http://aiocm.org/member/Immunity,%20Inflammation,%20and%20Cancer.pdf

Immunity, Inflammation, and Cancer

Improved approaches for the detection of common epithelial malignancies are urgently needed to reduce the worldwide morbidity and mortality caused by cancer. MicroRNAs (miRNAs) are small (≈22 nt) regulatory RNAs that are frequently dysregulated in cancer and have shown promise as tissue-based markers for cancer classification and prognostication. We show here that miRNAs are present in human plasma in a remarkably stable form that is protected from endogenous RNase activity. miRNAs originating from human prostate cancer xenografts enter the circulation, are readily measured in plasma, and can robustly distinguish xenografted mice from controls. This concept extends to cancer in humans, where serum levels of miR-141 (a miRNA expressed in prostate cancer) can distinguish patients with prostate cancer from healthy controls. Our results establish the measurement of tumor-derived miRNAs in serum or plasma as an important approach for the blood-based detection of human cancer.

/pdf/circulating-micrornas-as-stable-blood-based-markers-for-3iuj944mh7.pdf

Circulating microRNAs as stable blood-based markers for cancer detection

BackgroundCoronavirus disease 2019 (Covid-19) is associated with diffuse lung damage. Glucocorticoids may modulate inflammation-mediated lung injury and thereby reduce progression to respiratory failure and death.MethodsIn this controlled, open-label trial comparing a range of possible treatments in patients who were hospitalized with Covid-19, we randomly assigned patients to receive oral or intravenous dexamethasone (at a dose of 6 mg once daily) for up to 10 days or to receive usual care alone. The primary outcome was 28-day mortality. Here, we report the final results of this assessment.ResultsA total of 2104 patients were assigned to receive dexamethasone and 4321 to receive usual care. Overall, 482 patients (22.9%) in the dexamethasone group and 1110 patients (25.7%) in the usual care group died within 28 days after randomization (age-adjusted rate ratio, 0.83; 95% confidence interval [CI], 0.75 to 0.93; P<0.001). The proportional and absolute between-group differences in mortality varied considerably according to the level of respiratory support that the patients were receiving at the time of randomization. In the dexamethasone group, the incidence of death was lower than that in the usual care group among patients receiving invasive mechanical ventilation (29.3% vs. 41.4%; rate ratio, 0.64; 95% CI, 0.51 to 0.81) and among those receiving oxygen without invasive mechanical ventilation (23.3% vs. 26.2%; rate ratio, 0.82; 95% CI, 0.72 to 0.94) but not among those who were receiving no respiratory support at randomization (17.8% vs. 14.0%; rate ratio, 1.19; 95% CI, 0.92 to 1.55).ConclusionsIn patients hospitalized with Covid-19, the use of dexamethasone resulted in lower 28-day mortality among those who were receiving either invasive mechanical ventilation or oxygen alone at randomization but not among those receiving no respiratory support. (Funded by the Medical Research Council and National Institute for Health Research and others; RECOVERY ClinicalTrials.gov number, NCT04381936. opens in new tab; ISRCTN number, 50189673. opens in new tab.)

/pdf/dexamethasone-in-hospitalized-patients-with-covid-19-2p8rrjo50m.pdf

Dexamethasone in Hospitalized Patients with Covid-19

MicroRNAs (miRNAs) circulate in the bloodstream in a highly stable, extracellular form and are being developed as blood-based biomarkers for cancer and other diseases. However, the mechanism underlying their remarkable stability in the RNase-rich environment of blood is not well understood. The current model in the literature posits that circulating miRNAs are protected by encapsulation in membrane-bound vesicles such as exosomes, but this has not been systematically studied. We used differential centrifugation and size-exclusion chromatography as orthogonal approaches to characterize circulating miRNA complexes in human plasma and serum. We found, surprisingly, that the majority of circulating miRNAs cofractionated with protein complexes rather than with vesicles. miRNAs were also sensitive to protease treatment of plasma, indicating that protein complexes protect circulating miRNAs from plasma RNases. Further characterization revealed that Argonaute2 (Ago2), the key effector protein of miRNA-mediated silencing, was present in human plasma and eluted with plasma miRNAs in size-exclusion chromatography. Furthermore, immunoprecipitation of Ago2 from plasma readily recovered non–vesicle-associated plasma miRNAs. The majority of miRNAs studied copurified with the Ago2 ribonucleoprotein complex, but a minority of specific miRNAs associated predominantly with vesicles. Our results reveal two populations of circulating miRNAs and suggest that circulating Ago2 complexes are a mechanism responsible for the stability of plasma miRNAs. Our study has important implications for the development of biomarker approaches based on capture and analysis of circulating miRNAs. In addition, identification of extracellular Ago2–miRNA complexes in plasma raises the possibility that cells release a functional miRNA-induced silencing complex into the circulation.

https://www.pnas.org/content/pnas/108/12/5003.full.pdf

Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma

Remdesivir in adults with severe COVID-19: a randomised, double-blind, placebo-controlled, multicentre trial.

