MicroRNA-199a-3p在肾癌中的表达及作用

目的检测microRNA-199a-3p（miR-199a-3p）在肾癌细胞株和组织中的表达情况并探究miR-199a-3p在肾癌细胞中的作用。方法利用实时定量RT-PCR检测miR-199a-3p在肾癌细胞和组织中的表达水平;利用miR-199a-3p模拟物转染肾癌细胞786-0上调miR-199a-3p后,通过CCK-8、克隆形成、Transwell以及细胞周期检测来探究其在肾癌细胞中的作用。结果 miR-199a-3p在肾癌细胞中明显低表达,在78%（14/18）的肾癌组织中亦明显低表达;上调miR-199a-3p可显著抑制肾癌细胞的增殖、存活和侵袭并能诱导细胞周期G1期阻滞。结论我们的研究显示在肾癌中miR-199a-3p明显低表达并参与肾癌的发生、发展,这表明miR-199a-3p具有作为肾癌诊断和治疗靶点的潜能。     

lncRNA RP5-916L7.2 correlates with advanced tumor stage,and promotes cells proliferation while inhibits cells apoptosis through targeting miR-328 and miR-939 in tongue squamous cell carcinoma

BackgroundThis study aimed to investigate the correlation of lncRNA RP5-916L7.2 with tumor features of tongue squamous cell carcinoma (TSCC), and its effect on cells proliferation and apoptosis as well as its target miRNAs in TSCC cells.Methods30 TSCC patients underwent surgery were consecutively enrolled, tumor tissue and paired adjacent tissue were obtained for lncRNAs determination. Blank mimic (NC(+)), lncRNA RP5-916L7.2 mimic (RP5-916L7.2(+)), blank inhibitor (NC(−)), lncRNA RP5-916L7.2 inhibitor (RP5-916L7.2(−)), lncRNA RP5-916L7.2 inhibitor/miR-328-5p inhibitor (RP5-916L7.2(−)/miR-328(−)) and lncRNA RP5-916L7.2 inhibitor/miR-939-5p inhibitor (RP5-916L7.2(−)/miR-939(−)) plasmids were transfected into Tca-8113 cells. qPCR assay, CCK-8 assay, AV/PI assay were performed to detect the miRNA/lncRNA expression, cells proliferation and cells apoptosis, respectively.ResultslncRNA RP5-916L7.2 was increased in tumor tissue compared with paired adjacent tissue, and correlated with higher T stage, N stage as well as TNM stage in TSCC patients. In vitro experiments revealed that lncRNA RP5-916L7.2 promoted cells proliferation and repressed cells apoptosis in Tca-8113 cells. Subsequently, we selected top five potential target miRNAs of lncRNA RP5-916L7.2, and found that lncRNA RP5-916L7.2 reversely regulated the levels of miR-328-5p and miR-939-5p in Tca-8113 cells. Thus, we conducted rescue experiments, which showed that lncRNA RP5-916L7.2 enhanced cells proliferation and inhibited cells apoptosis through targeting miR-328-5p and miR-939-5p in Tca-8113 cells.ConclusionslncRNA RP5-916L7.2 was up regulated in tumor tissue and positively correlated with tumor stage, and promoted cells proliferation while inhibited cells apoptosis by targeting miR-328-5p and miR-939-5p in TSCC.     

MicroRNA-21在肾细胞癌中高表达的研究

目的 本研究旨在检测MicroRNA-21(miR-21)在肾细胞癌及其相应癌旁肾实质组织中的表达,并分析其与临床病理指标的关联.方法 应用实时荧光定量PCR 技术(RT-qPCR)对miR-21 在48 例不同阶段肾细胞癌和癌旁组织中的相对表达量进行检测,并分析其表达量与临床病理指标之间的关联.结果 与相应癌旁组织比较,miR-21 在肾细胞癌中存在显著高表达(P ＜0.05),平均升高倍数约13.miR-21 表达水平上调与患者年龄、性别、病理类型、病理分期无关联(P 均＞0.05).结论 miR-21 在肾细胞癌组织中显著高表达,提示其可能在肾细胞癌的早期诊断、治疗、判断预后等方面具有重要意义.     

