Role of Non-Coding RNAs in Pathogenesis, Diagnosis, and Therapy of Legg-Calvé-Perthes Disease: A Systematic Review

Introduction

Legg-Calvé-Perthes disease (LCPD) is a complex disorder involving unilateral or bilateral idiopathic sterile necrosis of the femoral head [1]. It is suggested that it usually occurs in boys aged 4–8 years (sex ratio 5: 1) and is most common in European populations, especially Scandinavian ones [1,2]. The largest epidemiological study of LCPD has been conducted among this group. Johannson et al determined the prevalence of the disease as 9.3 per 100 000 people during a 20-year follow-up [3]. The main symptoms of the disease include primarily hip pain, sometimes with radiation to the knee, and lameness, with no general symptoms [4]. Typically, the course of the disease can be divided into several stages (Waldenström, 1922): I – initial or avascular stage, II – fragmentation stage, III – re-ossification or healing stage, and IV - healed stage [5,6]. Although the etiology of the disease remains unexplained, it is suggested that genetic (higher incidence in monozygotic twins, association with trisomy 21 syndrome, collagenopathies associated with COL2A1 gene mutation, delayed ossification in the femoral head); individual (low birth weight, excessive body weight, abnormal blood supply to the femoral heads, thrombophilias, attention deficit hyperactivity disorder [ADHD]); environmental (exposure to secondhand smoke at the child’s parents); or socioeconomic (malnutrition, socioeconomic deprivation) factors are involved [1,2,4,5,7]. There is a growing body of evidence indicating that non-coding RNAs play a significant role in the pathogenesis of LCPD.

Non-coding RNAs, including microRNA (miRNA), circular RNA (circRNA), and long-non-coding RNA (lncRNA), among others, are transcripts that have no protein-coding potential [9]. Their exact number and function are still being discovered, but it is already known that they play a key role in the process of cell cycle regulation and the pathogenesis of many diseases. Moreover, their function as diagnostic, predictive, and prognostic markers of multiple disease entities has been demonstrated [9,10]. These regulatory molecules influence key biological processes involved in the development and progression LCPD, such as chondrocyte apoptosis, angiogenesis, and inflammatory responses. Dysregulation of miR-214-3p, miR-206, and various lncRNAs may contribute to ischemia and cartilage degeneration, highlighting their potential etiological and diagnostic relevance.

The main aim of the present article is to present the role of non-coding RNA molecules in the pathogenesis, diagnosis, and treatment of LCPD.

A systematic review of scientific publications available in the PubMed, Google Scholar, Scopus, and Web of Science databases was performed. The keywords used were “Perthes disease”, “miRNA”, “lncRNA”, “circRNA”, and “non-coding RNA.” The review was conducted simultaneously by 2 reviewers independently to find publications that met the inclusion criteria. Only full-text original publications, meta-analyses, and review publications published in English were included in the review. Abstracts, editorials, and letters to the editor were excluded. Pre-print publications were also not included. Due to the small number of results and their recent publication, the time limit was the discovery of miRNAs, which was 1993 [11]. Initially, 23 publications were obtained. A substantive evaluation of titles and abstracts was performed, and we decided to exclude publications not directly related to LCPD and non-coding RNAs. Ultimately, 8 publications were included in the analysis. To the best of our knowledge, this paper is one of the few systematic reviews available in the literature on the role of non-coding RNA molecules in the pathogenesis, diagnosis, and treatment of LCPD.

LCPD is characterized by an intricate, incompletely understood etiology, with many suggested factors leading to its development, including numerous molecular pathways involving, among others, abnormalities in the life cycle and apoptosis of chondrocytes [12].

Diagnostic Factors

MIR-206:

