Identifying MiR-140-3p as a stable internal reference to normalize MicroRNA qRT-PCR levels of plasma small extracellular vesicles in lung cancer patients.

Exploration of a stably expressed gene as a reference is critical for the accurate evaluation of miRNAs isolated from small extracellular vesicles (sEVs). In this study, we analyzed small RNA sequencing on plasma sEV miRNAs in the training dataset (n = 104) and found that miR-140-3p was the most stably expressed candidate reference for sEV miRNAs. We further demonstrated that miR-140-3p expressed most stably in the validation cohort (n = 46) when compared to two other reference miRNAs, miR-451a and miR-1228-3p, and the commonly-used miRNA reference U6. Finally, we compared the capability of miR-140-3p and U6 as the internal reference for sEV miRNA expression by evaluating key miRNAs expression in lung cancer patients and found that miR-140-3p was more suitable as a sEV miRNA reference gene. Taken together, our data indicated miR-140-3p as a stable internal reference miRNA of plasma sEVs to evaluate miRNA expression profiles in lung cancer patients.

Identification and evaluation of circulating small extracellular vesicle microRNAs as diagnostic biomarkers for patients with indeterminate pulmonary nodules.

BACKGROUND: The identification of indeterminate pulmonary nodules (IPNs) following a low-dose computed tomography (LDCT) is a major challenge for early diagnosis of lung cancer. The inadequate assessment of IPNs' malignancy risk results in a large number of unnecessary surgeries or an increased risk of cancer metastases. However, limited studies on non-invasive diagnosis of IPNs have been reported. METHODS: In this study, we identified and evaluated the diagnostic value of circulating small extracellular vesicle (sEV) microRNAs (miRNAs) in patients with IPNs that had been newly detected using LDCT scanning and were scheduled for surgery. Out of 459 recruited patients, 109 eligible patients with IPNs were enrolled in the training cohort (n = 47) and the test cohort (n = 62). An external cohort (n = 99) was used for validation. MiRNAs were extracted from plasma sEVs, and assessed using Small RNA sequencing. 490 lung adenocarcinoma samples and follow-up data were used to investigate the role of miRNAs in overall survival. RESULTS: A circulating sEV miRNA (CirsEV-miR) model was constructed from five differentially expressed miRNAs (DEMs), showing 0.920 AUC in the training cohort (n = 47), and further identified in the test cohort (n = 62) and in an external validation cohort (n = 99). Among five DEMs of the CirsEV-miR model, miR-101-3p and miR-150-5p were significantly associated with better overall survival (p = 0.0001 and p = 0.0069). The CirsEV-miR scores were calculated, which significantly correlated with IPNs diameters (p < 0.05), and were able to discriminate between benign and malignant PNs (diameter ≤ 1 cm). The expression patterns of sEV miRNAs in the benign, adenocarcinoma in situ/minimally invasive adenocarcinoma, and invasive adenocarcinoma subgroups were found to gradually change with the increase in aggressiveness for the first time. Among all DEMs of the three subgroups, five miRNAs (miR-30c-5p, miR-30e-5p, miR-500a-3p, miR-125a-5p, and miR-99a-5p) were also significantly associated with overall survival of lung adenocarcinoma patients. CONCLUSIONS: Our results indicate that the CirsEV-miR model could help distinguish between benign and malignant PNs, providing insights into the feasibility of circulating sEV miRNAs in diagnostic biomarker development. TRIAL REGISTRATION: Chinese Clinical Trials: ChiCTR1800019877. Registered 05 December 2018, https://www.chictr.org.cn/showproj.aspx?proj=31346 .

Detection and characterization of pancreatic and biliary tract cancers using cell-free DNA fragmentomics.

