A mutator phenotype due to a DNA mismatch repair deficiency is usually detected by typing a number of microsatellite markets. Here, eight hereditary nonpolyposis colon cancer patients with microsatellite instability were investigated by inter-Alu PCR, known to amplify DNA segments that may represent preferential targets of replication errors. Among 40-60 bands revealed in a single PCR experiment, more than 20% were found altered in tumoral DNA samples compared to matched normal samples from the same patient. Shifts and changes in signal intensity accounted for most of the alterations, whereas gains or losses of bands were rare. Certain bands were affected only in a single patient, whereas the instabilities in others were common. These results suggest that some genomic regions are more susceptible than others to the expression of a mutator phenotype. Four such bands altered in at least five patients were characterized further and shown to be unstable because of contractions of the Alu poly(A) tails. Interestingly, none of the bands representing loci shown previously to be polymorphic in the population displayed instability in the tumoral samples. Inter-Alu PCR appears to be a robust, cost-effective, and sensitive technique for revealing the mutator phenotype in cancer cells.
Background: Hypertension (HTN) causes vascular injury identified by endothelial dysfunction, vascular stiffening, and remodeling, which contributes to kidney damage leading to chronic kidney disease (CKD). MicroRNAs (miRNAs) repress/degrade target mRNAs. Their role in vascular injury in HTN remains unclear. We aimed to identify differentially expressed (DE) miRNAs in gluteal subcutaneous arteries of patients with HTN associated or not with CKD to shed light on the pathophysiological molecular mechanisms. Methods: Normotensive subjects and patients with HTN associated or not with CKD grades 3-4 were studied (n=15-16). Small arteries were isolated from gluteal subcutaneous biopsies, RNA extracted and small and total RNA sequencing performed by Illumina HiSeq-2500. DE genes were identified with a P <0.05 and fold change (FC) >1.3. Top 3 DE miRNAs ( P <0.001, FC>2, mean read count number (MRCN) >3,000 in all groups and having predicted mRNA targets) were selected for validation by reverse transcription-quantitative PCR (RT-qPCR). The mRNA targets of the top selected miRNA were predicted by TargetScan with P <0.01, FC>1.5 and MRCN>150 and the top 9 targets were validated by RT-qPCR using gain- and loss-of-function in human aortic endothelial cells (HAECs). Gene ontology enrichment analysis (GOEA) was done in Cytoscape. Results: DE miRNAs and mRNAs were identified uniquely associated with HTN (miRNAs: 10, mRNAs: 68), CKD (miRNAs: 68, mRNAs: 395), and in both groups (miRNAs: 2, mRNAs: 32). miR-338-3p presented the best correlation between RNA sequencing and RT-qPCR (R 2 =0.328, P <0.001) among the top 3 DE miRNAs. Two of the selected top 9 miR-338-3p predicted targets were validated in HAECs. Protein tyrosine phosphatase receptor type S ( PTPRS , FC: 0.80±0.08 vs 1.00±0.00) and glutathione peroxidase 3 ( GPX3 , FC: 0.88±0.04 vs 1.00±0.00) were down-regulated in HAECs transfected with miR-338-3p mimics ( P <0.05). GOEA showed association of GPX3 with oxidative stress detoxification ( q <0.05), and of PTPRS with the immune system, neuronal system and developmental process ( q <0.001). Conclusion: Down-regulated miR-338-3p in gluteal subcutaneous small arteries of hypertensive patients with CKD targets PTPRS and GPX3 that may play a role in vascular injury in HTN.
Background: Rat chromosome (RNO) 2 introgression from normotensive Brown Norway (BN) rats into hypertensive Dahl salt sensitive (SS) background (consomic SB2) reduced vascular inflammation. We hypothesized that the BN-RNO2 contains genes that reduce vascular inflammation, which could be identified using microRNA (miRNA) and total RNA expression profiling in aorta of congenic rats containing different portions of BN-RNO2 on the SS background. Methods and Results: Twelve-to-13-week-old male SS rats and congenic rats containing the distal portion of BN-RNO2 (SB2a), the middle segment (SB2b) and the proximal segment (SB2e) on the SS background, fed a normal-salt diet, were studied. Systolic blood pressure (SBP) was measured by telemetry. SBP was lower in SB2a and SB2b but not SB2e compared to SS (125±3, 127±6, 138±4 vs 146±2 mm Hg, P <0.05). Total RNA was extracted from aorta and used to construct libraries for small and total RNA sequencing using Illumina HiSeq-2500. The bioinformatics pipeline included: FastQC for quality control, STAR for genome alignment to Rattus norvegicus release-86, mirdeep2 for miRNA annotation and counting, Htseq-count for mRNA and long non-coding RNA annotation and counting; R for differential expression analysis. Differentially expressed miRNAs and genes (mRNA and non-coding RNA) were identified in SB2a vs SS (miRNAs: 3 up and 2 down, genes: 1 up and 3 down), SB2b vs SS (miRNAs: 2 up and 3 down, genes: 67 up and 112 down) and SB2e vs SS (miRNAs: 29 up and 25 down, genes: 12 up and 35 down), with FDR<0.05. Differentially expressed genes encoded within different BN-RNO2 congenic portions were identified in SB2a vs SS (2 down), SB2b vs SS (14 up and 18 down) and SB2e vs SS (1 down). Conclusions and Perspectives: Differentially expressed BN-RNO2 encoded genes were identified in aorta of congenic SB2a, SB2b and SB2e rats. Whether these genes play a role in inflammation or vascular injury remains to be determined.
