Marwa Matboli

Clinical verification of a novel urinary microRNA panal: 133b, -342 and -30 as biomarkers for diabetic nephropathy identified by bioinformatics analysis

BACKGROUND Because microvascular disease is one of the major drivers of diabetic complications, early detection of diabetic nephropathy (DN) by assessing the expression of exosomal microRNAs (miRNAs) in DN patients and healthy controls, may be of clinical value. The aim of this study wasto identify a novel miRNA panel of DN by combining bioinformatics analysis of miRNA databases and clinical verification to evaluate the significance of this panel as urine biomarkers for type 2 diabetic nephropathy (T2DN). PATIENTS AND METHODS Public miRNA databases e.g miro-Ontology and miRWalk were analyzed and a novel panel of 3 microRNAs was retrieved. Meanwhile, combinatorial target prediction algorithms were applied. Multiple case-matched normal were examined by quantative RT-PCR for differential expression in urine exosomes from 210 participants, and the three identified miRNAs were validated as DN biomarkers. RESULTS We found urinary exosomalmiR-133b, miR-342, and miR-30a were expressed at significantly elevated levels in T2DN patients (P<0.001) compared to normal. Furthermore, high-level expression of the 3 miRNAs was associated withHbA1c,systolic-diastolic blood pressure, LDL, serum creatinine, urinary albumin creatinine ratio and estimated glomerular filtration rate(eGFR). Moreover, 39.3%, 19.6% and 17.9% of patients with normo-albuminuria had positive (miR-133b, miR-342 and miR-30a, respectively); indicating the possibility of molecular changes in these patients before onset of albuminuria. CONCLUSION We have identified novel urinary exosomal miRNA biomarkers of DN which were altered not only in micro and macroalbuminuric groups but also in some normoalbuminuria cases prior to albuminuria.

Urinary exosomal microRNA panel unravels novel biomarkers for diagnosis of type 2 diabetic kidney disease.

BACKGROUND A potential approach adopted in the current study is to design a panel based on in silico retrieval of novel miRNAs related to diabetic kidney disease and to evaluate its usefulness in disease diagnosis. PATIENT AND METHODS In the current study, we measured the differential expression of a 6 miRNA panel in urine pellet and exosome in an initial screening group using syber green-based PCR array. Also, we performed pathway enrichment analysis of the key target genes of these miRNAs. Finally, we selected the most significantly up-regulated miRNAs in DKD, exosomal miR-15b, miR-34a and miR-636, that were measured by real-time PCR in a larger independent set of 180 participants to evaluate their usefulness as novel urine biomarkers for diagnosis diabetic kidney disease. RESULTS PCR array analysis showed that miR-15b, miR-34a, and miR-636 were upregulated in both urine pellet and exosome of type 2DKD patients. qRT-PCR validation in the larger independent set of participants confirmed the significant up-regulation of these urinary exosomal miRs (P<0.001). Notably, a positive correlation was found between these miRs, serum creatinine and urinary protein creatinine ratio. The sensitivity of this miRs based panel in urine exosomes reached 100% in diagnosis of DKD. CONCLUSION We identified urinary exosomal miR-15b, miR-34a, and miR-636 as a novel diagnostic panel and a major contributor in the pathogenesis of diabetic kidney disease.

Association of long noncoding RNA and c-JUN expression in hepatocellular carcinoma

ABSTRACT Background: Long noncoding RNAs(lncRNAs) have emerged as key elements in modulating gene expression in different biological contexts. Patients and methods: We used quantitative real-time PCR (Qpcr) to evaluate the expression of lncRNA-UCA1 and C-JUN in serum of 70 patients with hepatocellular carcinoma (HCC), 32 patients chronic hepatitis C (CHC) and 38 healthy subjects and their correlation with different clinicopathological factors. Results: The expression of lncRNA-UCA1 and C-JUN was positive in 91.4%HCC patients with strong discriminating power between HCC and healthy subjects and CHC patients as well. The median follow up period was 29 months. The survival analysis showed that both lncRNA-UCA1 and C-JUN were independent prognostic factors. Of note, we identified C-JUN expression changes consistent with the lncRNA-UCA1 target regulation. Conclusion: This information sheds light on the possible role of lncRNA-UCA1 and C-JUN mRNA as promising diagnostic and prognostic markers as well as potential therapeutic targets in HCC.

Rapid detection of urinary long non-coding RNA urothelial carcinoma associated one using a PCR-free nanoparticle-based assay.

Abstract We developed a specific hybridization assay for direct detection of long non-coding RNA urothelial carcinoma associated-1 (lncRNA-UCA1). Total RNA was extracted from urine pellet samples (bladder carcinoma patients and controls). Then, we compared the developed nanoassay with quantitative real time polymerase chain reaction (qRT-PCR) results in detection of urine UCA1 in bladder cancer and control samples. The sensitivity and the specificity of UCA1 nanoassay were 92.1% and 93.3%, respectively. The concordance of the two methods was 98%. Interestingly, all bilharzial benign cases showed negative lncRNA-UCA1 using both methods. UCA1-nanoassay is a valid test for direct detection of urine UCA1 for bladder cancer detection.

