Therapeutic combination silencing VEGF and SOX10 increases the antiangiogenic effect in the mouse melanoma model B16-F10 - in vitro and in vivo studies.

INTRODUCTION: Gene therapy is an innovative form of treatment of genetic diseases, in which psiRNA molecules silencing specific genes are applied. AIM: The study evaluated the anti-tumour effect of psiRNA silencing preparations of the vascular endothelial growth factor (VEGF) and Sry-related HMG-Box gene 10 (SOX10) on melanoma (B16-F10) by inhibiting angiogenesis. MATERIAL AND METHODS: The preparations based on plasmid vectors psiRNA silencing the gene SOX10 and VEGF that form complexes with cationic lipid (psiRNA/carrier) have been developed. psiRNA preparations were tested on the mouse melanoma cell line B16-F10, both in vitro and in vivo. The silencing activity of transfected melanoma cells with the obtained psiRNA preparations was examined using the qPCR and Western blot methods. The anti-tumour activity of psiRNA preparations on melanoma tumour cells was then evaluated in a mouse in vivo model. RESULTS: In vitro studies have shown that the B16-F10 cells efficiently transfect non-viral preparations - psiRNA: Lyovec (74-89%). Worth mentioning is the fact that silencing SOX10 in B16-F10 melanoma cells increases the expression of the COL18A1 gene (compared to the preparation inhibiting only VEGF), which codes the endostatin to stop angiogenesis. In vivo results show that the level of haemoglobin in tumours of mice treated with psiRNA formulations was over 6 times lower than controls and tumour mass was 60-80% lower. CONCLUSIONS: The novel study proves that simultaneous inhibition of SOX10 and VEGF enhances the antiangiogenic action and thus contributes to a significant halt of disease development. In addition, these data expand knowledge about SOX10 regulation and functions.

Profiling of microRNA as a tool to introduce rAAV vectors in gene therapy of breast cancer: A preliminary report.

BACKGROUND: Despite the wide range of diagnostic and therapeutic methods, breast cancer is responsible for many deaths each year. One of the original and novel cancer therapeutic approaches is gene therapy based on recombinant adeno-associated viral vectors. Among the molecular factors with the potential to become useful diagnostic biomarkers, microRNA (miRNA) molecules are being considered for personalized therapies. OBJECTIVES: The aim of the study was to examine the utility of miRNA profiling in the design of personalized recombinant adeno-associated virus (rAAV)-based gene therapy for breast cancer patients. MATERIAL AND METHODS: The analysis of 754 miRNAs in 7 breast cancer samples and control samples was performed using real-time polymerase chain reaction (PCR) based on TaqMan® Low-density Array (TLDA) cards. Online repositories were used to explore the relationship between miRNAs and genes encoding rAAV receptors (KIAA0319L, HSPG2, FGFR1, c-MET, PDGFRA, ITGB5, and RPSA). Then, we performed a comparative analysis of the results to examine the possibility of using miRNA profiling in the design of rAAV-based therapeutic protocols. RESULTS: Fifty-two percent of tested miRNAs were noted in at least 1 analyzed breast cancer and control tissue. Thirteen miRNAs were selected due to being outliers in the tested samples. In total, 155 miRNAs targeted genes encoding rAAV receptors in the tested samples (29 miRNAs for KIAA0319L, 60 miRNAs for c-MET, 31 miRNAs for HSPG2, 43 miRNAs for FGFR1, 36 miRNAs for PDGFRA, 18 miRNAs for RPSA, and 25 miRNAs for ITGB5). The expression of the selected miRNAs was not homogeneous across the 7 samples. CONCLUSION: Profiling of microRNA could be a significant factor in the design of rAAV-based personalized gene therapy for breast cancer patients.

Gestational weight gain and blood pressure control in physiological pregnancy and pregnancy complicated by hypertension.

