Epigenetic and Posttranscriptional Regulation in Retinoblastoma ()
1. Introduction
Retinoblastoma (RB), which accounts for 6% of pediatric cancer cases and occurs in one in every 15,000 - 20,000 live births, is the most prevalent primary intraocular malignant tumor in pediatric patients worldwide [1] . Pediatric cancer can be hereditary or spontaneous, and RB is known to be caused by the biallelic inactivation of the RB1 gene, which is situated at the 13q14 region of chromosome 13 [2] . Due to somatic and germline mutations in the tumor suppressor RB1 gene, RB can be inherited or acquired. The functional retinoblastoma protein (pRB), which is necessary for chromosomal integrity, is evidenced by studying the process that grants RB an infinite capacity for proliferation [3] .
Studies have demonstrated that RB1 knockdown induced cone precursors’ development, proliferation, and malignancy in dissociated retinal cultures [4] . These cone precursors formed tumors in orthotopic xenografts with histologic characteristics and protein expression profiles typical of differentiated human RB. Some other oncogenes and suppressors have been discovered. The genes that promote RB progression are the chromatin remodeling factors such as murine double minute 4 (MDM4), kinesin family member 14 (KIF14), DNA factor remodeling (DFR), transcription factor E2F3, and the tumor suppressor cadherin 11 (CDH11) [5] .
2. Epigenetic Biomarkers in Retinoblastoma
Epigenetic regulation’s crucial significance in Retinoblastoma is a newly discovered field. Retinoblastoma could develop and spread due to separate or combined genetic and epigenetic processes. There has been remarkable progress in understanding the genetic changes that lead to retinoblastoma pathogenesis, but less in epigenetic modification. Site-specific DNA methylation, histone modifications, long non-coding RNA, modification of microRNA (miRNA) mediated gene silencing, and ATP-dependent chromatin remodeling are examples of epigenetic modifications that silence tumor suppressor genes and activate oncogenes. It has been shown that the inactivation of RB1 can lead to dysregulation of the tumor suppressor and oncogenic pathways through epigenetic mechanisms. Future therapies and diagnoses will be possible with a better understanding of the epigenetic alterations in retinal fibroblastoma [1] .
3. DNA Methylation Biomarkers of Retinoblastoma
Although the gene’s promoter region remains unmethylated despite having a high density of CpG islands, it is well known that methylation occurs mainly in the gene’s CpG-poor coding region in normal tissue. In an ideal situation, this methylation pattern persists, but when it becomes unbalanced across the genome, it turns healthy cells into malignant ones. DNA methyltransferase expression is elevated in cancerous cells, and the unmethylated CpG island in the promoter region of the gene is the site of localized hypermethylation. The gene’s transcription is repressed as a result of this hypermethylation. Tumor suppressor genes are hypermethylated in malignant cells, while oncogenes are hypomethylated. Hypermethylation of the CpG island in the gene’s promoter region is one of the primary mechanisms by which cancer-related genes like RB1, cyclin-dependent kinase inhibitor 2A (CDKN2A/p16), von Hippel-Lindau tumor suppressor (VHL), MutL homolog 1 (MLH), O6-methylguanine-DNA methyltransferase (MGMT), and ras-associated domain family 1A (RASSF1A) transformed the cells [6] .
3.1. Histone Modification Biomarkers of Retinoblastoma
One of the most crucial processes for controlling the expression of genes in eukaryotes is the posttranslational modification of nucleosomal histones, which changes chromatin into active or inactive chromatin. Histone posttranslational modifications can take many forms, including methylation, acetylation, phosphorylation, and ubiquitination.
3.2. Biomarkers from Non-Coding RNA Regulation in Retinoblastoma
Many processes, including control by non-coding RNAs, are part of epigenetics and posttranscriptional processes, generally considered vital in ocular diseases (ncRNAs). Despite being unable to be translated into proteins, non-coding RNAs (ncRNAs) constitute the most significant portion of the human genome and have a greater impact on many biological functions than coding RNAs [7] .
Long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and circular RNAs (circRNAs) are among the different classes of non-coding RNAs that have been discovered. These ncRNAs play a role in the onset, pathogenesis, and development of many eye diseases, including cataracts, premature retinopathy, age-related macular degeneration, eye tumors, etc. Recent studies examining ncRNAs’ function in RB have seen an increase in recent years. In this context, ncRNAs are regarded as relevant and innovative biomarkers not only for the early diagnosis of this tumor but also for providing a therapeutic and prognostic approach to improving the survival and quality of life of RB patients [8] .