Luo et al conducted a study in a group of subjects with LCPD (n=20) and healthy controls (n=20), taking chondrocytes from the femoral head. They observed overexpression of miR-206 in chondrocytes taken from the patients. Moreover, this overexpression was associated with increased apoptosis in the chondrocytes studied and appeared in cells of dexamethasone-treated cell lines. This suggests an effect of glucocorticosteroids in the developmental pathway of LCPD [13]. Such an effect was possible through the inhibition of the target gene, SOX9, which is crucial in the process of chondrocyte differentiation and formation of normal cartilage and ensuring homeostasis of the extracellular matrix [14–16]. This occurs through its promotion of type II collagen expression [16]. In addition, it affects the expression of collagen types IX, XI, aggrecan, SOX5, SOX6, WP2 protein, and FGFR4 factor [17]. SOX9 is also involved in the pathogenesis of LCPD, probably through alterations associated with ischemia and thinner articular cartilage [15]. Its expression is influenced by lnc-RNA DA12594 and miRNAs miR-548, miR-101, miR-1/205, miR-590/590-3p, miR-145, miR-300, and miR-384-5p, although their association with LCPD pathogenesis has not been established [17]. The above results suggest the involvement of miR-206 in the pathogenesis of LCPD, and they indicate the role of its overexpression as a potential diagnostic factor in this disease.

MIR-214 AND CONNECTED PATHWAYS:

The best-described miRNA with the best-known role in LCPD is miR-214. In a study involving 20 subjects with LCPD and 20 healthy controls, Zhu et al observed significantly lower expression of miR-214 in extracted hip cartilage cells and serum from patients with LCPD compared to biological material from healthy subjects. Similar observations were made in dexamethasone-treated cells. The authors further suggested that miR-214 could be an effective diagnostic tool in LCPD [18]. Further analysis by the authors showed that the direct target of miR-214 is the Bax protein. Its high expression and low disrupted Bcl-2/Bax ratio in the cells studied were induced by low expression of miR-214. The above observations proved the pro-apoptotic effect of miR-214 on cells through upregulation of the mitochondrial apoptosis pathway dependent on the Bcl-2 family of proteins [18]. Moreover, apoptosis of chondrocytes and chondroblasts is one of the suggested mechanisms of idiopathic non-traumatic necrosis of the femoral head [19]. Other researchers also suggest a negative involvement of this miRNA in chondrogenesis by affecting chondrocyte differentiation (inhibition of the activity of Atf4, Sox9 and col2a1) and inhibition of osteogenic differentiation of periodontal ligament stem cells (through Atf4) [20,21]. This demonstrates the pivotal role of miR-214 in the development of the cartilage and skeletal system of the human body and its involvement in the pathogenesis of LCPD.

Lan et al (n=20) made similar observations on the same study material. They noted that reduced expression of exos-miR-214-3p affected the inhibition of cartilage cell proliferation and increased their apoptosis. In addition, overexpression of this molecule affected macrophage M2 polarization and enhanced angiogenesis, while reducing chronic inflammation of the synovial membrane of the hip joint [22]. Accordingly, another pathogenetic mechanism of miRNA-stimulated LCPD has been suggested – disruption of the blood supply to the articular cartilage and increased inflammation induced by decreased macrophage M2 activity [22,23].

The same authors in another study (n=20), observed increased expression of circCDR1as in material collected from LCPD subjects compared to healthy controls, and they found an inverse correlation with the level of previously-studied, downregulated miR-214-3p [24]. The correlation between the 2 factors occurs at the epigenetic level through recruitment of the PRC2 complex and its subunits EZH2, SUZ12, and EED. Accordingly, it has been observed that miR-214-3p is a direct effector of this circRNA, and it affects cell apoptosis through a Bax-dependent pathway, as well as by downregulating the expression of COL1A1 and RUNX2 genes [24]. The circCDR1as/miR214-3p pathway also affects chondrocyte cells through reduced expression of VEGFA, as well as downregulation of macrophage M2 polarization, producing the effects described above [21–23]. The authors also noted that suppression of circCDR1as expression could be a therapeutic target of LCPD, producing beneficial effects [24]. Other authors have noted the role of circCDR1as as an inhibitor of bone marrow mesenchymal stem cell differentiation in steroid-induced non-traumatic necrosis of the femoral head, and its high expression may be a potential marker of the severity of this disease [25,26].