Objective: MicroRNAs (miRNAs) have been shown to offer great potential in the diagnosis of cancer. We investigated whether plasma miRNAs could discriminate between patients with and without colorectal cancer (CRC). Methods: This study was divided into three phases: (1) marker discovery using real-time PCR-based miRNA profiling on plasma, corresponding cancerous and adjacent non-cancerous colonic tissues of five patients with CRC, along with plasma from five healthy individuals as controls; (2) marker selection and validation by real-time quantitative RT-PCR on a small set of plasma; and (3) independent validation on a large set of plasma from 90 patients with CRC, 20 patients with gastric cancer, 20 patients with inflammatory bowel disease (IBD) and 50 healthy controls. Results: Of the panel of 95 miRNAs analysed, five were upregulated both in plasma and tissue samples. All the five miRNAs were validated on the plasma of 25 patients with CRC and 20 healthy controls. Both miR-17-3p and miR-92 were significantly elevated in the patients with CRC (p Conclusion: MiR-92 is significantly elevated in plasma of patients with CRC and can be a potential non-invasive molecular marker for CRC screening.

/pdf/differential-expression-of-micrornas-in-plasma-of-patients-2g746o6ohl.pdf

Differential expression of microRNAs in plasma of patients with colorectal cancer: a potential marker for colorectal cancer screening.

Background: Circulating RNA in plasma/serum is an emerging field for noninvasive molecular diagnosis. Because RNA is widely thought to be labile in the circulation, we investigated the stability and various preanalytical factors that may affect RNA concentrations in blood specimens.

Methods: Blood samples were collected from 65 healthy volunteers. The effects of two preanalytical variables were studied: ( a ) time delay in processing of EDTA blood and clotted blood after venesection, and ( b ) freezing and thawing of plasma and serum. The lability of free added RNA in plasma was also investigated. Plasma/serum RNA was measured by a real-time quantitative reverse transcription-PCR assay for glyceraldehyde 3-phosphate dehydrogenase mRNA, whereas DNA was measured by a real-time quantitative PCR assay for the β-globin gene.

Results: No significant difference was found for plasma RNA concentrations obtained from uncentrifuged EDTA blood that had been left at 4 °C for 0, 6, and 24 h ( P =0.182). On the other hand, the serum RNA concentrations increased significantly over 24 h when uncentrifuged clotted blood was stored at 4 °C ( P 99% of the free added RNA could no longer be amplified after incubation in plasma for 15 s. Never-frozen plasma, freeze-thawed plasma, and thawed plasma left at room temperature for 1 h showed no significant differences in RNA concentration ( P =0.465). No significant difference was observed for freeze-thawed serum ( P = 0.430).

Conclusions: Plasma RNA is stable in uncentrifuged EDTA blood stored at 4 °C, but to obtain a stable serum RNA concentration, uncentrifuged clotted blood should be stored at 4 °C and processed within 6 h. A single freeze/thaw cycle produces no significant effect on the RNA concentration of plasma or serum.

Stability of Endogenous and Added RNA in Blood Specimens, Serum, and Plasma

https://escholarship.org/content/qt3147b77b/qt3147b77b.pdf?t=qafxta

Effects of early corticosteroid treatment on plasma SARS-associated Coronavirus RNA concentrations in adult patients.

The discovery of circulating fetal nucleic acid in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis. Thus far, a gender- and polymorphism-independent fetal-specific target that can be used for prenatal screening and monitoring in all pregnant women has not been reported. In addition, the origin of such circulating nucleic acid has remained unclear. Here we provide direct evidence that the placenta is an important source of fetal nucleic acid release into maternal plasma by demonstrating that mRNA transcripts from placenta-expressed genes are readily detectable in maternal plasma. The surprising stability of such placental mRNA species in maternal plasma and their rapid clearance after delivery demonstrate that such circulating mRNA molecules are practical markers for clinical use. The measurement of such plasma mRNA markers has provided a gender-independent approach for noninvasive prenatal gene expression profiling and has opened up numerous research and diagnostic possibilities.