BACKGROUND: Obesity is a widely recognised risk factor for chronic and gestational hypertension. Influence of gestational weight gain on blood pressure control throughout the pregnancy is not well characterised. MATERIAL AND METHODS: Women in the third trimester of a singleton pregnancy were recruited to the study. Medical records were analysed and a special survey was conducted to obtain history on hypertensive disorders in pregnancy and weight changes during pregnancy. Blood pressure measurements were taken during the office visit in line with international guidelines. Relationships between gestational weight gain and maximal and office values of systolic and diastolic blood pressure values were analysed. RESULTS: Data of 90 women in normal pregnancy, 40 with gestational hypertension and 21 with chronic hypertension were analysed. Gestational weight gain was 11.9 ± 4.6 kg in the normal pregnancy group, 13.0 ± 5 kg in the gestational hypertension group and 10.6 ± 3.4 kg in the chronic hypertension group. Gestational weight gain positively correlated with both office (r = 0.48; p < 0.001) and maximal blood pressure values (r = 0.34; p = 0.004) in normal pregnancy and with maximal blood pressure values (r = 0.57; p = 0.02) in women with chronic hypertension. No correlation was observed between gestational weight gain and blood pressure values among women with gestational hypertension. CONCLUSION: In normal pregnancy and in women with chronic hypertension greater gestational weight gain is related to higher blood pressure values in the third trimester.

Mosaic Recombinant Adeno-associated Virus Vector rAAV/DJ/CAG for Targeted Gene Delivery to Melanoma Cells Metastasized to the Lung.

BACKGROUND/AIM: Patients with metastasized melanoma have limited treatment options and poor diagnosis. Therefore, the development of treatments requires a new therapeutic approach, of which gene therapy using rAAV vectors can be proposed. The aim of the study was to examine the efficiency of the rAAV vector to transduce mouse melanoma cells both in vitro and in vivo. MATERIALS AND METHODS: Different rAAV serotypes encoding GFP under the control of both chicken beta-actin and cytomegalovirus promoters were used in the experiments. Intranasal, intraperitoneal, intravenous and intratumoral pathways of administration of rAAV vectors were tested using quantitative-PCR and immunohistochemical staining. RESULTS: The highest transduction efficiency in metastatic cells in vivo was observed 7 days after intranasal administration of a 1010 gc/0.03 ml dose of rAAV/DJ-CAG. CONCLUSION: Melanoma gene therapy based on rAAV vectors is a possible treatment option.

Downregulated expression of microRNAs associated with cardiac hypertrophy and fibrosis in physiological pregnancy and the association with echocardiographically-evaluated myocardial function.

The aim of the present study was to analyze the profiles of cardiac microRNAs (miRNAs/miRs) in healthy pregnant women and non-pregnant controls. A total of 61 healthy women >18 years of age with singleton pregnancies in the third trimester were compared with 19 non-pregnant controls. Specifically, expression of miRNAs associated with cardiac hypertrophy (miR-1, miR-17-5, miR-22, miR-34a, miR-124, miR-133a, miR-195, miR-199a-3p, miR-199b, miR-210, miR-222 and miR-1249) and miRNAs associated with cardiac hypertrophy and fibrosis (miR-15b, miR-21, miR-26a, miR-29-a, miR-29c, miR-30c, miR-101, miR-146a, miR-191, miR-208a-5p and miR-328) were analyzed and compared with echocardiographic examination results. Both groups had similar cardiac miRNA expression profiles, but differed in quantitative evaluation. Women in the third trimester of physiological pregnancy exhibited downregulation of certain profibrotic miRNAs (miR-21, miR-30c and miR-328), decreased expression of a hypertrophic and antimetabolic miRNAs (miR-146a), downregulation of an antifibrotic miRNA (miR-222), and downregulation of a hypertrophic miRNA (miR-195). In pregnant women, the indices of systolic function were associated with miR-195 expression, and an interplay between miR-17-5p and diastolic function was observed. While the profiles of cardiac miRNAs expressed in healthy pregnant women and healthy non-pregnant controls were similar, these two groups differed in terms of expression of specific miRNAs. In the third trimester of physiological pregnancy, a downregulation of miR-17-5p, miR-21, miR-30c, miR-146a, miR-195, miR-222 and miR-328 was observed. The differences in the association between echocardiographic indices with miRNAs in pregnant and non-pregnant women suggest that miRNAs regulate both the structure and function of the pregnant heart, influencing cardiac muscle thickness as well as systolic and diastolic function.

