Urinary MicroRNA as Biomarker in Renal Transplantation

Urine represents a noninvasive source in which proteins and nucleic acids can be assessed. Such analytes may function as biomarkers to monitor kidney graft pathology at every desired frequency, thereby providing a time window to prevent graft damage by therapeutic intervention. Recently, several proteins have been measured in urine as markers of graft injury. However, the specificity is limited, and measuring urinary proteins generally lacks the potential to predict early kidney graft damage. Currently, urinary mRNA and microRNA are being investigated to evaluate the prognostic value of changes in gene expression during the initial stages of graft damage. At such time point, a change in treatment regimen and dosage is expected to have maximum potency to minimize future decline in graft function. Both mRNA and microRNAs have shown promising results in both detection and prediction of graft injury. An advantage of microRNAs compared to mRNA molecules is their stability, a characteristic that is beneficial when working with urine samples. In this review, we provide the current state of urinary biomarkers in renal transplantation, with a focus on urinary microRNA. In addition, we discuss the methods used to study urinary microRNA expression.     

MicroRNA调控成骨细胞增殖、凋亡的研究进展

微小RNA( microRNA)是一类非编码小分子RNA,在基因表达调控中起重要作用,它通过与靶mRNA的特异性结合,导致靶mRNA降解或者抑制其翻译,对基因进行转录后调控,从而控制细胞的增殖、分化、凋亡等,参与疾病的发生发展。成骨细胞是骨形成过程中的重要细胞,其数量或功能的改变明显影响骨代谢。近年来.microRNA与骨代谢的关系备受关注,诸多研究表明microRNA在成骨细胞的分化中发挥重要调控作用,但其调节成骨细胞增殖和凋亡的研究相对较少。本文就 microRNA调控成骨细胞增殖、凋亡的研究进展进行综述。     

人类白细胞抗原-G与早期胚胎生长发育和着床

人类白细胞抗原-G(HLA-G)是人类组织相容性抗原复合物,在母胎免疫耐受中起作用。近年的研究发现,HLA-G在着床前胚胎表达。我们和国际上其他的临床研究显示,分泌可溶性HLA-G的胚胎在体外受精和胚胎移植中的临床妊娠率和种植率较高。进一步的研究发现,HLA-G与胚胎的免疫耐受、生长、着床相关的粘附和浸润相关,并通过MEK1/2信号传导通路促进人绒毛膜促性腺激素的分泌。我们还发现,子宫内膜细胞与可溶性HLA-G结合,并表达其受体;可溶性HLA-G还促进子宫内膜细胞与滋养细胞的粘附;白血病抑制因子和miR-152参与HLA-G在滋养细胞表达的调控。HLA-G表达和功能相关的分子机制研究显示,HLA-G在胚胎生长和着床上起重要作用,可能是胚胎发育潜能的标志分子。     

HLA‐G in Transplantation: A Relevant Molecule for Inhibition of Graft Rejection?

The human MHC class I molecule HLA-G has long been known as a molecule selectively expressed by cytotrophoblastic cells. By inhibiting the cytolytic function of decidual NK cells, HLA-G protects the HLA-A and -B negative semiallogeneic embryonic tissue against the mother's immune system. In the light of this immuno-suppressive function, the role of HLA-G in transplantation was investigated. We will review here recently published data on this topic, showing that expression of HLA-G affects the responsive capacity of the immune system, might directly participate in graft acceptation, and should be taken into account for the monitoring of transplantation patients.     

Expression of Human Leukocyte Antigen G (HLA-G) Correlates with Poor Prognosis in Gastric Carcinoma

Objective We had previously demonstrated that human leukocyte antigen G (HLA-G) was expressed in a majority of primary colorectal carcinomas and that the detection of HLA-G expression had a strong and independent prognostic value for that cancer. Currently, we investigate whether or not HLA-G is also expressed in patients with gastric carcinoma and whether the expression has any clinical application value. Methods The expression of HLA-G was investigated immunohistochemically in 160 patients with gastric carcinoma. The correlation between HLA-G status and various clinicopathological parameters was analyzed with the levels of HLA-G expression used to compare the survival length amongst patients. Results HLA-G protein expression was observed in 71% (113 of 160) of the primary site of gastric carcinomas, but not in the normal stomach tissues. HLA-G expression in the tumors was significantly correlated with the tumor location, histological grade, depth of invasion, lymph nodal metastasis, clinical stages of the disease, and host immune response (P = .012, .008, .001, .038, .030, and .016, respectively). Patients with HLA-G positive tumors had a significantly shorter survival time than those patients with tumors that were HLA-G negative (P = .001). As well, in multivariate analysis, HLA-G demonstrated an independent prognostic factor (P = .0001, relative risk 9.08; 95% confidence interval, 3.44–24.0). Conclusions Overall, our results indicated that the expression of HLA-G is a characteristic feature of gastric carcinoma and that immunostaining by anti-HLA-G antibody may be a potentially useful prognostic indicator.     