Expression profile of plasma microRNAs in nonsyndromic cleft lip and their clinical significance as biomarkers

ObjectiveThe aim of this study was to determine the expression profile of plasma microRNAs in nonsyndromic cleft lip (NSCL) and their clinical significance as biomarkers.MethodsAgilent human miRNA microarray chips were used to analyze three NSCL plasma samples (mixed as CL group) and three normal plasma samples (mixed as Control group). Six selected plasma miRNAs were validated using qRT-PCR between another 13 CL and 11 healthy children. The receiver operating characteristic (ROC) curve analysis was applied for three elevated miRNAs, miR-16-2-3p, miR-365a-3p and miR-877-5p. Their target genes were further assessed using gene ontology and pathway analysis.ResultsThe plasma miRNA differentially expressed (fold change ≥2) amounted to 305. In particular, it had been validated that miR-16-2-3p, miR-365a-3p and miR-877-5p were elevated in NSCL plasma samples. ROC curve analysis revealed that each microRNA was able to significantly discriminate NSCL subjects from normal controls. Gene ontology and pathway analysis revealed that many processes over-represented in CL are related to system development process, regulation of nitrogen compound metabolic process, FoxO signaling pathway and the ErbB signaling pathway.ConclusionOur study demonstrated that plasma miR-16-2-3p, miR-365a-3p and miR-877-5p might become biomarkers to diagnose NSCL and dysregulation of these miRNAs might be involved in the progression of NSCL.     

A miRNA Signature of Prion Induced Neurodegeneration

MicroRNAs (miRNAs) are small, non-coding RNA molecules which are emerging as key regulators of numerous cellular processes. Compelling evidence links miRNAs to the control of neuronal development and differentiation, however, little is known about their role in neurodegeneration. We used microarrays and RT-PCR to profile miRNA expression changes in the brains of mice infected with mouse-adapted scrapie. We determined 15 miRNAs were de-regulated during the disease processes; miR-342-3p, miR-320, let-7b, miR-328, miR-128, miR-139-5p and miR-146a were over 2.5 fold up-regulated and miR-338-3p and miR-337-3p over 2.5 fold down-regulated. Only one of these miRNAs, miR-128, has previously been shown to be de-regulated in neurodegenerative disease. De-regulation of a unique subset of miRNAs suggests a conserved, disease-specific pattern of differentially expressed miRNAs is associated with prion–induced neurodegeneration. Computational analysis predicted numerous potential gene targets of these miRNAs, including 119 genes previously determined to be also de-regulated in mouse scrapie. We used a co-ordinated approach to integrate miRNA and mRNA profiling, bioinformatic predictions and biochemical validation to determine miRNA regulated processes and genes potentially involved in disease progression. In particular, a correlation between miRNA expression and putative gene targets involved in intracellular protein-degradation pathways and signaling pathways related to cell death, synapse function and neurogenesis was identified.     

Non‐invasive urine markers for the differentiation between RCCs and oncocytoma

BackgroundRecently, our group showed that Vim3 is overexpressed in tissue samples of renal oncocytomas and Mxi‐2 in clear cell renal carcinoma (ccRCC). The mechanism leading to the truncation of both proteins is known and involves with two miRs, both detectable in urine. Since the analysis of miRs is time‐consuming, our aim was to identify the truncated proteins in urine instead. Furthermore, urine samples from small renal masses (SRMs) (n = 45, <4 cm) were analyzed to get a pre‐surgical differentiation of the cancer subtypes.MethodsUrines were accessed from the urological biobank (n = 350). Proteins were isolated from urine samples, and Western blots were performed. Each sample was analyzed with ELISA for the expression of Vim3 and Mxi‐2. A lateral flow assay was established. For the detection of SRMs, the miRs were isolated and qRT‐PCR was performed.ResultsA significant increase of Vim3 in urines from patients with oncocytoma (n = 20) was detectable with ELISA compared to all other subtypes of RCCs (chromophobe (n = 50), papillary (n = 40), ccRCC (n = 200), and controls (n = 40) (***p < 0.0001)). Mxi‐2 was predominantly overexpressed in ccRCCs (***p < 0.0001). Lateral flow assay of Vim3 and Mxi‐2 shows two bands in the case of oncocytoma and ccRCC indicating the specificity of this test.For SRMs, an overexpression of miR‐15a/Mxi2 was detectable in urine samples from ccRCC and chromoRCC patients. In contrast to that, miR‐498/Vim3 were predominantly overexpressed in oncocytoma patients.ConclusionBoth proteins (Vim3 and Mxi‐2) were detectable in patients’ urines and can be used for the non‐invasive differentiation of kidney cancers.     

