Long Non-Coding RNAs Gene Variants as Molecular Markers for Diabetic Retinopathy Risk and Response to Anti-VEGF Therapy.

BACKGROUND: Long non-coding RNAs (lncRNAs) play essential roles in molecular diagnosis and therapeutic response in several diseases. PURPOSE: For the first time, we aimed to evaluate the association of four lncRNAs TUG1 (rs7284767G/A), MIAT (rs1061540T/C), MALAT1 (rs3200401C/T), and SENCR (rs12420823C/T) variants with susceptibility to diabetic retinopathy (DR), disease severity, and early therapeutic response to intravitreous anti-vascular endothelial growth factor aflibercept therapy. PATIENTS AND METHODS: This case-control study enrolled 126 adult patients with type 2 diabetes. TaqMan assays using Real-Time PCR were run for genotyping. Multivariable regression analyses were applied to assess the role of each polymorphism after the adjustment of covariates. RESULTS: Carriers of TUG1 A/G and MIAT T/C and C/C genotypes were more likely to develop DR [OR=3.15 (95% CI=1.15-8.64), and OR=4.31 (95% CI=1.78-10.47)], while MALAT1 T/C conferred protection (OR=0.40, 95% CI=0.16-0.99). For TUG1, MALAT1, MIAT, and SENCR genotype combinations, GTCT and GCCC had a higher disease risk (P=0.012). For disease severity, MIAT T/T homozygosity was associated with higher DR grade [33.3% (T/T) vs 10% (C/C) and 4.2% (C/T) carriers, P=0.012]. Otherwise, patients with the SENCR T variant exhibited better pre-treatment best-corrected visual acuity level (p=0.021). Following aflibercept administration, carrying the TUG1 A or MIAT T/C was associated with a poor therapeutic response (OR=5.02, 95% CI=1.60-15.76, and OR=10.23, 95% CI=1.51-69.15, respectively). CONCLUSION: The lncRNAs TUG1 (rs7284767G/A) and MIAT (rs1061540T/C) were associated with increased DR susceptibility and poor response to aflibercept treatment, while MALAT1 (rs3200401C/T) conferred protection to DR. These genetic determinants could be useful in DR risk stratification and pharmacogenetics after validation in large-scale studies.

Chronic valproic acid administration enhances oxidative stress, upregulates IL6 and downregulates Nrf2, Glut1 and Glut4 in rat's liver and brain.

Valproic acid (VPA) is a powerful antiepileptic drug that was associated with several neurological and hepatic problems especially with increasing its dose and duration. These problems may be metabolic in origin and related to glucose homeostasis. So, the present study investigated the effect of different doses and durations of VPA on the expression of glucose transporters (Glut1 and Glut4), oxidative stress and inflammatory cytokine (IL-6) in the liver and specific brain regions. Seventy-two male Sprague-Dawley rats were randomly allocated into three equal groups: (1) saline group, (2) 200 mg VPA group and (3) 400 mg VPA group. By the end of experiments, the expressions of Glut1, Glut4 nuclear factor erythroid-like 2 related factor (Nrf2), IL-6 and oxidative stress markers [malondialdehyde (MDA) and glutathione (GSH)] in the liver, corpus striatum, prefrontal cortex (PFC) and cerebellum were assessed. We found that administration of VPA (200 mg and 400 mg) caused a significant decrease in the Glut1 and Glut4 expression in different tissues in a dose- and time-dependent manner (P < 0.01). Also, VPA (200 and 400 mg) caused a significant increase in MDA with a decrease in GSH in tissues at different times. Moreover, VPA (200 and 400 mg) caused significant upregulation in IL-6 expression and downregulation in Nrf2 expression (P < 0.01). The results suggest that increasing the dose and time of VPA therapy downregulates Glut1 and Glut4 in the liver and brain which may impair glucose uptake in these tissues. This effect was associated with enhanced oxidative stress, downregulation of nrf2 and upregulation of IL-6 in liver and brain tissues.

Association of polymorphisms in metastasis suppressor genes NME1 and KISS1 with breast cancer development and metastasis.

