There are multiple studies suggesting a possible association of miRs with ES. The functional study of miR-30a showed a functional correlation between EWS-FLI1 and CD99 membrane glycoprotein which are two significant biomarkers and therapeutic targets for ES ^[1][44]. Moreover, there is also a report that miR-20b, miR-130b, and miR-34b act as oncomiRs (miRs associated with cancer) in ES progression ^[2][9]. According to Kawano et al. (2017), the modulation of Transforming Growth Factor β Receptor II (TGFBR2) by miRs contributes significantly to the advancement of ES. They observed an antagonistic association between the downregulation of TGFBR2 and the overexpression of miR-20b in humans. This clearly indicates the oncomiRic character of miR-20b and its potential to regulate the TGFRB2 pathway ^[3][45].

miR-130b has a direct association with ES metastasis and found that its overexpression leads to enhanced cellular invasion and migration ability ^[4][46]. Additionally, the molecular evidence demonstrates that miR-130b promotes the Cell Division Cycle 42 (CDC42) pathway by suppressing Rho GTPase Activating Protein 1 (ARHGAP1), which results in the constitutive activation of CDC42. The activation of P21 (RAC1) Activated Kinase 1 (PAK1), which positively targets gene expression and is responsible for sarcoma invasion and metastasis and creates a positive feedback loop by promoting miR-130b expression, is furthermore caused by the knockdown of ARHGAP1 gene expression and prolonged activation of CDC42 ^[4][46]. miR-34b is also involved in promoting proliferation, migration, and invasion of Ewing’s sarcoma cells by decreasing the levels of mRNA and Notch homolog protein 1 (Notch 1), respectively ^[5][47]. The prior data indicated that the Notch signaling pathway can act as an oncogene in human cancers ^[6][48]. Another miR linked with ES is miR-708-5p through a differential expression pattern. miR-708-5p is downregulated in ES cells and its overexpression is correlated with reduced proliferation of various tumours ^[7][8][49,50]. The overexpression of miR-708-5p suppresses the tumor invasiveness and migration, thanks to positively targeting Rho-GTPase Rho C effectors (ROCK1 and ROCK2) and Matrix Metallopeptidase 12 (MMP-12), respectively ^[9][51]. On the contrary, another oncomiR miR-181c acts as an ES tumor promotor by negatively targeting the Tumour Necrosis Factor Receptor Super Family Member 6 (TNFRSF6) in ES cells ^[10][52].

One prior report showed that both miR-193b and miR-124 can act as tumour suppressors and specifically, miR-193b has been shown its associated with tumorigenesis and inhibit tumour growth and metastasis ^[11][53]. The functional data reported that miR-124 target Zinc Finger Protein SNAI2 (SLUG), which regulate cellular motility, inducing epithelial to mesenchymal transition (EMT) and overexpression of the cell cycle Cyclin D2 regulator (encoded by the CCND2 gene). Moreover, miR-124 overexpression inhibits cell motility and block the cell cycle progression ^[12][54]. The miR-193 negatively correlated with receptor tyrosine-protein kinase erbB-4 (ErbB4 gene), which has been identified as a metastasis promoter in ES ^[13][14][55,56]. Similarly, miR-30d is associated with growth, malignant progression, and apoptosis in ES. The miR-30d is also considered a tumor suppressor and its overexpression is linked with poor prognosis in prostate cancer ^[15][16][57,58]. The mechanistic study also investigated how downregulating the production of matrix metalloproteinases-9 and -2 (MMP-9/-2) in ES cell lines led to an increase in miR-30d in those cells.