The clinical utility of miR-21 as a diagnostic and prognostic marker for renal cell carcinoma

Renal cell carcinoma (RCC) is the most common neoplasm of the kidney. Increasing evidence suggests that microRNAs are dysregulated in RCC and are important factors in RCC pathogenesis. miR-21 is a known oncogene with tumor-promoting effects in many types of cancer. In this study, we analyzed miR-21 in 121 cases of healthy kidney and different RCC subtypes, including clear cell (ccRCC), papillary (pRCC), chromophobe (chRCC), and oncocytoma. Total RNA was extracted, and the expression of miR-21 was measured with real-time quantitative RT-PCR using miR-21-specific probes. The expression of miR-21 was significantly up-regulated in RCC compared with healthy kidney. There was a significant difference in the expression levels between RCC subtypes, with the highest levels of expression in ccRCC and pRCC subtypes. miR-21 expression distinguished ccRCC and pRCC from chRCC and oncocytoma with 90% specificity (95% CI, 63.9% to 98.1%) and 83% sensitivity (95% CI, 53.5% to 97.6%). Significantly higher miR-21 levels were associated with higher stage and grade. Patients who were miR-21 positive had statistically significant shorter disease-free and overall survival rates. Thus, miR-21 is up-regulated in RCC, and its expression levels can be used as a diagnostic marker to distinguish ccRCC and pRCC from chRCC and oncocytoma. Moreover, it has potential as a prognostic marker in RCC, although it is not independent of tumor stage and grade.     

Serum exosomal miR-377-3p inhibits retinal pigment epithelium proliferation and offers a biomarker for diabetic macular edema

ObjectivesDiabetic macular edema (DME) is a complication of diabetes mellitus that leads to diabetic retinopathy. Thus far, the role of serum exosomal microRNAs (miRNAs) in DME progression remains elusive. This study investigated serum exosomal miRNAs from patients with type 2 diabetes (T2D) and DME to identify miRNAs associated with expression of vascular endothelial growth factor (VEGF), a pivotal component in DME progression; it also evaluated the diagnostic values of these miRNAs for DME.MethodsSerum was collected from patients with T2D who did (n = 20) and did not have DME (n = 24). Exosomes were isolated from serum and subjected to real-time polymerase chain reaction, western blotting, luciferase reporter, and miRNA profiling analyses.ResultsVEGF was significantly upregulated in ARPE-19 cells treated with exosomes from patients with T2D and DME, compared with exosomes from patients with T2D alone. Among the top 10 downregulated miRNAs identified during exosomal miRNA profiling, miR-377-3p inhibited the expression of VEGF. Luciferase reporter assays confirmed that miR-377-3p could directly regulate VEGF expression. Receiver operating characteristic analysis identified serum exosomal miR-377-3p as a potential biomarker for DME.ConclusionSerum exosomal miR-377-3p inhibits VEGF expression to suppress retinal pigment epithelium proliferation and offers a diagnostic biomarker for DME.     

Prognostic significance of subclassifying stage pT3a renal tumors with fat invasion: a retrospective study of 99 patients

PurposeTo analyze the recurrence in patients with clinic stage T1 renal cell carcinoma (RCC) who were upstaged to stage T3a after partial nephrectomy (PN) using a new sub-classification criterion.MethodsA retrospective study of pathological characteristics was performed in patients who were upstaged to pT3a on the basis of fat invasion (FI).ResultsAfter analyzing the pathological findings, we proposed the following new sub-classification criteria for pT3a RCC with FI: (1) renal tumor invades the pseudo-capsule and contacts the perinephric adipose tissue directly or the tumor protrudes into the perinephric adipose tissue like a tongue (Type A); and (2) tumor nodules are distributed in perinephric adipose tissues (Type B). A significant difference was observed in the recurrence rate between the two subtypes A and B. For Type B, the recurrence rate after radical nephrectomy (RN) and PN was 15.79% and 63.64%, respectively. The recurrence rates for Types A and B after PN were 11.11% and 63.64%, respectively.ConclusionsT3a RCC with tumor nodules in perinephric adipose and/or an irregular tumor protruding into the adipose tissues lead to a higher recurrence rate. We recommend that T3a RCC be carefully analyzed and patients be treated on an individual basis.     

