression of HBV infection, while none of the three SNPs was relevant to HBV susceptibility. The minor allele “C” of rs31223 was found to be associated with an increased probability of HBsAg seroclearance (P = 0.033) and genotype “CC” of rs246871 to be associated with an increased probability of HBV-associated HCC (P = 0.007). In accordance, haplotypic analysis of the three polymorphisms also showed that the haplotype block CGC* and TGC* were significantly associated with HBsAg seroclearance (P<0.05) while haplotype block CAT*, CGT*, TAC* and TGT* were significantly associated with HBV-associated HCC (all P<0.05).

Conclusions

Genetic variants of Tim-3 have an important impact on disease progression of HBV infection. With specific Tim-3 polymorphisms, patients infected with HBV could be potential candidates of HCC and HBsAg seroclearance.     

Serum Sphingolipids Reflect the Severity of Chronic HBV Infection and Predict the Mortality of HBV-Acute-on-Chronic Liver Failure

Patients with HBV-acute-on-chronic liver failure (HBV-ACLF) have high mortality and frequently require liver transplantation; few reliable prognostic markers are available. As a class of functional lipids, sphingolipids are extensively involved in the process of HBV infection. However, their role in chronic HBV infection remains unknown. The aim of this study was to determine the serum sphingolipid profile in a population of patients with chronic HBV infection, paying special attention to exploring novel prognostic markers in HBV-ACLF. High performance liquid chromatography tandem mass spectrometry was used to examine the levels of 41 sphingolipids in 156 serum samples prospectively collected from two independent cohorts. The training and validation cohorts comprised 20 and 28 healthy controls (CTRL), 29 and 23 patients with chronic hepatitis B (CHB), and 30 and 26 patients with HBV-ACLF, respectively. Biometric analysis was used to evaluate the association between sphingolipid levels and disease stages. Multivariate analysis revealed difference of sphingolipid profiles between CHB and HBV-ACLF was more drastic than that between CTRL and CHB, which indicated that serum sphingolipid levels were more likely to associate with the progression HBV-ACLF rather than CHB. Furthermore, a 3-month mortality evaluation of HBV-ACLF patients showed that dhCer(d18∶0/24∶0) was significantly higher in survivors than in non-survivors (including deceased patients and those undergoing liver transplantation, p<0.05), and showed a prognostic performance similar to that of the MELD score. The serum sphingolipid composition varies between CTRL and chronic HBV infection patients. In addition, dhCer(d18∶0/24∶0) may be a useful prognostic indicator for the early prediction of HBV-ACLF.     

Frequency and Absolute Number of FoxP3^＋ Regulatory T Cells Correlate with Disease Progression of Chronic HIV-1 Infection

CD4 CD25 Regulatory T cells (Treg) have been found to down-regulate immune activation in HIV-1 infection. However,whether the depletion of Treg benefits to the disease status of HIV infection remains undefined. To address this issue,we enumerated the Treg absolute counts and frequency in 75 antiviral-nave HIV-1-infected individuals in this study. It was found that HIV-infected patients displayed a significant decline in Treg absolute counts but a significant increase in Treg frequency. In addition,with disease progression indicated by CD4 T-cell absolute counts,circulating Treg frequency gradually increased; while Treg absolute counts were gradually decreased,suggesting that the alteration of Treg number closely correlated with disease progression in HIV infection. Functional analysis further showed that Treg efficiently inhibit both CD4 and CD8 T cell proliferation in vitro. Thus,our findings indicates that Treg actively participate in pathogenesis of chronic HIV infection,influencing the disease progression.     

Circulating miR-3613-5p but not miR-125b-5p,miR-199a-3p,and miR-451a are biomarkers of endometriosis

ObjectiveThis study aimed to assess the utility of circulating miR-125b-5p, miR-199a-3p, miR-451a, and miR-3613-5p as biomarkers of endometriosis.Study designPatients with stage III or IV of endometriosis according to the revised American Society of Reproductive Medicine (rASRM) staging classification, as well as control women, were recruited. We created a prospective study conducted on a group of 48 patients (n = 25 controls, n = 24 endometriosis) who had laparoscopic surgery. Blood samples were taken and plasma miRNA levels were measured by quantitative real-time polymerase chain reaction (RT-qPCR) and assessed with AUC and ROC curves.ResultsMiR-451a and miR-3613-5p were significantly decreased in the plasma of endometriosis patients. miR-451a had a receiver-operating characteristic (ROC) area under the curve 0.8283 and miR-3613-5p had a ROC area under the curve 0.7617. The concentration of circulating miR-125b-5p and miR-199-3p did not differ between endometriosis patients and controls. Plasma miRNA levels did not change with BMI, smoking status, fertility problems, or menstrual pain according to the VAS scale (p > 0.05).ConclusionCirculating miR-451a and miR-3613-5p levels significantly differed between endometriosis and controls. However, the levels of miR-451a were discordant with previous studies. Therefore, miR-3613-5p may have better potential as the endometriosis biomarker. Circulating miR-125b-5p and miR-199a-3p cannot be used as reliable markers of endometriosis.     

Frequency and Absolute Number of FoxP3+ Regulatory T Cells Correlate with Disease Progression of Chronic HIV-1 Infection

CD4+CD25+ Regulatory T cells (Treg) have been found to down-regulate immune activation in HIV-1 infection. However, whether the depletion of Treg benefits to the disease status of HIV infection remains undefined. To address this issue, we enumerated the Treg absolute counts and frequency in 75 antiviral-na(i)ve HIV-1-infected individuals in this study. It was found that HIV-infected patients displayed a significant decline in Treg absolute counts but a significant increase in Treg frequency. In addition, with disease progression indicated by CD4 T-cell absolute counts, circulating Treg frequency gradually increased; while Treg absolute counts were gradually decreased, suggesting that the alteration of Treg number closely correlated with disease progression in HIV infection.Functional analysis further showed that Treg efficiently inhibit both CD4 and CD8 T cell proliferation in vitro. Thus, our findings indicates that Treg actively participate in pathogenesis of chronic HIV infection,influencing the disease progression.