Our previous studies have demonstrated that stable microRNAs (miRNAs) in mammalian serum and plasma are actively secreted from tissues and cells and can serve as a novel class of biomarkers for diseases, and act as signaling molecules in intercellular communication. Here, we report the surprising finding that exogenous plant miRNAs are present in the sera and tissues of various animals and that these exogenous plant miRNAs are primarily acquired orally, through food intake. MIR168a is abundant in rice and is one of the most highly enriched exogenous plant miRNAs in the sera of Chinese subjects. Functional studies in vitro and in vivo demonstrated that MIR168a could bind to the human/mouse low-density lipoprotein receptor adapter protein 1 (LDLRAP1) mRNA, inhibit LDLRAP1 expression in liver, and consequently decrease LDL removal from mouse plasma. These findings demonstrate that exogenous plant miRNAs in food can regulate the expression of target genes in mammals.

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Exogenous plant MIR168a specifically targets mammalian LDLRAP1: Evidence of cross-kingdom regulation by microRNA

Efficient patient management relies on early diagnosis of disease and monitoring of treatment. In this regard, much effort has been made to find informative, blood-based biomarkers for patients with cancer. Owing to their attributes-which are specifically modulated by the tumour-circulating cell-free microRNAs found in the peripheral blood of patients with cancer may provide insights into the biology of the tumour and the effects of therapeutic interventions. Moreover, the role of microRNAs in the regulation of different cellular processes points to their clinical utility as blood-based biomarkers and future therapeutic targets. MicroRNAs are optimal biomarkers owing to high stability under storage and handling conditions and their presence in blood, urine and other body fluids. In particular, detection of levels of microRNAs in blood plasma and serum has the potential for an earlier cancer diagnosis and to predict prognosis and response to therapy. This Review article considers the latest developments in the use of circulating microRNAs as prognostic and predictive biomarkers and discusses their utility in personalized medicine.

Clinical relevance of circulating cell-free microRNAs in cancer

Circulating, cell-free microRNAs (miRNAs) hold great promise as a new class of cancer biomarkers due to their surprisingly high stability in plasma, association with disease states, and ease of sensitive measurement. Yet little is known about the origin of circulating miRNAs in either healthy or sick people, or what factors influence levels of circulating miRNA biomarkers. Of 79 solid tumor circulating miRNA biomarkers reported in the literature, we found that fifty-eight percent (47/79) are highly expressed in one or more blood cell type. Plasma levels of miRNA biomarkers expressed by myeloid (e.g., miR-223, miR-197, miR-574-3p, let-7a) and lymphoid (e.g., miR-150) blood cells tightly correlated with corresponding white blood cell counts. Plasma miRNA biomarkers expressed by red blood cells (e.g., miR-486-5p, miR-451, miR-92a, miR-16) could not be correlated to red cell counts due to limited variation in hematocrit in the cohort studied, but were significantly increased in hemolyzed specimens (20-30 fold plasma increase; p<0.0000001). Finally, in a patient undergoing autologous hematopoietic cell transplantation, plasma levels of myeloid- and lymphoid-expressed miRNAs (miR-223 and miR-150, respectively) tracked closely with changes in corresponding blood counts. We present evidence that blood cells are a major contributor to circulating miRNA, and that perturbations in blood cell counts and hemolysis can alter plasma miRNA biomarker levels by up to 50-fold. Given that a majority of reported circulating miRNA cancer biomarkers are highly expressed in blood cells, we suggest caution in interpretation of such results as they may reflect a blood cell-based phenomenon rather than a cancer-specific origin.

https://cancerpreventionresearch.aacrjournals.org/content/canprevres/5/3/492.full.pdf

Blood cell origin of circulating microRNAs: a cautionary note for cancer biomarker studies.

Extracellular microRNAs (miRNA) are present in most biological fluids, relatively stable, and hold great potential for disease biomarkers and novel therapeutics. Circulating miRNAs are transported by membrane-derived vesicles (exosomes and microparticles), lipoproteins, and other ribonucleoprotein complexes. Evidence suggests that miRNAs are selectively exported from cells with distinct signatures that have been found to be altered in many pathophysiologies, including cardiovascular disease. Protected from plasma ribonucleases by their carriers, functional miRNAs are delivered to recipient cells by various routes. Transferred miRNAs use cellular machinery to reduce target gene expression and alter cellular phenotype. Similar to soluble factors, miRNAs mediate cell-to-cell communication linking disparate cell types, diverse biological mechanisms, and homeostatic pathways. Although significant advances have been made, miRNA intercellular communication is full of complexities and many questions remain. This review brings into focus what is currently known and outstanding in a novel field of study with applicability to cardiovascular disease.