BACKGROUND: Plasma cell-free DNA (cfDNA) fragmentomics has demonstrated significant differentiation power between cancer patients and healthy individuals, but little is known in pancreatic and biliary tract cancers. The aim of this study is to characterize the cfDNA fragmentomics in biliopancreatic cancers and develop an accurate method for cancer detection. METHODS: One hundred forty-seven patients with biliopancreatic cancers and 71 non-cancer volunteers were enrolled, including 55 patients with cholangiocarcinoma, 30 with gallbladder cancer, and 62 with pancreatic cancer. Low-coverage whole-genome sequencing (median coverage: 2.9 ×) was performed on plasma cfDNA. Three cfDNA fragmentomic features, including fragment size, end motif and nucleosome footprint, were subjected to construct a stacked machine learning model for cancer detection. Integration of carbohydrate antigen 19-9 (CA19-9) was explored to improve model performance. RESULTS: The stacked model presented robust performance for cancer detection (area under curve (AUC) of 0.978 in the training cohort, and AUC of 0.941 in the validation cohort), and remained consistent even when using extremely low-coverage sequencing depth of 0.5 × (AUC: 0.905). Besides, our method could also help differentiate biliopancreatic cancer subtypes. By integrating the stacked model and CA19-9 to generate the final detection model, a high accuracy in distinguishing biliopancreatic cancers from non-cancer samples with an AUC of 0.995 was achieved. CONCLUSIONS: Our model demonstrated ultrasensitivity of plasma cfDNA fragementomics in detecting biliopancreatic cancers, fulfilling the unmet accuracy of widely-used serum biomarker CA19-9, and provided an affordable way for accurate noninvasive biliopancreatic cancer screening in clinical practice.

PGE2 activates EP4 in subchondral bone osteoclasts to regulate osteoarthritis.

Prostaglandin E2 (PGE2), a major cyclooxygenase-2 (COX-2) product, is highly secreted by the osteoblast lineage in the subchondral bone tissue of osteoarthritis (OA) patients. However, NSAIDs, including COX-2 inhibitors, have severe side effects during OA treatment. Therefore, the identification of novel drug targets of PGE2 signaling in OA progression is urgently needed. Osteoclasts play a critical role in subchondral bone homeostasis and OA-related pain. However, the mechanisms by which PGE2 regulates osteoclast function and subsequently subchondral bone homeostasis are largely unknown. Here, we show that PGE2 acts via EP4 receptors on osteoclasts during the progression of OA and OA-related pain. Our data show that while PGE2 mediates migration and osteoclastogenesis via its EP2 and EP4 receptors, tissue-specific knockout of only the EP4 receptor in osteoclasts (EP4LysM) reduced disease progression and osteophyte formation in a murine model of OA. Furthermore, OA-related pain was alleviated in the EP4LysM mice, with reduced Netrin-1 secretion and CGRP-positive sensory innervation of the subchondral bone. The expression of platelet-derived growth factor-BB (PDGF-BB) was also lower in the EP4LysM mice, which resulted in reduced type H blood vessel formation in subchondral bone. Importantly, we identified a novel potent EP4 antagonist, HL-43, which showed in vitro and in vivo effects consistent with those observed in the EP4LysM mice. Finally, we showed that the Gαs/PI3K/AKT/MAPK signaling pathway is downstream of EP4 activation via PGE2 in osteoclasts. Together, our data demonstrate that PGE2/EP4 signaling in osteoclasts mediates angiogenesis and sensory neuron innervation in subchondral bone, promoting OA progression and pain, and that inhibition of EP4 with HL-43 has therapeutic potential in OA.

Comprehensive Evaluation and Application of a Novel Method to Isolate Cell-Free DNA Derived From Bile of Biliary Tract Cancer Patients.

Cell-free DNA (cfDNA) exists in various types of bodily fluids, including plasma, urine, bile, and others. Bile cfDNA could serve as a promising liquid biopsy for biliary tract cancer (BTC) patients, as bile directly contacts tumors in the biliary tract system. However, there is no commercial kit or widely acknowledged method for bile cfDNA extraction. In this study, we established a silica-membrane-based method, namely 3D-BCF, for bile cfDNA isolation, exhibiting effective recovery of DNA fragments in the spike-in assay. We then compared the 3D-BCF method with four other commercial kits: the BIOG cfDNA Easy Kit (BIOG), QIAamp DNA Mini Kit (Qiagen), MagMAXTM Cell-Free DNA Isolation Kit (Thermo Fisher), and NORGEN Urine Cell-Free Circulating DNA Purification Mini Kit (Norgen Biotek). The proposed 3D-BCF method exhibited the highest cfDNA isolation efficiency (p < 0.0001) from patient bile samples, and bile cfDNA of short, medium or long fragments could all be extracted effectively. To test whether the extracted bile cfDNA from patients carries tumor-related genomic information, we performed next-generation sequencing on the cfDNA and verified the gene-mutation results by polymerase chain reaction (PCR)-Sanger chromatograms and copy-number-variation (CNV) detection by fluorescence in situ hybridization (FISH) of tumor tissues. The 3D-BCF method could efficiently extract cfDNA from bile samples, providing technical support for bile cfDNA as a promising liquid biopsy for BTC patient diagnosis and prognosis.