Rapid Generation of MicroRNA Sponges for MicroRNA Inhibition

MicroRNA (miRNA) sponges are transcripts with repeated miRNA antisense sequences that can sequester miRNAs from endogenous targets. MiRNA sponges are valuable tools for miRNA loss-of-function studies both in vitro and in vivo. We developed a fast and flexible method to generate miRNA sponges and tested their efficiency in various assays. Using a single directional ligation reaction we generated sponges with 10 or more miRNA binding sites. Luciferase and AGO2-immuno precipitation (IP) assays confirmed effective binding of the miRNAs to the sponges. Using a GFP competition assay we showed that miR-19 sponges with central mismatches in the miRNA binding sites are efficient miRNA inhibitors while sponges with perfect antisense binding sites are not. Quantification of miRNA sponge levels suggests that this is at least in part due to degradation of the perfect antisense sponge transcripts. Finally, we provide evidence that combined inhibition of miRNAs of the miR-17∼92 cluster results in a more effective growth inhibition as compared to inhibition of individual miRNAs. In conclusion, we describe and validate a method to rapidly generate miRNA sponges for miRNA loss-of-function studies

Bulged miRNA sponges can effectively inhibit miRNA function.

<p>(<b>A</b>) GFP competition assay in WEHI-231 B-cell lymphoma cells infected with empty vector (EV), bulged and perfect miR-19 sponges. Bulged MBS sponges have mild (2 MBS) or strong effects (6 and 20 MBS) on cell growth while perfect MBS sponges and the empty vector control have no effect. Legend: x = EV; Δ = P-2MBS; □ = P-6MBS; ○ = P-18MBS; ▴ = B-2MBS; ▪ =  = B-6MBS; ▾ = B-20MBS. (<b>B</b>) From the experiment shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029275#pone-0029275-g003" target="_blank">Fig. 3a</a>, EV, perfect en bulged MBS sponge infected GFP+ cells were sorted at day 14. Quantification of sponge (LTR driven) and GFP transcript levels (PGK driven) by qRT-PCR revealed lowered sponge/GFP transcript ratios for the perfect sponges but not for the bulged sponges as compared to the empty vector control. The number of MBS per sponge vector is indicated on the x-axis. (<b>C</b>) Quantification of sponge transcript levels in the AGO2-immunoprecipitated (IP) fraction of miR-155 sponge infected KM-H2 cells. AGO2-IP of miR-155 sponge infected KM-H2 cells was performed and followed by quantification of miR-155 sponge transcript levels in total, flowthrough (FT) and IP fraction of AGO2 and IgG IP experiments. MiR-155 sponge transcripts are highly enriched in the AGO2-IP fraction but not in the IgG-IP control fraction. In the inset of the graph a Western Blot which shows that AGO2 levels are enriched in the IP fraction upon AGO2-IP but not in the IgG-IP control. (<b>D</b>) GFP competition assay in WEHI-231 cells infected with individual bulged miRNA sponges against each member of the miR-17∼92 cluster and combination (combi-sp) sponges against each miRNA family of the miR-17∼92 cluster. As a control, empty vector and a combi-sp vector with seed scrambled miRNA BS (combi-scr) were used. All individual miRNA sponges show negative effects on cell growth, however, the strongest effects are observed when all miRNAs of the miR-17∼92 cluster are inhibited simultaneously. No effects on proliferation were observed with the empty vector and combi-scr vector controls. Legend: x = EV; □ = miR-17-5p-sp (12MBS); Δ = miR-18a-sp (10MBS); * = miR-19-sp (20MBS); ı = miR-20a-sp (7MBS); ○ = miR-92a-sp (10MBS); ▪ = Combi-sp1 (4×3MBS); ▴ = Combi-sp2 (4×3MBS); • = Combi-scr (4×3MBSscr). * = p-value <0.05; ** = p-value <0.01; *** = p-value <0.001.</p

Confirmation of miR-19 binding to miR-19 sponge constructs.

<p>(<b>A</b>) Luciferase reporter assays in HEK293 cells reveal that repression of Renilla activity is more prominent in reporter vectors that contain perfect MBS sequences as compared to reporter vectors that encode bulged MBS sequences and is in both cases dependent on the number of MBS. (<b>B</b>) Release of miR-19 specific repression of Renilla luciferase activity by anti-miR-19a/b oligos confirms that miR-19 binds to the miR-19 MBS sequences. No release of luciferase activity is observed with a control anti-miR-16 oligo. Open bars: Mock, grey bars: miR-16 inhibitor and black bar: miR-19 inhibitor mix. For each graph the number of MBS per reporter vector is indicated on the x-axis and on the y-axis the ratio of Renilla (R) over Firefly (F) luciferase is depicted. * = p-value <0.05; ** = p-value <0.01; *** = p-value <0.001.</p

The rapid generation of miRNA sponges.