Intercellular Transport of MicroRNAs

The tumor suppressor p53 is one of the most frequently mutated genes in human cancers. MicroRNAs (miRNAs) are small non-protein coding RNAs that regulate gene expression on the post-transcriptional level. Recently, it was shown that p53 regulates the expression of several miRNAs, thereby representing an important mechanism of p53 signaling. Several independent studies identified the members of the miR-34 family as the most prevalent p53-induced miRNAs. miR-34s are frequently silenced in variety of tumor entities, suggesting that they are important tumor suppressors. Indeed, ectopic expression of miR-34s inhibits proliferation, epithelial to mesenchymal transition, migration, invasion, and metastasis of various cancer cell entities. Moreover, delivery or re-expression of miR-34 leads to notable repression of tumor growth and metastasis in cancer mouse models, and may therefore represent an efficient strategy for future cancer therapeutics. Besides their crucial functions in cancer, members of the miR-34 family also play important roles in spermatogenesis, stem cell differentiation, neuronal development, aging, and cardiovascular functions. Consequently, miR-34 has also been implicated in various non-cancerous diseases, such as brain disorders, osteoporosis, and cardiovascular complications.

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The p53/miR-34 axis in development and disease

BACKGROUND: Sensitive and specific biomarkers for the early detection of esophageal squamous cell carcinoma (ESCC) are urgently needed to reduce the high morbidity and mortality of the disease. The discovery of serum microRNAs (miRNAs) and their unique concentration profiles in patients with various diseases makes them attractive, novel noninvasive biomarkers for tumor diagnosis. In this study, we investigated the serum miRNA profile in ESCC patients to develop a novel diagnostic ESCC biomarker.

METHODS: Serum samples were taken from 290 ESCC patients and 140 age- and sex-matched controls. Solexa sequencing technology was used for an initial screen of miRNAs in serum samples from 141 patients and 40 controls. A hydrolysis probe–based stem–loop quantitative reverse-transcription PCR (RT-qPCR) assay was conducted in the training and verification phases to confirm the concentrations of selected miRNAs in serum samples from 149 patients and 100 controls.

RESULTS: The Solexa sequencing results demonstrated marked upregulation of 25 serum miRNAs in ESCC patients compared with controls. RT-qPCR analysis identified a profile of 7 serum miRNAs (miR-10a, miR-22, miR-100, miR-148b, miR-223, miR-133a, and miR-127-3p) as ESCC biomarkers. The area under the ROC curve for the selected miRNAs ranged from 0.817 to 0.949, significantly higher than for carcinoembryonic antigen (0.549; P < 0.0005). More importantly, this panel of 7 miRNAs clearly distinguished stage I/II ESCC patients from controls.

CONCLUSIONS: This panel of 7 serum miRNAs holds promise as a novel blood-based biomarker for the diagnosis of ESCC.

Expression Profile of MicroRNAs in Serum: A Fingerprint for Esophageal Squamous Cell Carcinoma

BACKGROUND: Although microRNAs (miRNAs) play essential roles in spermatogenesis, little is known about seminal plasma miRNAs in infertile men. We investigated the profile of seminal plasma miRNAs in infertile men to identify miRNAs that are altered in infertility; we then evaluated their diagnostic value.

METHODS: Seminal plasma samples were obtained from 289 infertile men and 168 age-matched fertile control individuals. The stability of the miRNAs was first assessed by time-course and freeze–thaw cycle analyses. The Solexa sequencing technology was used for an initial screen of the miRNAs in samples pooled from 45 patients with nonobstructive azoospermia, 58 patients with asthenozoospermia, and 100 fertile controls. A stem–loop quantitative reverse-transcription PCR (RT-qPCR) assay was conducted in the training and verification sets to confirm the concentrations of the altered miRNAs in 73 patients with nonobstructive azoospermia, 79 patients with asthenozoospermia, 34 patients with oligospermia, and 68 fertile controls.

RESULTS: The miRNAs in seminal plasma were stable. The Solexa sequencing analysis demonstrated 19 markedly altered miRNAs in the patient groups, compared with the control group. RT-qPCR analysis identified 7 miRNAs (miR-34c-5p, miR-122, miR-146b-5p, miR-181a, miR-374b, miR-509–5p, and miR-513a-5p) as markedly decreased in azoospermia but increased in asthenozoospermia. The area under the ROC curve for these miRNAs ranged from 0.733 to 0.921, markedly higher than for routine biochemical parameters (0.510–0.622). Moreover, the concentrations of some selected miRNAs were also increased in the semen sperm of the asthenozoospermia patients.

CONCLUSIONS: The measurement of miRNAs in seminal plasma provides a novel, noninvasive approach for diagnosing male infertility.

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Chen Yang

Papers

Expression Profile of MicroRNAs in Serum: A Fingerprint for Esophageal Squamous Cell Carcinoma

Altered Profile of Seminal Plasma MicroRNAs in the Molecular Diagnosis of Male Infertility