BACKGROUND: NME1 and KISS1 genes are two tumor metastasis suppressor genes, mapped to chromosomes 17q21.3 and 1q32 respectively. Here, we analyzed the association of EcoR1 (rs34214448-G/T) polymorphism in NME1 gene and 9 del T (rs5780218-A/-) polymorphism in KISS1 gene with breast cancer development and metastasis. RESULTS: The study included 75 women newly diagnosed with breast cancer recruited from Oncology Center at Mansoura University Hospitals and 37 age-matched healthy female volunteers as a control group. DNA was extracted from peripheral blood samples and genotyping of rs34214448 and rs5780218 SNPs was carried out by PCR-RFLP technique. NME1 EcoR1 (rs34214448) polymorphism has a statistically significant association with breast cancer risk (P < 0.001). Most of breast cancer group (55%) had heterozygous (G/T) genotype while most of control group (95%) had homozygous wild (G/G) genotype (P < 0.0005). Also, KISS1 rs5780218 polymorphism has a statistically significant association with breast cancer risk. The wild (A/A) genotype was associated with lower risk of breast cancer (A/- + -/- vs. A/A: OR = 3.1, 95% CI = 1.15-8.36, P = 0.025). EcoR1 (rs34214448) polymorphism revealed a significant association with tumor stage and distant metastasis as patients. Carriers of the wild (G/G) genotype were more likely to present with advanced disease stages and distant metastasis. CONCLUSIONS: Both EcoR1 (rs34214448) polymorphism of NME1 gene and rs5780218 polymorphism of KISS1 gene revealed significant association with increased risk of breast cancer development. The (G/G) genotype of EcoR1 polymorphism was associated with higher risk of breast cancer metastasis.

Netrin-1: A new promising diagnostic marker for muscle invasion in bladder cancer.

BACKGROUND: Bladder cancer is the most common urological malignancy with a high tendency for progression and recurrence. So far, no reliable diagnostic marker is present with 100% sensitivity and specificity. Netrins are related to laminin proteins, and were first discovered to be involved in neural development. After that, they were found in other organs of the body and several studies stated that they have implicated in cancer progression. PURPOSE: This study aimed at investigating the netrin-1 gene expression in bladder cancer tissues, in addition to the possibility of using urinary netrin-1 as a marker for muscle invasion diagnosis in bladder cancer cases. METHODS: Netrin-1 gene expression in bladder cancer tissue was detected in this study by real-time polymerase chain reaction. Moreover, netrin-1 protein was measured in tissue and urinary deposit samples by western blotting. RESULTS: The results of this study revealed that netrin-1 is expressed in bladder cancer and control tissues, with a strong positive correlation between netrin-1 in tissues and urinary netrin-1 (rs = 0.762, P < 0.0005). Receiver operating characteristic curve analysis confirmed the muscle-invasion diagnostic value of urinary netrin-1 with bladder cancer cases, providing an area under the curve equals to 0.758 (95% confidence interval, 0.630-0.886, P < 0.0005), with 96% sensitivity and 67% specificity. Bladder cancer patients had been included to examine risk factors for local recurrence, distant metastasis, and death. Cox regression models showed that netrin-1 gene expression, tumor size, and age are positive predictor markers for local tumor recurrence. Age is a predictor for distant metastasis, and tumor stage is a predictor for death. CONCLUSION: Urinary netrin-1 can be used as a promising biomarker for diagnosis of muscle invasion, which may help in the follow up of non-invasive tumors. In addition, tissue netrin-1 expression may serve as a predictor of local tumor recurrence.

Molybdenum bupropion combined neurotoxicity in rats.

