An intestinal 70/30 Caco2/HT-29 co-culture was set up starting from the parental populations of differentiated cells to mimic the human intestinal epithelium. Co-culture was harvested at confluence 0 (T0) and at 3, 6, 10, and 14 days post confluence after plating (T3, T6, T10, and T14, respectively) for morphological and functional analysis. Transmission electron microscopy revealed different features from T0 to T14: microvilli and a complete junctional apparatus from T6, mucus granules from T3, as also confirmed by PAS/Alcian Blue staining. The specific activity of alkaline phosphatase (ALP), aminopeptidase N (APN), and dipeptidyl peptidase IV (DPPIV) progressively increased after T0, indicating the acquirement of a differentiated and digestive phenotype. Transepithelial electrical resistance (TEER), indicative of the barrier properties of the monolayer, increased from T0 up to T6 reaching values very similar to the human small intestine. The apparent permeability coefficient for Lucifer Yellow (LY), along with morphological analysis, reveals a good status of the tight junctions. At T14, HT-29 cells reduced to 18.4% and formed domes, indicative of transepithelial transport of nutrients. This Caco2/HT-29 co-culture could be considered a versatile and suitable in vitro model of human intestinal epithelium for the presence of more than one prevalent intestinal cell type, by means of a minimum of 6 to a maximum of 14 post-confluence days obtained without the need of particular inducers of subclones and growth support to reach an intestinal differentiated phenotype.

An early cancer diagnosis is essential to treat and manage patients, but it is difficult to achieve this goal due to the still too low specificity and sensitivity of classical methods (imaging, actual biomarkers), together with the high invasiveness of tissue biopsies. The discovery of novel, reliable, and easily collectable cancer markers is a topic of interest, with human biofluids, especially blood, as important sources of minimal invasive biomarkers such as circulating microRNAs (miRNAs), the most promising. MiRNAs are small non-coding RNAs and known epigenetic modulators of gene expression, with specific roles in cancer development/progression, which are next to be implemented in the clinical routine as biomarkers for early diagnosis and the efficient monitoring of tumor progression and treatment response. Unfortunately, several issues regarding their validation process are still to be resolved. In this review, updated findings specifically focused on the clinical relevance of circulating miRNAs as prognostic and diagnostic biomarkers for the most prevalent cancer types (breast, lung, and prostate cancers in adults, and osteosarcoma in children) are described. In addition, deep analysis of pre-analytical, analytical, and post-analytical issues still affecting the circulation of miRNAs’ validation process and routine implementation is included.

Aging is associated with an increased incidence of age-related bone diseases. Current diagnostics (e.g., conventional radiology, biochemical markers), because limited in specificity and sensitivity, can distinguish between healthy or osteoporotic subjects but they are unable to discriminate among different underlying causes that lead to the same bone pathological condition (e.g., bone fracture risk). Among recent, more sensitive biomarkers, miRNAs — the non-coding RNAs involved in the epigenetic regulation of gene expression, have emerged as fundamental post-transcriptional modulators of bone development and homeostasis. Each identified miRNA carries out a specific role in osteoblast and osteoclast differentiation and functional pathways (osteomiRs). miRNAs bound to proteins or encapsulated in exosomes and/or microvesicles are released into the bloodstream and biological fluids where they can be detected and measured by highly sensitive and specific methods (e.g., quantitative PCR, next-generation sequencing). As such, miRNAs provide a prompt and easily accessible tool to determine the subject-specific epigenetic environment of a specific condition. Their use as biomarkers opens new frontiers in personalized medicine. While miRNAs circulating levels are lower than those found in the tissue/cell source, their quantification in biological fluids may be strategic in the diagnosis of diseases that affect tissues, such as bone, in which biopsy may be especially challenging. For a biomarker to be valuable in clinical practice and support medical decisions, it must be (easily) measurable, validated by independent studies, and strongly and significantly associated with a disease outcome. Currently, miRNAs analysis does not completely satisfy these criteria, however. Starting from in vitro and in vivo observations describing their biological role in bone cell development and metabolism, this review describes the potential use of bone-associated circulating miRNAs as biomarkers for determining predisposition, onset, and development of osteoporosis and bone fracture risk. Moreover, the review focuses on their clinical relevance and discusses the pre-analytical, analytical, and post-analytical issues in their measurement, which still limits their routine application. Taken together, research and clinical findings may be helpful for creating miRNA-based diagnostic tools in the diagnosis and treatment of bone diseases.