4. microRNA Biogenesis
Small non-coding RNAs called microRNAs (miRNA) function as negative posttranscriptional regulators by degrading or suppressing the translation of messenger RNAs (mRNAs) [9] . MicroRNAs have between 18 and 24 nucleotides. Generating miRNAs begins with DNA sequences known as miRNA genes or gene clusters that are only transcripted as miRNA molecules or collectively as polycistronic transcripts, respectively. A protein-coding gene’s intron or untranslated region (UTR) may also be where miRNAs are found. The primary miRNA-generating processes are canonical and non-canonical biogenesis pathways (Figure 1).
5. Canonical miRNA Biogenesis
RNA polymerase II and III can perform the conventional transcription of miRNA genes. Whereas RNA polymerase III transcribes miRNA genes in repetitive DNA sequences, RNA polymerase II transcribes miRNA genes in intergenic or intronic regions. Pre-miRNAs are released into the cytoplasm after being transcribed by the protein Exportin-5 (EXP5) [10] .
The RNase III endonuclease DROSHA and its cofactor DGCR8 convert the pre-miRNA into a shorter miRNA precursor in the cytoplasm (pre-miRNA).
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Figure 1. Biogenesys non-coding RNA. circRNAs can be classified into intronic circRNA (ciRNA), exonic-circRNA (ecircRNA), and exon-intron circRNA (ElciRNA). IncRNA can be generated in various types: (A) Sense; (B) Antisense; (C) Divergent; (D) Intergenic; (E) Intronic, and miRNAs, which are processed and secreted by exosomes. Created with BioRender.com.
Pre-miRNA is transformed into a mature and functional miRNA once it reaches the cytoplasm by the RNase III endonuclease Dicer and its cofactor TRBP [11] . The hairpin structure of pre-miRNA includes a stem and a loop. Pre-miRNA size and length can differ based on the species and cell type. The pre-miRNA is exported to the cytoplasm by Exportin-5 following processing by DROSHA.
In the RNA-induced silencing complex, the mature miRNA attaches to effector proteins that silence genes, such as the Argonaute protein (AGO) (RISC). The RISC complex binds to the 3’UTR region of target mRNAs to silence gene expression by mRNA degradation or translation repression [10] .
In conclusion, the synthesis of miRNAs is a multistage, tightly controlled process involving several protein components. Creating miRNA-based therapeutics and discovering new therapeutic targets depend on our ability to comprehend miRNA production.
6. Non-Canonical miRNA Biogenesis
In addition to the canonical pathway, unexpected non-coding RNAs may be converted into miRNAs through different mechanisms depending on the cell type and cellular state. The same proteins from the canonical pathway are combined differently to produce functionally equivalent miRNAs in the non-canonical pathway. Drosha/DGCR8-independent and Dicer-independent are the two main non-canonical miRNA biogenesis mechanisms that are distinguished [12] .
MiRtrons biogenesis is an illustration of the first. By examining critical characteristics such as guanine concentration, hairpin length, free energy, and bulges in the stem region, miRtrons may be separated from conventional miRNAs [12] . The whole intronic sequence of the mRNA-expressing genes in which they are found is matched by miRtron-derived pri-miRNAs. Splicing is used to process them rather than the microprocessor complex. After splicing, pre-miRNAs produced by the miRtron are exported to the cytoplasm by XPO5, where Dicer completes the process to create a mature miRNA.
Pre-miR-451 is an illustration of a non-canonical Dicer-independent mechanism for miRNA production. Drosha first breaks it down to release the pre-miRNA, and then it is loaded into Ago2 in the cytoplasm to stimulate the maturation of this microRNA. Because Pre-stem-loop miR-451’s structure is too short to detect and process effectively, Dicer does not digest it. As a result, it appears that Ago2 is essential in this pathway for efficient guide strand selection and RISC loading. This miRNA plays a role in the development and growth of tumors in several malignancies. It is downregulated in epithelial cancers that become invasive adenocarcinomas after losing their basolateral polarity, as shown in non-small cell lung carcinoma, colorectal cancer, and gastric cancer (NSCLC) [12] .