miR‐30b‐5p up‐regulation related to the dismal prognosis for patients with renal cell cancer

The diagnosis of renal cell carcinoma (RCC) is often made late since there is no early symptom, which thus results in dismal patient prognosis. As a result, new biomarkers are urgently needed and efforts should be made to identify their functions in predicting RCC prognosis. microRNAs (miRNAs) are a class of small noncoding RNAs that are about 20‐22 nucleotides in length, and they have been demonstrated to function as prognostic markers in numerous tumors. This study aimed to assess the role of miR‐30b‐5p in predicting the prognosis of RCC postoperatively. In this study, RNA was extracted from 284 formalin‐fixed and paraffin‐embedded kidney cancer tissue samples. After cDNA synthesis, real‐time quantitative PCR (RT‐qPCR) was adopted for detecting the relative miR‐30b‐5p level. Then, the Kaplan‐Meier method, Cox regression analysis, and the receiver operating characteristic curve analysis were applied in analyzing the miR‐30b‐5p effect on the prognosis for patients. Our findings indicated that, following adjustment for age, gender, tumor stage, and tumor size, patients with low miR‐30b‐5p expression had remarkably longer overall survival. Thus, the miR‐30b‐5p level might be related to RCC prognosis.     

CD98hc (SLC3A2), a novel marker in renal cell cancer

Background  In a variety of malignant diseases, molecular targeting represents a therapeutic option, whereby, when compared with chemotherapy, fewer side effects are thought to be expected. Especially in renal cell cancer (RCC), tyrosine kinase-inhibitors have been established as useful and highly effective therapy. However, tyrosine kinase-inhibitors currently approved for RCC treatment lack single molecule specificity and bear a variety of side effects of the gastro-intestinal tract, skin, heart and haematopoietic system. Therefore, the identification of novel cell surface markers is sought, which might lead to novel diagnostic and therapeutic strategies in cancer.
Material and methods  Paraffin-embedded RCCs from a well characterized tissue bank were immunohistochemically quantified for embryonic transmembrane antigen CD98hc (SLC3A2) expression and semi-quantitative analyses were correlated with subtype or grade of differentiation.
Results  We found increased CD98hc expression in different types of malign RCCs, among them clear cell (cc)RCC, papillary (p)RCC and chromophobe (ch)RCC, but lack of expression in the benign renal oncocytoma. Thereby, the extent of CD98hc expression directly complies with grade of malignancy. Furthermore, the more malignant type II pRCC significantly higher expressed CD98hc than the less malignant and more differentiated type I pRCC (type II 83·34%, type I 4·76% CD98hc positive, P  < 0·00001; n  = 51). The established marker for type I pRCC, Cytokreatin 7, showed 95·24% expression in type I and 26·67% expression in type II pRCC ( P  < 0·00001, n  = 51).
Conclusions  From these data, we conclude that CD98hc is expressed in RCCs, whereby the extent of expression is likely to correlate directly with grade of malignancy. In pRCCs, CD98hc might represent a novel and reliable marker for type II pRCC.     

Three new circulating microRNAs may be associated with wet age-related macular degeneration

Abstract

This study investigates the circulating microRNA (miRNA) expression profiles in patients with age-related macular degeneration (AMD) and the role of miRNA in wet AMD and its pathways. Exosomes were extracted from serum samples of AMD patients (n?=?70) and a control group (n?=?50). After isolating miRNA from the exosomes, miRNAs were transformed into cDNA. In the control and AMD samples, the expression was compared with a panel including 175 genes using the PCR array method. Target genes and pathways of miRNAs were detected by KEGG and Biocarta signaling pathway enrichments. Comparing the serum samples between groups revealed that the expression levels of 15 microRNAs within 175 genes had significantly changed. In the validation studies, miR-129-3p and miR-132-3p had no significant expression in AMD group compared to the controls. miR-486-5p and miR-626 had higher expression in AMD patients compared to the control group, while miR-885-5p showed significantly lower expression. Pathway analysis revealed that these miRNAs may have critical roles in the apoptosis and neovascularization pathways. The data suggest that some miRNAs within the serum may have a role in the pathogenesis of wet AMD. Further studies are needed to examine the use of these miRNAs as biomarkers.     