Additionally, miR-30d overexpression results in the accumulation of apoptotic cells, which increases the expression of the Bcl-2-like protein 4 (Bax) and concurrently decreases the levels of the B-cell lymphoma 2 (Bcl-2) proteins. Furthermore, the MEK/ERK and PI3K/Akt signaling pathways, which are involved in cell survival, proliferation, cell cycle progression, metabolism, and protein expression in ES cells, have been connected to miR-30d and other key signaling pathways in the evolution of tumours ^[17][18][59,60]. The functional study reported that miR-107 overexpression is correlated with cell cycle progression, apoptosis, and restrained cell cycle arrest. The molecular mechanism behind these effects is probably mediated by the negative target of miR-107 on the Hypoxia-Inducible factor-1β (HIF-1β) gene ^[19][61]. Another miR-185 is also known as oncomiR due to its low expression in ES tissues and cells and tumour suppressor activity in various cancers ^[20][21][62,63]. The prior study explores the role of miR-185 in ES progression by its overexpression in ES cell lines which reduces cell proliferation and colony formation ability through Bcl-2 and the E2F Transcription Factor 6 (E2F6) targets ^[22][64].

In ES, miRs such as miR-683 and miR-107 prevent tumor progression and cell proliferation. The ES cells demonstrated that miR-638 is downregulated, and its overexpression inhibits vascular endothelial growth factor A (VEGFA) function by suppressing mRNA and VEGFA protein expression. It also induces cell death and inhibits cell proliferation. While miR-107 appears to suppress angiogenesis by inhibiting the development of new blood vessels ^[23][65]. In another bioinformatic study, it has been reported that miR-21, upon expression in ES cells, works as a tumour suppressor by targeting and downregulating the expression of Activated Leucocyte Cell Adhesion Molecule (ALCAM) ^[24][66]. Whereas the downregulation of miR-199b-5p has been reported in ES cell lines in comparison with human MSCs (Table 1) ^[25][67].

The miR-181c is linked with cancer development and manifested as an oncogene in ES ^[30][72]. The miR-214-3p is exhibited oncomiR behaviour and is constitutively suppressed in cell lines. However, when the EWS-FLI1 and/or CD99 genes are silenced, it is once again expressed, and miR-214-3p function as a marker of tumour aggressiveness by restricting ES cell proliferation and migration and suppressing the expression of its target HMGA1 ^[31][73].

miRs have altered the expression levels in cancer and this changed miRs expression profiles have been identified in several malignancies, particularly in osteosarcoma and EWS, respectively. It has been studied that EWS/FLI1 makes changes in miR-145 and let-7a expression profiles in EWS. When EWS-FLI1 is knocked down, the miR-145 expression increases rapidly. It is markedly decreased in EWS cell lines ^[32][74]. In contrast, miR-145 transfection raised EWS-FLI1 levels while ectopic expression of miR-145 in Ewing’s sarcoma cell lines significantly decreased EWS-FLI1 protein. Like let-7a, HMGA2 is a direct EWS-FLI-1 target implicated in the tumorigenicity of EFT cells because of its impact on the target oncogene’s expression. A signature of five miRs (miR-34a, miR-23a, miR-92a, miR-490-3p, and miR-130b) was found to be an independent predictor of risk for disease progression and survival after microarray analysis of data from 49 individuals with primary ES ^[33][75].