https://www.pnas.org/content/pnas/100/8/4748.full.pdf

mRNA of placental origin is readily detectable in maternal plasma

H19 is an imprinted oncofetal non-coding RNA recently shown tobe the precursor of miR-675. The pathophysiological roles of H19and its mature product miR-675 to carcinogenesis have, however,notbeendeﬁned.Byquantitativereversetranscription–polymerasechain reaction, both H19 and miR-675 were found to be upregu-lated in human colon cancer cell lines and primary humancolorectal cancer (CRC) tissues compared with adjacent non-cancerous tissues. Subsequently, the tumor suppressorretinoblas-toma (RB) was conﬁrmed to be a direct target of miR-675 as themicroRNA suppressed the activity of the luciferase reporter car-rying the 3#-untranslated region of RB messenger RNA that con-tains the miR-675-binding site. Suppression of miR-675 bytransfection with anti-miR-675 increased RB expression and atthe same time, decreased cell growth and soft agar colony forma-tionin human coloncancercells. Reciprocally, enhanced miR-675expression by transfection with miR-675 precursor decreased RBexpression, increased tumor cell growth and soft agar colony for-mation. Moreover, the inverse relationship between the expres-sions of RB and H19/miR-675 was also revealed in human CRCtissues and colon cancer cell lines. Our ﬁndings demonstrate thatH19-derived miR-675, through downregulation of its target RB,regulates the CRC development and thus may serve as a potentialtarget for CRC therapy.IntroductionColorectal cancer (CRC) is the third most common cancer worldwidewith an estimated 1 million new cases and a half million deaths eachyear (1). Screening for CRC from curable early stages has the poten-tial to reduce both the incidence and mortality of the disease (2).Although 5 year mortality rates of CRC have slightly declined overthe last three decades, there is still a pressing need to identify newprognostic biomarkers and therapeutic targets for this disease. Fur-thermore, the underlying pathophysiological mechanisms of CRC de-velopment remain elusive (1–4).MicroRNAs (miRNAs) are 19- to 25-nucleotide regulatory non-codingRNAsthatareinitiallyexpressedashairpintranscriptsofprimarymiRNA.TheseprimarymiRNAhairpinsarecleavedbytwoRNAaseIIIenzymes, Drosha and Dicer, to generate mature miRNAs. MiRNAsregulate the expressions of a wide variety of genes by translation re-pression or promoting RNA degradation and are important in the regu-lation of various cellular processes, such as cellular proliferation,differentiation and apoptosis (5–7). To date, .723 human miRNAsare annotatedinthe miRBase registry(miRBaseversion 11.0), but mostof the genes regulated by human miRNAs are not well deﬁned.Dysregulation of a speciﬁc spectrum of miRNAs in human malig-nancies is frequently observed. Emerging evidence suggests miRNAsfunction as both tumor suppressors and oncogenes. About 50% ofannotated human miRNAs located at chromosomal regions involvedin loss of heterozygosity, ampliﬁcation or breakpoints that are asso-ciated with cancers (5,7,8). The miRNAs downregulated in humancancers indicate that they may function as tumor suppressors. Let-7,which targets the oncogene RAS, has shown to be downregulated inBCL2, are downregulated in chronic lymphocytic leukemias (10).Expression levels of miR-143 that targets ERK5 and miR-145 werefound to be decreased in colon cancer (11). In contrast, the miRNAsupregulated in cancers may function as oncogenes. MiR-155 and itshost gene BIC are highly expressed in several types of B-cell lym-phoma (12). The miR-17-92 cluster, which is located on chromosome13q31, is activated by the oncogene c-Myc and is highly expressed inB-cell lymphoma and lung cancer (13). Therefore, the importance ofmiRNAs acting as a new layer of gene regulation in tumorigenesis isemerging.H19 is a paternally imprinted (maternally expressed) oncofetalgene and is located on chromosome 11p15.5, close to the IGF II genelocus. The H19 gene does not encode for a protein but instead codesfor a capped, spliced and polyadenylated 2.7 kb RNA (14–16). H19 ishighly expressed from the early stages of embryogenesis to fetal lifein many organs including the fetal adrenal, liver and placenta but isnearly completely downregulated postnatally (17).Emerging evidence showed that H19 expression was upregulated inmany cancers including CRC (18,19), hepatocellular carcinoma(20), testicular cancer (21), choriocarcinoma (22), esophageal cancer(18), ovarian cancer(23), breast cancer (24) and bladder cancer (25,26),with or without the loss of imprinting. Patients with more H19-positive bladder cancer cells are potentially at higher risk of recurrentdisease (27). In the tumor formed by the injection of choriocarcinomaJar and JEG-3 cells into the nude mice, the H19 RNA level is higherthan those cells before the injection (28). Similarly, the H19 RNAlevel is greatly enhanced in tumor of human bladder carcinoma cellsformed in nude mice (25). The overexpression of H19 in cancertissues hints for its oncogenic function, but the exact underlyingmechanism is still not clear. Recently, H19 was reported to be theprimary miRNA precursor of miR-675 in both human and mice (29).As both H19 and miRNAs are believed to be involved in tumorio-genesis, this prompted us to speculate that the tumoriogenesis processinduced by H19 may be mediated through miR-675. Therefore, in thisstudy, we investigated the pathophysiological roles of H19 and miR-675 in CRC carcinogenesis. Furthermore, using in silico predictionand in vitro functional assays, we conﬁrmed retinoblastoma (RB)protein as a putative direct target of miR-675. This veriﬁcation ofthe oncogenic function of H19-miR-675-RB in CRC suggests thatthis pathway may serve as the potential target for cancer therapy.Materials and methods

Enders K.O. Ng

Papers

Differential expression of microRNAs in plasma of patients with colorectal cancer: a potential marker for colorectal cancer screening.

Stability of Endogenous and Added RNA in Blood Specimens, Serum, and Plasma

Effects of early corticosteroid treatment on plasma SARS-associated Coronavirus RNA concentrations in adult patients.

mRNA of placental origin is readily detectable in maternal plasma

Oncofetal H19-derived miR-675 regulates tumor suppressor RB in human colorectal cancer.