<p>(<b>A</b>) Schematic overview of the strategy to ligate miRNA sponge oligo duplexes into the pMSCV-PIG-sp vector. Each oligo duplex contains 2 miRNA binding sites (MBS) and phosphorylated SanDI restriction enzyme compatible overhangs to enable miRNA sponge generation in one single ligation reaction. SanDI overhangs are non-palindromic allowing for directional cloning of the oligo duplexes. Arrows indicate transcription start sites. (<b>B</b>) Overview of the number of MBS per clone generated from ligation reactions with vector/duplex ratios varying from 1∶3 up to 1∶1000. Per ratio the mean is indicated.</p

COVID-19-related mortality in kidney transplant and dialysis patients: Results of the ERACODA collaboration

Background. Patients on kidney replacement therapy comprise a vulnerable population and may be at increased risk of death from coronavirus disease 2019 (COVID-19). Currently, only limited data are available on outcomes in this patient population. Methods. We set up the ERACODA (European Renal Association COVID-19 Database) database, which is specifically designed to prospectively collect detailed data on kidney transplant and dialysis patients with COVID-19. For this analysis, patients were included who presented between 1 February and 1 May 2020 and had complete information available on the primary outcome parameter, 28-day mortality. Results. Of the 1073 patients enrolled, 305 (28%) were kidney transplant and 768 (72%) dialysis patients with a mean age of 60 6 13 and 67 6 14 years, respectively. The 28-day probability of death was 21.3% [95% confidence interval (95% CI) 14.3\u201330.2%] in kidney transplant and 25.0% (95% CI 20.2\u201330.0%) in dialysis patients. Mortality was primarily associated with advanced age in kidney transplant patients, and with age and frailty in dialysis patients. After adjusting for sex, age and frailty, in-hospital mortality did not significantly differ between transplant and dialysis patients [hazard ratio (HR) 0.81, 95% CI 0.59\u20131.10, P \ubc 0.18]. In the subset of dialysis patients who were a candidate for transplantation (n \ubc 148), 8 patients died within 28 days, as compared with 7 deaths in 23 patients who underwent a kidney transplantation &lt;1 year before presentation (HR adjusted for sex, age and frailty 0.20, 95% CI 0.07\u20130.56, P &lt; 0.01). Conclusions. The 28-day case-fatality rate is high in patients on kidney replacement therapy with COVID-19 and is primarily driven by the risk factors age and frailty. Furthermore, in the first year after kidney transplantation, patients may be at increased risk of COVID-19-related mortality as compared with dialysis patients on the waiting list for transplantation. This information is important in guiding clinical decision-making, and for informing the public and healthcare authorities on the COVID-19-related mortality risk in kidney transplant and dialysis patients

Recovery of dialysis patients with COVID-19: health outcomes 3 months after diagnosis in ERACODA

© The Author(s) 2022.Background. Coronavirus disease 2019 (COVID-19)-related short-term mortality is high in dialysis patients, but longer-term outcomes are largely unknown. We therefore assessed patient recovery in a large cohort of dialysis patients 3 months after their COVID-19 diagnosis. Methods. We analyzed data on dialysis patients diagnosed with COVID-19 from 1 February 2020 to 31 March 2021 from the European Renal Association COVID-19 Database (ERACODA). The outcomes studied were patient survival, residence and functional and mental health status (estimated by their treating physician) 3 months after COVID-19 diagnosis. Complete follow-up data were available for 854 surviving patients. Patient characteristics associated with recovery were analyzed using logistic regression. Results. In 2449 hemodialysis patients (mean ± SD age 67.5 ± 14.4 years, 62% male), survival probabilities at 3 months after COVID-19 diagnosis were 90% for nonhospitalized patients (n = 1087), 73% for patients admitted to the hospital but not to an intensive care unit (ICU) (n = 1165) and 40% for those admitted to an ICU (n = 197). Patient survival hardly decreased between 28 days and 3 months after COVID-19 diagnosis. At 3 months, 87% functioned at their pre-existent functional and 94% at their pre-existent mental level. Only few of the surviving patients were still admitted to the hospital (0.8–6.3%) or a nursing home (∼5%). A higher age and frailty score at presentation and ICU admission were associated with worse functional outcome. Conclusions. Mortality between 28 days and 3 months after COVID-19 diagnosis was low and the majority of patients who survived COVID-19 recovered to their pre-existent functional and mental health level at 3 months after diagnosis