MIRNA IN EXOSOMES:

Huang et al described the overexpression of miRNAs in serum exosomes in subjects with LCPD (n=13) and healthy controls (n=13). The expression of hsa-miR-3133, hsa-miR-4644, hsa-miR-150-5p, hsa-miR-4693-3p, hsa-miR-4693-5p, hsa-miR-141-3p, hsa-miR-7154-5p, hsa-miR-4709-5p, and hsa-miR-30a-3p was significantly increased in serum exosomes of LCPD patients. Moreover, miR-3133, miR-4644, miR-4693-3p, and miR-4693-5p played a role in the pathogenesis of LCPD as factors promoting vascular endothelial dysfunction. In addition, miR-3133, miR-4693-3p, miR-4693-5p, miR-141-3p, and miR-30a were promoters of osteoclastogenesis [27]. Interestingly, miR-141-3p negatively affects osteogenesis stimulated by oxidative stress, including by downregulating the activity of SDF-1 factor, and through this, downregulating the expression of BMP-2 and Runx2 [28]. miR-30a, which inhibits osteoblast differentiation and stimulates inflammation, also acts by negatively affecting BMP-2 and Runx2 [29]. In addition, the role of miR-3133 in inhibiting osteogenesis is based on disruption of Runx2 and TGF-β1 pathway activity [30]. It also affects the disruption of proliferation and angiogenesis by inhibiting activity of the JUNB/VEGF pathway [31]. mir-4693-5p has a proven role in joint pathologies, including rheumatoid arthritis, where, by influencing the overexpression of the factor HIF-1α, it induces apoptosis, stimulates inflammation, and increases oxidative stress [32].

MIR-12093-3P:

Zhang et al, in their study on a rabbit model, showed that in Perthes disease, some of the key pathogenetic elements are ischemia and impaired angiogenesis, as well as an increased inflammatory response. The entire network of 28 miRNAs and 29 lncRNAs, whose impaired expression affected the expression of genes responsible for angiogenesis and inflammation, played a role in these mechanisms. The authors observed a particularly strong role for miR-12093-3p, which targeted 293 genes [33]. The role of this miRNA includes receptor functions of the extracellular matrix of the musculoskeletal system by affecting the MAPK signaling pathway and linoleic acid metabolism [34].

LONG-NON-CODING RNAS:

Long-non-coding RNAs also play their part in the pathogenesis of LCPD, which was demonstrated in the aforementioned study. Among lncRNAs, the most important were HIF3A, LOC103350994, LOC108178457, LOC108176261, LOC108178064, and LOC103345386. Their involvement in the pathogenesis of LCPD was mainly based on disruption of immune function and stimulation of inflammation [33].

Another network of connections was discovered by Wang et al (n=9). In their study, they observed that 13 lncRNAs had a unique affinity for mRNAs potentially involved in LCPD pathogenesis. Three of them (n335645, n335724, and n339477) were at the center of this connection network and positively affected the expression of specific mRNAs. Among the key target mRNAs correlated with the expression of the aforementioned lncRNAs (and especially n335645) are: ILK (plays a role in the stimulation of angiogenesis, regulates vascular wall tension, and stimulates osteoblasts and bone remodeling through the Wnt pathway), VCL (responsible for adequate cell-cell cross-adhesion and provides adequate vascular tightness), and R-RAS (regulates angiogenesis and vascular remodeling by inhibiting the VEGF-dependent SAPK2/p38MAPK pathway) [35–39].

MIR-223:

In a recent study in animal models, Yang et al noted that miR-223-5p expression is significantly downregulated under hypoxic conditions in LCPD cells [40]. The direct target of this miRNA is CHAC2, which is a regulator of glutathione metabolism, one of the key factors responsible for cell apoptosis and cell cycle regulation [40]. The above mechanisms play a significant role in LCPD. The above data are summarized in Table 1.

Therapeutic Targets

LIMITATIONS OF THE STUDY:

This review yielded only 8 publications, all from recent years, and all were conducted on relatively small groups or only on animal models. This indicates research in this field is only beginning.

Future Directions

Further studies on ncRNAs in LCPD may contribute to more definitive clarification of the pathogenesis of the disease, and thus a more accurate understanding of risk factors and prevention options. Changes in their expression could be a potential diagnostic factor in LCPD. Some of the miRNAs are potential therapeutic targets, which could lay the groundwork for developing treatments.

Conclusions

Non-coding RNAs (miRNAs, lncRNAs, circRNAs) play a role in the pathogenesis of LCPD by affecting the expression of genes responsible for angiogenesis and the inflammatory response. Abnormal expression of some of them (especially miR-206 and miR-214) may be a good potential diagnostic and prognostic marker for the disease and a potential future therapeutic target. A detailed understanding of the genetic and epigenetic determinants of the disease, along with the implementation of appropriate prophylaxis, may help to achieve better treatment outcomes for LCPD in the future.