Rise in antifibrotic and decrease in profibrotic microRNA protect the heart against fibrosis during pregnancy: A preliminary study.

BACKGROUND: Physiological pregnancy is associated with volume overload. Unlike cardiac pathologies linked with volume overload, such as mitral or aortic regurgitation, pregnancy is thought to be unrelated to fibrosis of the heart. However, changes in the cardiac extracellular matrix during pregnancy remain poorly understood. OBJECTIVES: The aim of the study was to examine the expression of 11 microRNAs associated with cardiac fibrosis (miR-21, miR-26a, miR-26b-5p, miR-29b-3p, miR-29c-3p, miR-101a, miR-146a, miR-208a, miR-223 and miR-328) during pregnancy and to compare them with a healthy control group. MATERIAL AND METHODS: Six women in singleton pregnancy (30-36 weeks) and 6 non-pregnant women as a control group were included in the study. Each woman underwent an echocardiographic examination, and had blood pressure on both arms measured and a blood sample taken. MicroRNAs expression was analyzed using Custom TaqMan® Array MicroRNA Cards (Applied Biosystems, Foster City, USA). RESULTS: Median age of the pregnant women was 34 years (range 25-39 years) and of the control group 32 years (range 29-43 years). Median week of pregnancy was 34 years (range 31-36 years). Most of the examined microRNAs had a lower expression in the pregnancy group (fold change 1.0). CONCLUSIONS: In the 3rd trimester of physiological pregnancy, there is a 244% increase in expression of miR-101a and a decrease by 73% in expression of miR-328. Both of these changes can protect against fibrosis during volume overload occurring in physiological pregnancy.

Release kinetics of circulating miRNA-208a in the early phase of myocardial infarction.

BACKGROUND: The biochemical confirmation of myocardial infarction is based on cardiac troponin (cTnI or cTnT) determination. Recent scientific results suggested that microRNAs (miRNAs) might become a new biomarker of tissue injury. AIM: To evaluate the release kinetics of circulating heart-specific miRNA-208a and also to test the hypothesis that miRNA-208a can serve as an accessible, diagnostically sensitive plasma biomarker of ST-elevation acute myocardial infarction (STEMI). METHODS: Nineteen STEMI patients (four women and 15 men, aged 44-85 years), 12 patients with stable coronary artery disease (CAD), and eight patients with a negative observation of CAD as a control group were studied. Blood samples were collected on admission and at three, six, 12, 24, and 48 h afterwards; in the CAD and control group blood samples were taken only once. Plasma levels of miRNA-208a determined by real-time polymerase chain reaction and their relative fold changes were calculated. cTnI and creatinine kinase (CK)-MB mass were also measured in the patients' serum samples. RESULTS: miRNA-208a was increased in STEMI patients at the time of admission and nearly undetectable in CAD patients and controls. The peak of miRNA-208a was observed at 3 h after reperfusion (p < 0.001). The traditional biomarkers (cTnI and CK-MBmass), which increase later in comparison to miRNA-208a reaching the maximum concentrations 6 h after reperfusion, were observed. Circulating miRNA-208a levels strongly correlated with cTnI and CK-MBmass released from the infarcted area. CONCLUSIONS: These results demonstrate that plasma miRNA-208a is an interesting and promising candidate for a new biomarker released early after onset of myocardial infarction.