Background: Transgenic mice with tamoxifen-inducible endothelium-restricted human endothelin-1 overexpression (ieET-1) exhibited blood pressure (BP) elevation 3 weeks or 3 months after induction, and vascular injury was observed after 3 months. We hypothesized that 3-week or 3-month exposure to ET-1 overexpression leads to gene dysregulation in mesenteric arteries (MAs). Methods: Ten to 12-week old male ieET-1 mice and control ieCre mice expressing a tamoxifen-inducible Cre recombinase under the control of the endothelium-specific Tie2 promoter were treated with tamoxifen (1 mg/kg/day, s.c.) for 5 days and euthanized 16 days or 3 months later. RNA was extracted from MAs and used for total RNA-sequencing using Illumina HiSeq-2500. Differentially expressed (DE) genes were identified with fold change >1.3 and P <0.005. DE genes were validated by reverse transcription-quantitative PCR (RT-qPCR) using another set of mice. Alternative splicing was revealed by Spladder and validated by RT-qPCR in MAs. Results: RNA-sequencing revealed DE genes after 3-week (54) and 3-month ET-1 overexpression (7). Alternative splicing changes in mRNAs were revealed after 3-week (39) and 3-month ET-1 overexpression (21). One of the 3 genes validated by RT-qPCR, Khdrbs3, encodes KH domain containing RNA binding signal transduction associated 3 that regulates exon retention of some mRNAs including exon 7 of vascular endothelial growth factor A ( Vegfa ). Khdrbs3 was up-regulated after 3-week (fold change: 2.4±0.1 vs 1.0±0.1, P <0.05) and 3-month ET-1 overexpression (2.3±0.3 vs 1.3±0.1, P <0.05). Vegfa exon 7 retention was validated by demonstration of up-regulation of Vegfa 164 (1.3±0.1 vs 0.9±0.1, P<0.05) and Vegfa 188 isoforms (1.1±0.1 vs 0.9±0.1, P<0.05) after 3-month ET-1 overexpression ( P <0.05). Neuropilin 2 ( Nrp2 ) that interacts with VEGFA receptors was one of DE genes with alternative splicing in exon 17 ( Nrp2 a isoforms). Predicted Nrp2 a down-regulation ( P <0.05) was validated by RT-qPCR showing a decrease after 3-month ET overexpression (0.6±0.1 vs 1.1±0.2, P <0.05). Conclusions: This study demonstrated that 3-month ET-1 overexpression up-regulated Khdrbs3 and alternative splicing of Vegfa and Nrp2 , which may play a role in vascular injury in hypertension.
Vascular injury is an early manifestation in hypertension and a cause of end-organ damage. MicroRNAs play an important role in cardiovascular disease, but their implication in vascular injury in hypertension remains unclear. This study revealed using an unbiased approach, microRNA and mRNA sequencing with molecular interaction analysis, a microRNA-transcription factor coregulatory network involved in vascular injury in mice made hypertensive by 14-day Ang II (angiotensin II) infusion. A candidate gene approach identified upregulated miR-431-5p encoded in the conserved 12qF1 (14q32 in humans) microRNA cluster, whose expression correlated with blood pressure, and which has been shown to be upregulated in human atherosclerosis, as a potential key regulator in Ang II-induced vascular injury. Gain- and loss-of-function in human vascular smooth muscle cells demonstrated that miR-431-5p regulates in part gene expression by targeting ETS homologous factor. In vivo miR-431-5p knockdown delayed Ang II-induced blood pressure elevation and reduced vascular injury in mice, which demonstrated its potential as a target for treatment of hypertension and vascular injury.