Heavy metal toxicity is a common foodborne problem in Egypt, especially in combination. Molybdenum toxicity has been studied as a model of the heavy metal toxicity. Molybdenum could promote toxicity via oxidative-inflammatory mechanisms. Bupropion is a well-known antidepressant that has anti-oxidant mechanisms. It exerts a cytoprotective action against molybdenum induced metal toxicity. The aim of the study is to evaluate the effects of combined bupropion and molybdenum in a toxic animal model. The results showed that the combination of bupropion and high doses of molybdenum was extremely toxic with an evident animal fatality. Bupropion showed a clear anti-oxidant/anti-inflammatory profile detected by the ELISA assay of malondialdehyde (MDA), reduced glutathione, and interleukin -6 (IL-6), and real-time gene expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and tumor necrosis factor-α (TNF-α). The immunohistochemistry of nuclear factor Kappa Beta (NF-κB) showed that bupropion reduced the inflammatory response induced by the molybdenum neurotoxicity. Despite the improved laboratory profile, the animals were extremely intoxicated with recorded fatalities raising the question about other pathways and mechanisms explaining the drug metal interaction. Furthermore, Bupropion even in normal doses was toxic to the animals. Choroid plexus hyperplasia was reported in the histological examination of the animal brain loaded with bupropion, and choroid plexus papilloma was recorded in the combined drug metal group. More wide-scale studies are needed to verify the safety of the current antidepressant medications for the long-term therapy. It is important to focus on drug metal interaction as a possible cause of neuropathology.

The Role of Protamine 2 Gene Expression and Caspase 9 Activity in Male Infertility.

PURPOSE: Approximately 15% of couples are affected by infertility with the man responsible in almost half of the cases. PRMs (protamines) confer a higher order of DNA packaging in sperm than that in somatic cells. Because of the critical roles of PRMs in spermatid differentiation, aberrations in PRM expression or changes in protein structure could be causes of certain types of idiopathic human male infertility. The aim of this study was to give insight into the role of PRM2 gene expression and caspase 9 activity in the pathogenesis of male infertility. MATERIALS AND METHODS: The current study included 70 men with idiopathic infertility and 64 fertile men who attended the andrology outpatient clinic at Mansoura University Hospital. Semen sample analyses were done according to WHO recommendations. The acrosome reaction of spermatozoa recovered from each sample was assessed. Samples were separated using discontinuous gradient separation. From each semen sample mature sperm were separated from immature sperm. The resulting samples were divided into 2 parts, including one to determine caspase 9 activity and the other for RNA extraction and reverse transcriptase-polymerase chain reaction of PRM2 gene expression. The polymerase chain reaction product was electrophoresed on 2% agarose gel. RESULTS: PRM2 gene expression was significantly decreased in immature sperm extracted from the fertile and infertile groups. Caspase 9 activity was significantly increased in immature sperm extracted from both groups. CONCLUSIONS: Low levels of PRM2 may be associated with morphological abnormalities, initiation of the apoptotic pathway and decreasing sperm motility. PRM2 may be an important marker to better understand the key regulatory pathway of spermatogenesis and it may act as a crucial part of fertilization.

Androgen receptor expression relationship with semen variables in infertile men with varicocele.

PURPOSE: Androgen receptor, a member of the nuclear receptor superfamily, has important roles in male reproductive function. It is required for sexual differentiation, pubertal development, spermatogenesis regulation, meiosis completion and spermatocyte transition to haploid round spermatids. We assessed the association of androgen receptor expression and semen variables in infertile men with varicocele. MATERIALS AND METHODS: A total of 299 men were grouped into healthy, fertile controls, infertile men without varicocele and men with infertility associated with varicocele. A history was obtained, clinical examination and semen analysis were done and reproductive hormones were estimated. Androgen receptor expression and the acrosome reaction were determined in recovered spermatozoa. RESULTS: Androgen receptor expression was significantly decreased in infertile men with varicocele more than in infertile men without varicocele compared to fertile controls. Androgen receptor correlated positively with sperm count, motility, normal forms, velocity, linear velocity, acrosome reaction and α-glucosidase. It correlated negatively with serum follicle-stimulating hormone and estradiol. Multiple stepwise regression analysis of androgen receptor expression revealed that the sperm acrosome reaction and linearity index were the most affected independent variables. CONCLUSIONS: Androgen receptor expression was significantly decreased in infertile men with varicocele more than in infertile men without varicocele compared to fertile men. Androgen receptor expression correlated positively with sperm count, motility, normal forms, velocity, linear velocity and acrosome reaction.