Caffeic Acid Attenuates Diabetic Kidney Disease via Modulation of Autophagy in a High-Fat Diet/Streptozotocin- Induced Diabetic Rat

The aim of this study is to evaluate the anti-diabetic nephropathy effect of Caffeic acid and to prove our hypothesis for its mechanism of action that it may occur by reactivation of autophagy pathway via suppression of autophagy regulatory miRNAs. In vivo, high-fat diet and streptozotocin-induced (HFD-STZ) diabetic rats were treated with Caffeic acid once per day for 12 weeks before and after development of diabetic nephropathy. Blood and urine biochemical parameters, autophagy transcripts and their epigenetic regulators together with renal tissue morphology were investigated. In diabetic rats, Caffeic acid intake, caused improvement in albumin excretion,blood glucose, reduced renal mesangial matrix extension with increased vacuolation and reappearance of autophagosomes. Meanwhile, it resulted in autophagy genes up-regulation [RB 1-inducible coiled coil protein (RB1CC1), Microtubule-associated proteins 1A/1B light chain 3(MAP1LC3B), Autophagy related gene (ATG-12),] with simultaneous reduction in their epigenetic regulators; miRNA-133b, −342 and 30a, respectively. These above mentioned effects were more significant in the diabetic nephropathy Caffeic treated rats than in the prophylactic group. Based on our results we postulated that caffeic acid modulates autophagy pathway through inhibition of autophagy regulatory miRNAs, that could explain its curative properties against diabetic kidney disease.

Evaluation of Circulatory RNA-Based Biomarker Panel in Hepatocellular Carcinoma

BackgroundThe circulating transcriptome (coding and non-coding) plays a critical role in cancer. Novel accurate strategies for early detection of hepatocellular carcinoma (HCC) are strongly needed.Patients and MethodsWe chose an HCC-specific RNA-based biomarker panel based on the integration of differential lysosomal-associated membrane protein 2 (LAMP2) gene expression with its selected epigenetic regulators using bioinformatic methods. This was followed by RT-qPCR validation in serum of 78 patients with HCC, 36 patients with chronic hepatitis C (CHC) infection and 44 healthy volunteers. We used risk-score analysis to evaluate the diagnostic efficacy of the serum profiling system. Moreover, in twenty of the 78 HCC cases involved in the study we examined the expression of RNA-based biomarker panel in both HCC and adjacent non-tumor tissues and assessed their correlation with the serum level of this panel.ResultsThe four ribonucleic acid (RNA)-based biomarker panel [long non-coding RNA-C terminal binding protein, androgen responsive (lncRNA–CTBP), microRNA-16-2 (miR-16-2), microRNA-21-5-P (miR-21-5p) and LAMP2], had high sensitivity and specificity for discriminating HCC from healthy controls and also from CHC patients. Among these four RNAs, serum miR-16-2 and miR-21-5p were independent prognostic factors.ConclusionThe circulatory RNA-based biomarker panel can serve as a potential biomarker for HCC diagnosis and prognosis.

Breast tissue–based microRNA panel highlights microRNA-23a and selected target genes as putative biomarkers for breast cancer

We explored the differential expression of breast tissue-based panel of microRNAs (miRNAs) and their potential application as prognostic markers of breast cancer (BC). This study was divided into the following phases: (1) A panel of 6 BC characteristic miRNAs, which were retrieved based on the microarray signature profiling (released by miRWalk), was explored using SYBR Green-based polymerase chain reaction (PCR) array in 16 cancerous and 16 noncancerous breast tissue; (2) pathway enrichment analysis of the key miRNA target genes; (3) marker choice and validation by real-time PCR in a larger set of 76 patients with BC, 36 benign breast conditions, and 36 healthy volunteers; (4) validation of miRNA (miR)-23a target genes (forkhead box m [FOXM1] and histidine-rich glycoprotein [HRG]) by conventional reverse transcriptase (RT)-PCR; and (5) the prognostic significance of the investigated parameters in the BC validation group was explored. In PCR array-based miRNA expression analysis, 4 miRNAs were found to be altered more than twice (miR-96, miR-29c, miR-221, and miR-23a). Bioinformatic analysis of the target genes revealed enrichment for special biological process categories, that is, cell cycle, angiogenesis, apoptosis, cell proliferation, and cell adhesion. miR-23a, HRG messenger RNA, and FOX messenger RNA were positive in BC by 82.9%, 72.4%, and 71.1%, respectively. The overall concordance rates between miR-23a with HRG and FOXM1 tissue RNAs were 91% and 79%, respectively. The median follow-up period was 49 months. mi-23a and HRG RNA were significant independent prognostic markers in relapse-free survival. miR-23a may have an oncogenic function and enhance BC progression by directly activating FOXM1 and HRG at RNA level.