Immunohistochemical Study of HLA-G Expression in Lung Transplant Recipients

Human leukocyte antigen-G (HLA-G), a nonclassical HLA class I protein, promotes immune tolerance of solid-organ allografts, yet its role in lung transplantation (LTx) is unknown. We examined the expression of HLA-G in lung allografts through immunohistochemistry by a cross-sectional study of 64 LTx recipients, classified into four groups (stable patients, acute rejection [AR], bronchiolitis obliterans syndrome [BOS] and symptomatic viral shedders). A marked expression of HLA-G in bronchial epithelial cells (BEC) was frequently observed in stable recipients (n = 18/35 [51%]), but not in patients with AR (n = 14) or with BOS (n = 8). HLA-G was also expressed by 4 of 7 symptomatic viral shedders. In addition, HLA-G-positive patients from the stable group (n = 35) experienced lower incidence of resistant AR and/or BOS during long-term follow-up, as compared with their HLA-G-negative counterparts. Finally, in vitro data showed that interferon-γ, a cytokine present in lung allograft microenvironment, upregulated HLA-G mRNA and protein expression in primary cultured human BEC. We conclude that HLA-G expression in the bronchial epithelium of lung allograft is elevated in some LTx recipients in association with their functional stability, suggesting a potential role of HLA-G as a tolerance marker.     

胰腺癌外周血mdr1及其转录区域结合位点甲基化的检测和临床意义

目的：研究胰腺癌肿瘤组织及外周血有核细胞多药耐药基因1（mdr1）及其转录区域结合位点甲基化的表达及其相关性,探索适用于临床观察多药耐药性变化的有效手段。方法：应用免疫组织化学法和FQ-PCR技术、MSP方法分别检测胰腺癌肿瘤组织及其对应外周血标本中的mdr1的表达和转录区域结合位点甲基化程度,并通过Pearson统计学方法分析两者的相关性。结果：胰腺癌P-gp的表达与mdr1mRNA表达明显相关,并与mdr1基因转录区域结合位点甲基化程度显著相关。外周血中mdr1及其转录区域结合位点甲基化的表达与肿瘤组织中的表达存在显著相关性（P〈0.01）。结论：mdr1转录区域结合位点的甲基化与胰腺癌的多药耐药性密切相关;检测外周血mdr1及其甲基化的表达可动态监察胰腺癌病人化疗前后多药耐药性的变化,为临床治疗方案的决择提供依据。     

Local VEGF‐A blockade modulates the microenvironment of the corneal graft bed

The microenvironment plays an important role in several immunological processes. Vascular endothelial growth factor‐A (VEGF‐A) not only regulates angiogenesis, but is known as a modulator of the immune microenvironment. Modulating the site of transplantation might be beneficial for subsequent transplant survival. In this study, we therefore analyzed the effect that a local blockade of VEGF‐A in the inflamed cornea as the graft receiving tissue has on the immune system. We used the murine model of suture‐induced neovascularization and subsequent high‐risk corneal transplantation, which is an optimal model for local drug application. Mice were treated with VEGFR1/R2 trap prior to transplantation. We analyzed corneal gene expression, as well as protein levels in the cornea and serum on the day of transplantation, 2 and 8 weeks later. Local VEGF depletion prior to transplantation increases the expression of pro‐inflammatory as well as immune regulatory cytokines only in the corneal microenvironment, but not in the serum. Furthermore, local VEGFR1/R2 trap treatment significantly inhibits the infiltration of CD11c+ dendritic cells into the cornea. Subsequent increased corneal transplantation success was accompanied by a local upregulation of Foxp3 gene expression. This study demonstrates that locally restricted VEGF depletion increases transplantation success by modulating the receiving corneal microenvironment and inducing tolerogenic mechanisms.     

HLA-G-treated tolerogenic dendritic cells induce tolerogenic potential by increasing expression of B7-1 (CD80) molecules

BACKGROUND: By consensus, HLA-G has a role in the induction of tolerance via interaction with dendritic cells and manipulation of co-stimulatory molecule expression. The purpose of this study was to demonstrate that HLA-G modified dendritic cells exhibit a decreased ability to stimulate T-cells in vitro due to increased expression of B7-1, which provides regulatory signalling to T-cells as a consequence of binding CD28 and CD152 ligands. METHODS: Bone-marrow cells were cultured from Brown Norway (BN) rat femurs and sorted with anti-rat dendritic cell Ab (OX62) microbeads. Isolated dendritic cells (DCs) were treated with HLA-G tetramer for 3 days. Initially, cells were plated with media, alloantigenic splenocytes, and T-cells and then observed in mixed lymphocyte reaction for thymidine uptake. Also, the cells were analyzed by flow cytometry using antibodies for MHC-II (IA), CD80, and CD86. RESULTS: This study displays that HLA-G-modified DCs decrease induction of an alloproliferative response. In addition, this study demonstrated that HLA-G tetramer treatment decreases CD86 and permits expression of CD80 without altering MHC-II expression. DISCUSSION: This study specifically investigated the role of B7-1 (CD80), showing that HLA-G treatment of immature DCs allows expression of B7-1 (CD80) without altering MHC-II expression and simultaneously decreases B7-2 (CD86) expression. Therefore, a low level of B7-2 (CD86) allows attenuation of T-cell response after activation, both of which are beneficial to immune tolerance. The selective expression of B7-1 (CD80) is important and may serve as a guide for future therapies aimed at transplant tolerance.     

