Identification of miRNAs Associated With Gender Difference in Osteoarthritis Patients

Osteoarthritis (OA) is a chronic degenerative joint disease of considerable socio-economic impact, of which a primary and a secondary form are distinguished. Primary OA is a mild form that occurs mainly in the joints of the hands, feet, knee, and hip, and appears in young subjects. Secondary OA derives from different factors that modify the microenvironment of the cartilage such as trauma, congenital joint deformities, metabolic defects, infections that cause post-infectious arthritis, endocrine and neuropathic diseases and disorders that alter the normal structure and function of hyaline cartilage (eg . rheumatoid arthritis, gout, chondrocalcinosis), and is more prevalent in old age.

As regards the secondary form of OA, correlated with advancing age due to the natural aging process of the joint and to events of a traumatic nature, it is hypothesized that a key role in the etiology and progression of the disease may be played by the genre. Immune responses are known to differ in males and females. Some meta-analyzes reveal how the presence of some polymorphisms (ADAM12) in male patients predispose to the development of OA more than in women, especially in the knee joint. Furthermore, new molecular evidence suggests that in patients with OA there is a gender-specific activation of PI3K-AKT and hypoxia signaling, suggesting the presence of possible gender-specific protein factors that may modulate bone metabolism and disease progression. In this regard, studies evaluating the responses of joint chondrocytes to systemic factors suggest that there are differences in the biochemical and molecular characteristics of male and female cells, without prejudice to the fundamental role of hormones in the different gender-specific response to the development and progression of pathology.

It is widely known that the regulation of bone remodeling processes is entrusted to a hormonal mechanism, which mainly involves parathyroid hormone, vitamin D and calcitonin. Other hormone regulators involved are sex hormones (estrogen, testosterone), thyroid hormones, corticosteroids (including cortisone), insulin, and growth factors (including growth hormone).

In the development of OA it is thought that sex hormones may act in a prevalent way. In women of childbearing age, estrogen has a protective action on the bone, inhibiting bone remodeling, especially bone resorption; while in the post-menopausal period, circulating estrogens are reduced compared to testosterone, strongly contributing to an imbalance in bone remodeling, favoring the stiffening of the subchondral bone and the development of osteophytes at the joint margins. The osteophytes seem to be developed in an attempt to stabilize the joint damage. Consequently, the synovial membrane becomes inflamed and thickened, producing synovial fluid with less viscosity and in greater quantities and favoring the development of an inflammatory process on site. Obviously, the entire joint will be damaged up to the reduction of joint mobility and ankylosis.

Similarly men, even if they do not undergo a real andropause, starting from 45 years of age they suffer a progressive decline in the production and release of testosterone, whose levels after the age of 65 are no longer sufficient to maintain good efficiency of the bone tissue favoring its resorption and the possible onset of the pathology. However, it is not known whether the potential differences related to gender, in terms of hormonal release and alteration of bone homeostasis, may be related to the severity of the disease, that is to the development of the inflammatory condition associated with it.

It is therefore necessary to highlight another characteristic of OA, both in terms of development and progression, namely the different inflammatory response based on the patient's gender. In OA, as with other inflammatory diseases, the response of the male immune system induced by inflammation of the synovium and synovial fluid is different than in the female counterpart, suggesting that there could be differences between the two sexes in the levels of immunomodulators in joint tissues, including the synovial membrane, synovial fluid and cartilage itself. Recent studies attribute to the inflammatory microenvironment a key role in the prognosis of the disease.

Recently, the investigators obtained evidences regarding the role of microRNAs and lncRNAs as biomarkers of bone regeneration, directly involving specific cellular signalings (hypoxia, epithelium-mesenchyma transition and mechano-transduction), and representing, in some cases, a crucial point for the development of orthopedic diseases, such as osteoporosis, OA and forms of degeneration of the intervertebral discs. From an ongoing screening on tissue (joint) miRNA and lncRNA differently expressed and which could play a key role in the process of osseointegration of the prosthetic implant, miR-31-5p, miR-33a-5p, miR-133a, miR-675-5p and lncH19 were also evaluated. The correlation analysis between their expression profile and the patient's gender show a different expression of some of them and a gender-specific response on target genes and signaling.

The hypothesis of the study is that there are gender differences that contribute to the increase in the incidence and severity of OA, both in molecular terms (qualitative and quantitative differences in miRNAs involved in transcriptional and translational gene regulation) and inflammatory, such as differences in the development and progression of the lymphocyte population, involved in the OA-related inflammatory response. Therefore, the study aims to identify, characterize and evaluate an expression panel of tissue and circulating miRNAs isolated from patients with both mild and moderate-severe OA, who undergo prosthetic surgery, so as to be able to define possible gender-specific biomarkers of disease progression useful for controlling the osteointegration process of the prosthetic implant, and to be correlated with the pre- and post-operative inflammatory response.

The biological study involves the collection of samples from patients of both sexes suffering from OA, be it mild or moderate-severe, who undergo endo or arthro-prosthesis surgery, or arthroscopy. In particular, the following samples will be collected: 1) cells, such as synoviocytes, osteoblasts and chondrocytes deriving from operating waste tissues; 2) synovial fluid isolated during the operation; and 3) peripheral blood, from which to isolate the plasma and the lymphocyte component, taken 24 hours before surgery, and 72 hours and 1 month after surgery.

The miRNAs will be isolated from the samples isolated from tissues and plasma and will be molecularly characterized in order to identify a panel (also built starting from information obtained from the literature) expressed on the basis of the gender and severity of OA. The identified miRNAs will be validated, in vitro, using a specific molecular approach. The lymphocyte component of the blood and, isolated by means of special separation protocols through cell-specific magnetic beads, will be phenotypically characterized by a flow cytometric approach. The lymphocyte populations that will show a different phenotype between the male and female gender in correlation with the aggressiveness of the disease, will be analyzed and monitored over time up to 1 month of follow-up together with the analysis of synovial fluids, through in vitro studies of cytokine release and ELISA assays.