Association of obesity with 3-month mortality in kidney failure patients with COVID-19

Background: In the general population with coronavirus disease 2019 (COVID-19), obesity is associated with an increased risk of mortality. Given the typically observed obesity paradox among patients on kidney function replacement therapy (KFRT), especially dialysis patients, we examined the association of obesity with mortality among dialysis patients or living with a kidney transplant with COVID-19. Methods: Data from the European Renal Association COVID-19 Database (ERACODA) were used. KFRT patients diagnosed with COVID-19 between 1 February 2020 and 31 January 2021 were included. The association of Quetelet's body mass index (BMI) (kg/m2), divided into: <18.5 (lean), 18.5-24.9 (normal weight), 25-29.9 (overweight), 30-34.9 (obese I) and ≥35 (obese II/III), with 3-month mortality was investigated using Cox proportional-hazards regression analyses. Results: In 3160 patients on KFRT (mean age: 65 years, male: 61%), 99 patients were lean, 1151 normal weight (reference), 1160 overweight, 525 obese I and 225 obese II/III. During follow-up of 3 months, 28, 20, 21, 23 and 27% of patients died in these categories, respectively. In the fully adjusted model, the hazard ratios (HRs) for 3-month mortality were 1.65 [95% confidence interval (CI): 1.10, 2.47], 1 (ref.), 1.07 (95% CI: 0.89, 1.28), 1.17 (95% CI: 0.93, 1.46) and 1.71 (95% CI: 1.27, 2.30), respectively. Results were similar among dialysis patients (N = 2343) and among those living with a kidney transplant (N = 817) (Pinteraction = 0.99), but differed by sex (Pinteraction = 0.019). In males, the HRs for the association of aforementioned BMI categories with 3-month mortality were 2.07 (95% CI: 1.22, 3.52), 1 (ref.), 0.97 (95% CI: 0.78. 1.21), 0.99 (95% CI: 0.74, 1.33) and 1.22 (95% CI: 0.78, 1.91), respectively, and in females corresponding HRs were 1.34 (95% CI: 0.70, 2.57), 1 (ref.), 1.31 (95% CI: 0.94, 1.85), 1.54 (95% CI: 1.05, 2.26) and 2.49 (95% CI: 1.62, 3.84), respectively. Conclusion: In KFRT patients with COVID-19, on dialysis or a kidney transplant, obesity is associated with an increased risk of mortality at 3 months. This is in contrast to the obesity paradox generally observed in dialysis patients. Additional studies are required to corroborate the sex difference in the association of obesity with mortality

Clinical, Functional, and Mental Health Outcomes in Kidney Transplant Recipients 3 Months after a Diagnosis of COVID-19

Background. Kidney transplant patients are at high risk for coronavirus disease 2019 (COVID-19)-related mortality. However, limited data are available on longer-term clinical, functional, and mental health outcomes in patients who survive COVID-19. Methods. We analyzed data from adult kidney transplant patients in the European Renal Association COVID-19 Database who presented with COVID-19 between February 1, 2020, and January 31, 2021. Results. We included 912 patients with a mean age of 56.7 (±13.7) y. 26.4% were not hospitalized, 57.5% were hospitalized without need for intensive care unit (ICU) admission, and 16.1% were hospitalized and admitted to the ICU. At 3 mo follow-up survival was 82.3% overall, and 98.8%, 84.2%, and 49.0%, respectively, in each group. At 3 mo follow-up biopsy-proven acute rejection, need for renal replacement therapy, and graft failure occurred in the overall group in 0.8%, 2.6%, and 1.8% respectively, and in 2.1%, 10.6%, and 10.6% of ICU-admitted patients, respectively. Of the surviving patients, 83.3% and 94.4% reached their pre-COVID-19 physician-reported functional and mental health status, respectively, within 3 mo. Of patients who had not yet reached their prior functional and mental health status, their treating physicians expected that 79.6% and 80.0%, respectively, still would do so within the coming year. ICU admission was independently associated with a low likelihood to reach prior functional and mental health status. Conclusions. In kidney transplant recipients alive at 3-mo follow-up, clinical, physician-reported functional, and mental health recovery was good for both nonhospitalized and hospitalized patients. Recovery was, however, less favorable for patients who had been admitted to the ICU