HLA-G 14-bp Insertion/Deletion Polymorphism in Kidney Transplant Patients With Metabolic Complications

HLA-G, a nonclassical HLA molecule with limited polymorphism has immunomodulating/tolerogenic properties. The most common polymorphism of HLA-G is a deletion/insertion of 14 bp, located at the 3′UTR region of the gene (exon 8). This polymorphism is associated with modifications of mRNA stability that can lead to variations of membrane versus solubile HLA-G expression. HLA-G may be involved in the clinical outcomes of transplantation, as evidenced by studies in hematopoietic cell transplantation. We evaluated the possible prognostic importance of 14-bp polymorphisms of HLA-G among kidney transplantation patients. Using polymerase chain reaction amplification we genotyped 124 patients (mean organ survival: 878.95 ± 595.12 days; range = 1-2565) and 98 control individuals representative of the Italian population. Products were visualized by electrophoresis on agarose gels. The results showed no differences between patients and controls. Twenty-nine patients with acute or chronic rejection or in whom clinical conditions required the use of steroid bolus treatments also showed no association with HLA-G 14-bp genotypes or alleles. The subset of patients with dyslipidemia during follow-up showed a significant decrease among the HLA-G−14/−14 genotype, compared with heterozygous (+14/−14) and nondeleted homozygous (+14/+14) genotype patients (P_c = .03). These preliminary data showed that HLA-G 14-bp genotypes, although not predictive of rejection, may be useful to identify individuals at risk for the development of posttransplant complications.     

Dysregulation of collagen production in diabetes following recurrent skin injury: Contribution to the development of a chronic wound

Recurrent injury has been implicated in the development of chronic diabetic wounds. We have developed a chronic diabetic wound model based upon recurrent injury in diabetic mice. We hypothesized that dysregulation of collagen production at both the mRNA and microRNA levels contributes to the development of chronic diabetic wounds. To test this, both diabetic and nondiabetic mice were made to undergo recurrent injury. Real‐time PCR for TGF‐β1, SMAD‐3, Col1α1, Col3α1, microRNA‐25, and microRNA‐29a and Western blot for collagen I and III were performed 7 days following each injury. Diabetic wounds displayed decreased collagen at all time points. This was associated with dysregulated collagen production at both the gene and microRNA levels at all time points. Following the final injury, however, diabetic collagen production significantly improved. This appeared to be due to a substantial decrease in both microRNAs as well as an increase in the expression of collagen pathway genes. That dysregulated collagen production progressed throughout the course of wounding suggests that this is one factor contributing to the development of chronic diabetic wounds. Future studies using this model will allow for the determination of other factors that may also contribute to the development and/or persistence of chronic diabetic wounds.     

MiR-133a induces apoptosis through direct regulation of GSTP1 in bladder cancer cell lines

ObjectiveWe previously demonstrated that miR-133a is a tumor-suppressive microRNA (miRNA) and is commonly down-regulated in human bladder cancer (BC). The aim of this study is to determine a novel oncogenic gene targeted by miR-133a in BC.MethodsTo identify genes targeted by miR-133a, an oligo-microarray analysis was performed using the miR-133a-transfected BC cell lines. For gain/loss-of-function studies, miR-133a/si-glutathione S-transferase π1 (GSTP1)-transfectants were subjected to XTT assay and flow cytometry to evaluate their cell viability and apoptosis status. The luciferase reporter assay was used to confirm the actual binding sites between miR-133a and GSTP1 mRNA. The mRNA and protein expression of GSTP1 in BC cell lines and clinical samples were evaluated by real-time RT-PCR and Western blot, respectively.ResultsMiR-133a transfection induced cell viability inhibition and apoptosis in BC cell lines. We focused on the GSTP1 gene that was the top 7 down-regulated one in the gene profile from the miR-133a-transfectants. MiR-133a transfection repressed expression levels of mRNA and protein levels of GSTP1. A luciferase reporter assay suggested that the actual binding may occur between miR-133a and GSTP1 mRNA. Cell viability inhibition and apoptosis were induced in the si-GSTP1 transfectants compared with the controls (P < 0.005). GSTP1 mRNA expression levels in 43 clinical BCs were significantly higher than those in eight normal bladder epitheliums (P = 0.0277).ConclusionOur data suggest that tumor suppressive miR-133a directly regulated oncogenic GSTP1 gene in BC, and that an anti-apoptotic effect mediated by GSTP1 is maintained by miR-133a down-regulation in human BC.