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

MicroRNAs (miRNAs) are a class of non-coding RNAs that play important roles in regulating gene expression. The majority of miRNAs are transcribed from DNA sequences into primary miRNAs and processed into precursor miRNAs, and finally mature miRNAs. In most cases, miRNAs interact with the 3' untranslated region (3' UTR) of target mRNAs to induce mRNA degradation and translational repression. However, interaction of miRNAs with other regions, including the 5' UTR, coding sequence, and gene promoters, have also been reported. Under certain conditions, miRNAs can also activate translation or regulate transcription. The interaction of miRNAs with their target genes is dynamic and dependent on many factors, such as subcellular location of miRNAs, the abundancy of miRNAs and target mRNAs, and the affinity of miRNA-mRNA interactions. miRNAs can be secreted into extracellular fluids and transported to target cells via vesicles, such as exosomes, or by binding to proteins, including Argonautes. Extracellular miRNAs function as chemical messengers to mediate cell-cell communication. In this review, we provide an update on canonical and non-canonical miRNA biogenesis pathways and various mechanisms underlying miRNA-mediated gene regulations. We also summarize the current knowledge of the dynamics of miRNA action and of the secretion, transfer, and uptake of extracellular miRNAs.

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Overview of MicroRNA Biogenesis, Mechanisms of Actions, and Circulation.

MicroRNAs (miRNAs) are small noncoding RNAs that typically inhibit the translation and stability of messenger RNAs (mRNAs), controlling genes involved in cellular processes such as inflammation, cell-cycle regulation, stress response, differentiation, apoptosis, and migration. Thus, miRNAs have been implicated in the regulation of virtually all signaling circuits within a cell, and their dysregulation has been shown to play an essential role in the development and progression of cancer. Here, after a brief description of miRNA genomics, biogenesis, and function, we discuss the effects of miRNA dysregulation in the cellular pathways that lead to the progressive conversion of normal cells into cancer cells and the potential to develop new molecular miRNA-targeted therapies.

MicroRNAs in cancer.

The emergence of publications on extracellular RNA (exRNA) and extracellular vesicles (EV) has highlighted the potential of these molecules and vehicles as biomarkers of disease and therapeutic targets. These findings have created a paradigm shift, most prominently in the field of oncology, prompting expanded interest in the field and dedication of funds for EV research. At the same time, understanding of EV subtypes, biogenesis, cargo and mechanisms of shuttling remains incomplete. The techniques that can be harnessed to address the many gaps in our current knowledge were the subject of a special workshop of the International Society for Extracellular Vesicles (ISEV) in New York City in October 2012. As part of the “ISEV Research Seminar: Analysis and Function of RNA in Extracellular Vesicles (evRNA)”, 6 round-table discussions were held to provide an evidence-based framework for isolation and analysis of EV, purification and analysis of associated RNA molecules, and molecular engineering of EV for therapeutic intervention. This article arises from the discussion of EV isolation and analysis at that meeting. The conclusions of the round table are supplemented with a review of published materials and our experience. Controversies and outstanding questions are identified that may inform future research and funding priorities. While we emphasize the need for standardization of specimen handling, appropriate normative controls, and isolation and analysis techniques to facilitate comparison of results, we also recognize that continual development and evaluation of techniques will be necessary as new knowledge is amassed. On many points, consensus has not yet been achieved and must be built through the reporting of well-controlled experiments. Keywords: extracellular vesicle; exosome; microvesicle; standardization; isolation (Published: 27 May 2013) Citation: Journal of Extracellular Vesicles 2013, 2 : 20360 - http://dx.doi.org/10.3402/jev.v2i0.20360

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Standardization of sample collection, isolation and analysis methods in extracellular vesicle research

Secretory Mechanisms and Intercellular Transfer of MicroRNAs in Living Cells

Dysregulated expression of microRNAs (miRNAs) in various tissues has been associated with a variety of diseases, including cancers. Here we demonstrate that miRNAs are present in the serum and plasma of humans and other animals such as mice, rats, bovine fetuses, calves, and horses. The levels of miRNAs in serum are stable, reproducible, and consistent among individuals of the same species. Employing Solexa, we sequenced all serum miRNAs of healthy Chinese subjects and found over 100 and 91 serum miRNAs in male and female subjects, respectively. We also identified specific expression patterns of serum miRNAs for lung cancer, colorectal cancer, and diabetes, providing evidence that serum miRNAs contain fingerprints for various diseases. Two non-small cell lung cancer-specific serum miRNAs obtained by Solexa were further validated in an independent trial of 75 healthy donors and 152 cancer patients, using quantitative reverse transcription polymerase chain reaction assays. Through these analyses, we conclude that serum miRNAs can serve as potential biomarkers for the detection of various cancers and other diseases.

/pdf/characterization-of-micrornas-in-serum-a-novel-class-of-3urw6sycxl.pdf

Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases

DNA damage repair (DDR) plays a role in the tumorigenesis and progression of lung squamous cell carcinoma (LUSC), but the predictive value of DDR in LUSC has not been fully elucidated.

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1111/1759-7714.14370

Identification of a DNA damage repair gene‐related signature for lung squamous cell carcinoma prognosis

/pdf/correction-to-colorectal-cancer-organoid-models-uncover-2katuvbj.pdf

Correction to: Colorectal cancer organoid models uncover oxaliplatin-resistant mechanisms at single cell resolution.

Objective To evaluate clinical factors influencing the postoperative pulmonary sarcomatoid carcinoma (PSCs) prognosis. Methods We retrospectively evaluated patients with PSCs treated from October 2012 to October 2017. Kaplan–Meier survival curves were calculated using univariable analysis (log-rank test). Univariable/multivariable Cox regression analysis was also performed. Results Mixed PSCs were most common (64.10%). Pure PSCs occurred more often with large tumors compared with mixed PSCs. Patients with vs without pleural retraction, respectively, had significantly worse overall survival (OS; 16 vs 23 months) and disease-free survival (DFS; 11 vs 20 months), and patients with airway dissemination had significantly shorter OS (14 vs 21 months) and DFS (11 vs 20 months). Patients with PSC with an adenocarcinoma component had favorable OS. Airway dissemination, pleural retraction, metastatic mediastinal lymph node (LN) number, and pathological tumor-node-metastasis (pTNM) stage were risk factors for short OS. Neither adjuvant chemotherapy nor adjuvant radiotherapy provided a survival advantage. Airway dissemination was an independent prognostic factor (odds ratio, 1.87; 95% confidence interval, 1.04–3.36). Conclusion Pure PSCs were more likely with large tumors compared with mixed PSCs. Airway dissemination, pleural retraction, and metastatic mediastinal LN number were associated with OS. Airway dissemination was an independent prognostic factor.

https://doi.org/10.1177/03000605221128092

Clinical analysis of 78 pulmonary sarcomatoid carcinomas with surgical treatment

With more and more target medicine application in lung cancer, lots of patients take medicine at home, the treatment bone metastasis and screen of bone metastasis always has been neglected until skeletal‐related events (SREs) such as bone pain, hypercalcemia of malignancy and pathologic fractures emerging which significantly impairs the patients' daily activity ability, seriously lower quality of life.

Ting Gong

Papers

Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases

Identification of a DNA damage repair gene‐related signature for lung squamous cell carcinoma prognosis

Correction to: Colorectal cancer organoid models uncover oxaliplatin-resistant mechanisms at single cell resolution.

Clinical analysis of 78 pulmonary sarcomatoid carcinomas with surgical treatment

Retrospective study of EGFR‐mutant lung adenocarcinoma with bone metastatic clinical features