Downregulation of lncRNA-PVT1 participates in the development of progressive chronic kidney disease among patients with congestive heart failure

Patients and healthy controls

From June 2016 to June 2018, a total of 50 healthy controls (28 males and 22 females), 100 CHF patients with obvious complications (CHF group, 56 males and 44 females), and 50 CHF patients complicated with CKD (CHF + CKD group, 28 males and 22 females) at Affiliated Hospital of Shaoxing College of Arts and Sciences were included in the study. They were at the age of 46 to 68 years with a median of 57 years. All healthy controls showed normal physiological functions in systemic physiological exams. CKD was diagnosed based on urine and blood tests. CHF was diagnosed based on the fluid in the lungs on chest X-ray, heart size and/or blood flow to the heart muscle on electrocardiogram, heart rate, heart rhythm, and ventricle size. All participants signed informed consent. The study was approved by the Ethics Committee of our hospital.

Treatment and plasma preparations

The 50 patients in the CHF + CKD group were treated by optimized dialysis, angiotensin-converting enzyme inhibitors, or fluid overload control according to patients’ disease conditions and health conditions. Prior to therapy, fasting blood (2 ml) was extracted from all three groups of patients. During treatment, fasting blood was also extracted from CHF + CKD group at 1, 2, and 3 months after the treatment. All blood samples were mixed with citrate at a ratio of 1:10 in centrifugation tubes, followed by centrifugation at room temperature for 20 min at 1200 g. The supernatant (plasma) was collected and kept in liquid nitrogen.

Follow-up (2-year) of CHF group

To explore the predictive value of plasma PVT1 for CKD development in CHF patients, the 100 CHF patients were followed up for 2 years. Patients who died during the follow-up before the diagnosis of CKD were excluded. Follow-up was performed in a monthly manner through the outpatient visit.

RNA preparations

Total RNAs were extracted from all plasma samples using RNAzol (Sigma-Aldrich) and treated with DNase I (Invitrogen) for 2 h at 37 °C to remove genomic DNAs. RNA integrity was analyzed on a 5% urea-PAGE gel and Agilent 2100 Bioanalyzer. RNA purity was analyzed by OD26/280 ratio. Samples with RNA integrity value (RIN) higher than 9 and OD260/280 ratio around 2.0 were used in the subsequent experiments.

RT-qPCR assay

RNA samples with satisfactory quality were reverse transcribed into cDNA using SS-RT-IV kits (Invitrogen). To determine PVT1 expression, qPCRs were performed using SYBR Green Master Mix (Bio-Rad) with 18S rRNA as the internal control at conditions of denaturation at 95 °C for 1 min followed by 40 cycles of 10s at 95 °C and 55 s at 58 °C. Three technical replicates were included in each experiment. The relative Ct values of PVT1 were calculated as ΔCt = Ct (PVT1) – Ct (18S rRNA). The sample with the biggest ΔCt value was set to value “1”, and all other samples were normalized to this sample. Primer sequences used in PCR were PVT1 forward 5′-TGAGAACTGTCCTT ACGTGACC-3′ and reverse 5′-AGAGCACCAAGACTGGCTCT-3′ and 18S rRNA forward 5′-CTACCACATCCAAGGAAGC-3′ and reverse 5′-TTTTCGTCACTACCT CCCCG-3′.

Cardiomyocytes and treatment

Human AC16 cardiomyocytes (EMD Millipore, USA) were cultured in DMEM supplemented with 1% penicillin, 1% streptomycin, and 12% FBS at 37 °C in an incubator with 5% CO2. Cells were treated with 0, 1, 1, 2, and 5 μg/ ml LPS for 24 h prior to analyzing PVT1 expression.

Statistical analysis

PTV1 levels in plasma samples were expressed as mean values of three technical replicates. PVT1 expression levels during the treatment were represented by heatmaps plotted using Heml 1.0 software. The 100 CHF patients were divided into high and low PVT1 level groups (n = 50; cutoff value = the median PVT1 level in plasma samples of CHF patients). CKD-free curves were plotted and compared by log-rank test. P < 0.05 was considered statistically significant.

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