2. miRs as Therapeutic Agents and Applications

Due to their ability to target multiple genes and cellular pathways altered in pathological conditions, miRs could be good candidates for novel therapeutic agents. miRs are involved in various diseases and play important roles due to their involvement in mutations and downregulations of key enzymes. The dysregulation of the miR biogenesis pathway has been linked in several studies to the emergence of cancer. Additionally, it has been noted that a poor prognosis is associated with the downregulation of the drosha and dicer enzymes, which has been found in numerous cancers ^[34][35][87,88]. ES is a pediatric sarcoma that usually originates in bones. Genetically, it arises from chromosomal translocations that fuse the EWS protein to different transcription factor members of the ETS family, with EWS-ETS fusion genes acting as the pathogenic catalyst for ES ^[36][37][12,89]. Additionally, some miRs could potentially target insulin-like growth factor (IGF) which is a known player for ES ^[36][12]. Another study identified miR-145 as a top miR candidate that is repressed in ES ^[37][89]. The EWS/FLI1 fusion transcript may be directly targeted by miR-145, which suggests that this regulatory network should be further investigated as a possible target for miR-mediated therapeutics in this kind of cancer. Let-7a is also identified as a direct target for the EWS/FLI1 fusion transcript ^[38][77]. The role of miRs as biomarkers in ES has been investigated ^[39][40][90,91]. It has been shown that patients with high levels of miR-34a have better 5-year survival outcomes than patients with low levels of miR-34a ^[39][90]. An integrated analysis study of miRs and copy number changes revealed around 20 differentially expressed miRs in chromosomal regions with altered copy numbers. There are around 35 miRs have been shown to have distinct expression patterns in ES tissues and cell lines in comparison with MSCs ^[41][92]. Furthermore, the generated results showed that miR-31 act as a tumor suppressor that affected proliferation and invasion in Ewing cell lines. The expression levels of miR-100, miR-125b, miR-22, miR-221/222, miR-27a, and miR-29a are also suppressed upon suppression of EWS/FLI1 in ES cells. These miRs regulate the insulin-like growth factor (IGF) pathway and promote ES cell growth ^[36][12]. Moreover, miR-145 also controls the expression level of EWS/FLI1 and suppresses the ES cell growth ^[37][89]. The EWS/FLI1 fusion protein upregulates the EYA3 by suppressing miR-708 and as a result it may be considered as a drug target for the designing of novel chemical scaffolds against ES ^[42][93]. Another cell surface glycoprotein CD99 is present in ES tissue, and strongly related to ES malignancy and expression of EWS/FLI1 ^[43][94]. Expression of CD99 is regulated by miR-34a via the Notch signaling pathway and prevents ES differentiation. In addition, CD99 is also expressed in the membrane-bound extracellular vesicles, called exosomes derived from ES cells ^[43][94]. The analysis of 49 primary ES tumours revealed that miR-34a is related to ES progression and its low expression predicts poorer outcomes and overall worse survival. The IGF-1/AKT/mTOR pathway is also one of the key targets for ES treatment and IGF receptor 1 (IGF-1R) and mTOR inhibitors prevent tumour growth and improve ES survival rate in a mouse xenograft model ^[44][95]. However, besides these miRs, there are some other long non-coding RNAs (lncRNAs) that have been shown to affect various aspects of carcinogenesis mostly through modulation of the function of cancer-associated miRs ^[45][96]. Deregulation of the miRs is a frequent feature of several illnesses. The development of miR therapies for the treatment of a wide range of human illnesses is therefore the focus of many different sorts of study. The first-ever miRNA therapy is undergoing phase II clinical trials for the treatment of hepatitis C virus (HCV) infection. Miravirsen is a short-locked nucleic acid (LNA) medication targeting miR-122 that is quickly making its way from the lab to the clinic. Novel nucleic acid analogues called LNAs have some therapeutic potential. They are essentially modified or customized RNA nucleotides ^[46][97]. miRs have shown potential implications in the treatment of ES, a rare and aggressive form of bone and soft tissue cancer however, current research has explored different promising strategies and applications of miRs in ES therapy ^[47][98]. In tumor suppression, multiple miRs function as tumor suppressors by inhibiting the expression of genes involved in cancer growth and metastasis ^[48][99]. Researchers have identified miRs that are downregulated in ES and have explored the use of miR mimics to restore their levels. These miRNA mimics can potentially inhibit ES cell growth and induce apoptosis (cell death). Secondly, miRs target and downregulate oncogenes (genes that promote cancer), therefore, modulating the activity of miRs helps to suppress the expression of oncogenes that drive ES development and progression along with metastatic inhibition. Another significant application of miRs is chemotherapy sensitization; miRs modulate the sensitivity of cancer cells to chemotherapy drugs. Researchers have investigated the potential of miRs to sensitize ES cells to chemotherapy, making them more responsive to standard treatments. Most importantly, miR-based therapies also support traditional treatments such as chemotherapy and radiation therapy to achieve synergistic effects. Therefore, combinations of miR inhibitors with other drugs enhance the therapeutic response ^[49][100]. Moreover, miR profiling of ES tumors supports the identification of specific miR expression patterns associated with disease aggressiveness and prognosis. This information can guide treatment decisions and the development of personalized therapeutic strategies against ES. It’s noteworthy that miR-based therapies hold promise for ES treatment, further research is needed to fully understand the mechanisms and optimize their use in clinical settings for ES therapy in the future.