Identification of aberrantly expressed of serum microRNAs in patients with hormone-induced non-traumatic osteonecrosis of the femoral head

ObjectiveThe non-translation RNA-microRNA (miRNA) has been demonstrated to correlate to various disease occurrence in body. Serum miRNA was gradually considered as molecular markers for disease diagnosis. This study was designed to analyze differential serum miRNAs level in hormone-induced non-traumatic osteonecrosis of the femoral head (hormone-NOFH) patients.MethodsWe selected 30 patients with hormone-NOFH as case group, and 30 healthy volunteers were recruited as control group. miRCURYTM LNA miRNA chip and quantitative RT-PCR were used to examine differential miRNAs expression. Correlation assay was performed between miRNAs and NOFH trait.ResultsWe found that 9 miRNAs were upregulated while 3 miRNAs were downregulated in hormone-TOFH patient serum by result of miRNA chip. QRT-PCR assay revealed that the level of miR-423-5p was significantly increased and miR-10a-5p was significantly decreased. Using Spearman correlation analysis, we observed that miR-423-5p serum level is positive association to FHC levels whereas miR-10a-5p has no association with FHC levels. Furthermore, miR-423-5p is negatively correlated to its downstream molecule-adiponectin.ConclusionWe report a miRNA profile of hormone-NOFH and provide a new perspective to understand this intricate disease. This novel information suggests the potential roles of miR-423-5p in the diagnosis, prognosis biomarkers, or therapy targets of hormone-NOFH.     

不同临床分期卵巢浆液性癌的miRNA表达谱分析及其与临床预后的关系

目的：筛选在卵巢癌演进过程中起重要作用的 microRNA（miRNA）,检测关键 miRNA 与临床病理及预后的联系,找出与卵巢浆液性癌预后相关的 miRNA。方法 miRNA 芯片技术分别检测6例Ⅰ期浆液性癌,4例Ⅲ期浆液性癌的 miRNA 表达谱差异。统计分析,从中挑选4个 miRNA,miR-510、miR-509-5p、miR-508-3p、miR-483-5p,采用实时定量PCR方法检测其在16例Ⅰ期浆液性癌、35例Ⅲ期浆液性癌中的表达,对比分析。收集验证组患者的临床病理及预后资料。将miRNA实时定量PCR结果分为高表达组和低表达组,分别统计各miRNA的表达与临床病理参数及预后的关系。结果 miRNA芯片技术分析了768个miRNA在Ⅰ期和Ⅲ期的表达谱,其中26个miRNA在两组中的表达具有显著差异（P＜0.05）,差别在两倍以上。对比Ⅰ期,Ⅲ期中6个miRNA上调,20个miRNA下调。实时定量PCR结果与miRNA芯片结果一致,显示miR-510、miR-509-5p、miR-508-3p在Ⅰ期卵巢癌标本中显著高于Ⅲ期浆液性癌,miR-483在Ⅰ期中表达显著低于Ⅲ期浆液性卵巢癌。但4个miRNA的表达与其他临床病理参数无关。生存分析发现miR-510、miR-509-5p二者与预后有关,高表达组的预后显著优于低表达组。结论 miRNA参与卵巢浆液性癌的发展,miR-510及miR-509-5p可能在卵巢癌的侵袭和转移中起作用,有望成为预测卵巢癌预后的分子标记物。     

miR-16在肾癌中表达和临床意义的研究

目的检测肾癌组织及癌旁正常组织中miR-16的表达,分析miR-16表达水平与临床病理特征之间的关系,为深入研究miR-16在肾癌的发生发展中功能作用奠定基础。方法在标本库中随机挑选48例手术切除的肾癌及配对癌旁组织标本,提取总RNA并通过反转录获得cDNA,实时荧光定量PCR(qRT-PCR)检测标本中miR-16表达量,统计学分析其表达量与患者临床病理特征间的联系。结果 48对标本中41例(85.42%)肾癌标本miR-16表达上调,统计学分析证实肾癌组织中miR-16表达量明显高于配对的癌旁正常组织(P<0.01)。肾癌中miR-16表达量与患者性别、年龄、肾癌组织类型、TNM分期、AJCC临床分期无明显相关(P>0.05)。结论 miR-16在肾癌标本中明显表达上调,提示其可能与肾癌的发生发展有关。