Identification and validation of a novel autophagy gene expression signature for human bladder cancer patients.

We sought to identify and validate a novel urinary autophagy transcript signature in patients with bladder cancer and evaluate its clinical utility. We performed an initial screening for seven autophagy transcript–based panel (autophagy-related protein 12 (ATG12); WD repeat domain, phosphoinositide interacting 2 (WIPI2); FYVE and coiled-coil domain-containing protein 1 (FYCO1); microtubule-associated protein light chain (MAPLC3); RB1-inducible coiled-coil 1 (RB1CC1); tachylectin-II-like beta-propeller domain 1 (TECPR1); and Unc-51-like kinase (ULK1)) that was identified based on bioinformatics analysis followed by SYBR Green–based polymerase chain reaction array validation in paired tissue and urine samples. Afterward, we evaluated the expression of differentially expressed autophagy transcripts in an independent validation set with reverse transcription quantitative real-time polymerase chain reaction in urine sediments of 140 patients with bladder cancer, 68 patients with benign urological lesions, and 74 healthy controls (age and sex matched). The expression levels of ATG12, FYCO1, TECPR1, and ULK1 in paired bladder tissue and urine samples were significantly lower in bladder cancer than in control group (p < 0.001). In the validation set, the receiver-operating characteristic curve analyses demonstrated that each urinary autophagy transcripts showed high sensitivity and specificity for distinguishing bladder cancer from non–bladder cancer patients (ATG12, 75.4% and 86.1%; FYCO1, 87% and 75.7%; ULK1, 85.5% and 75.6%; and TECPR1, 90% and 81.9%). We document and validate a novel autophagy transcript signature for human bladder cancer diagnosis: bilharzial and non-bilharzial types.

Evaluation of MicroRNA-210 and protein tyrosine phosphatase, non receptor type 2 in pre-eclampsia.

BACKGROUND The precise origin of Pre-eclampsia (PE) remains elusive. Multiple pieces of evidence support the existence of hypoxia in PE. MiRNA-210 (miR-210), which is induced by Hypoxia-Inducible Factor-1α (HIF-1α) during hypoxia, is one of the most hypoxia sensitive miRNAs. MiR-210 mediates these functions by regulating a lot of target mRNAs. Protein tyrosine phosphatase, non-receptor type 2 (PTPN2) was one of miR-210 targets and was found to be down regulated by hypoxia. OBJECTIVE To assess the levels of relative expression of miR-210 and its target PTPN2 in Egyptian women with PE. This is in order to clarify their possible role in the progression of PE and their relation to each other and to different clinicopathological factors. STUDY DESIGN Group1 included 35 normal primigravida and group 2 included 35 primigravida patients with PE. PE group was subdivided into-mild and severe (PE). Total RNA was extracted from placental tissue samples and Real-Time PCR was performed on the extracted RNA. RESULTS There was a highly significant difference between the studied groups as regards fold change of placental miR-210 and PTPN2 (P<0.01). There was a highly significant negative correlation between miR-210 and PTPN2 RQ among the studied groups and among the preeclampsia group (P<0.01). CONCLUSION The results of this study demonstrated that placental expression of miR-210 was up regulated in pregnancies complicated with PE in comparison to normal pregnancies. This increase in miR-210 resulted in down regulation of its target PTPN2 mRNA and this can have a direct role in the pathogenesis of the PE disease. Additionally, both miR-210 & PTPN2 relative expression could differentiate between mild & severe PE.

Exploring the role of molecular biomarkers as a potential weapon against gastric cancer: A review of the literature.

Gastric cancer (GC) is a global health problem and a major cause of cancer-related death with high recurrence rates ranging from 25% to 40% for GC patients staging II-IV. Unfortunately, while the majority of GC patients usually present with advanced tumor stage; there is still limited evidence-based therapeutic options. Current approach to GC management consists mainly of; endoscopy followed by, gastrectomy and chemotherapy or chemo-radiotherapy. Recent studies in GC have confirmed that it is a heterogeneous disease. Many molecular characterization studies have been performed in GC. Recent discoveries of the molecular pathways underlying the disease have opened the door to more personalized treatment and better predictable outcome. The identification of molecular markers is a useful tool for clinical managementin GC patients, assisting in diagnosis, evaluation of response to treatment and development of novel therapeutic modalities. While chemotherapeutic agents have certain physiological effects on the tumor cells, the prediction of the response is different from one type of tumor to the other. The specificity of molecular biomarkers is a principal feature driving their application in anticancer therapies. Here we are trying to focus on the role of molecular pathways of GC and well-established molecular markers that can guide the therapeutic management.