7. microRNAs in RB
Using circulating miRNAs may have some benefits, such as being a non-invasive biomarker and being easily accessible and quick to detect, making them a novel platform for diagnostic and therapeutic use. Several lines of research suggested that miRNAs can alter the behavior of cells under different settings by selectively targeting a range of cellular and molecular targets. Several events may cause RB to initiate and develop when the expression of miRNAs is dysregulated. For instance, let-7b is among the let-7 family members whose deregulation is linked to the onset and development of RB. Many genes, including high-mobility group A1 (HMGA1) and high-mobility group A2 (HMGA2), play essential roles in RB development and may be upregulated due to let-7b’s downregulation [9] . Others have shown that miR-34 functions as a tumor suppressor in RB cells and is a potential therapeutic target [11] .
Zhao et al. evaluated the expression of several miRNAs in human RB tissues using the microarray approach. Specific miRNAs were validated using the in situ hybridization method and Northern blot analysis. According to their findings, patients with RB had higher levels of several tissue-specific miRNAs, including miR-129-1, miR-494, miR-198, miR-492, miR-513-2, let-7e, miR-513-1, miR-503, miR-518c, miR-129-2, miR-498, miR-320, and miR-373, than healthy individuals. According to these results, multiple miRNAs might be used as novel diagnostic biomarkers for patients with different stages of RB [14] .
It has been discovered that RB growth and progression may be influenced by a particular class of microRNA called miRNA-22. In 2012, researchers analyzed the expression and use of miRNA-22 in retinoblastoma cells. When retinoblastoma cells were compared to healthy retinal cells, they discovered that miRNA-22 was dramatically downregulated. Moreover, overexpressing miRNA-22 prevented retinoblastoma cells from proliferating and growing, indicating that this microRNA may have a tumor-suppressive function. In RB cells, the researchers also discovered two putative miRNA-22 targets, which may shed light on the molecular processes underlying the genesis of this disease [15] .
The present work examined curcumin’s impact on the expression of miRNAs in human Y79 retinoblastoma cells. In a functional search for miRNAs that support in vitro cell migration and have prometastatic potential, miR-373 was found. MiR-373 expression is higher in RB tumors than in normal retinas, indicating a potential function in the tumor pathway. The study discovered that curcumin treatment changed the expression of several miRNAs, including the overexpression of miR-22, which has previously been demonstrated to have tumor-suppressive effects in RB. Moreover, miR-21 expression was reduced by curcumin treatment, which has been linked to carcinogenic features in many malignancies [15] .
Beta et al. looked at the expression of several serum miRNAs in RB patients. Pooled serums from 14 patients with advanced RB and 14 normal volunteers were used to provide miRNAs. According to their findings, children with RB had higher levels of 21 serum miRNAs and lower levels of 24 serum miRNAs than the healthy group. They demonstrated that deregulating different serum miRNAs, such as miR-17, miR-18a, and miR-20a, might stimulate cell proliferation and prevent apoptosis in RB cells by influencing many cellular and molecular targets. Hence, it appears that the discovery of novel circulating miRNAs and their cellular and molecular targets may offer a new diagnostic tool for the early identification of RB patients. Also, finding new biomarkers may help us understand the biological mechanisms involved in the pathogenesis of RB. Several studies evaluated circulating miRNAs in RB patients as diagnostic, prognostic, and therapeutic biomarkers. Hence, we proposed that these molecules may be employed as a new diagnostic and therapeutic platform in RB patients [16] .
Liu and Col. evaluated circulating miRNAs as a diagnostic biomarker in RB patients. They demonstrated that the downregulation of several plasma miRNAs, such as miR-320, let-7e, and miR-21, is linked to the development of RB. The study used 65 plasma samples from patients with RB and 65 samples from healthy volunteers as controls. These results indicated that circulating miRNAs might be used as diagnostic biomarkers in RB patients [17] .
Also linked to tumor suppressor properties is miR-34. Studies have shown that the miR-34 family is transcriptionally activated by p53. Variable levels of miR-34a expression have been noted, and it has been proposed that miR-34a knockdown by anti-miR compounds may increase RB cell proliferation and chemotherapy resistance. Two RB cell lines are frequently employed (Y79 and Weri-Rb1). When topotecan and miR-34a were coupled, these scientists also noticed significant RB cell growth suppression. This finding is consistent with earlier research showing a comparable growth suppression with a combination of topotecan and the p53 activator nutlin-3. Overall, the data are consistent with miR-34a acting as a tumor suppressor in the healthy retina [18] .
Several malignancies have been investigated concerning the miR17-92 cluster (OncomiR-1). In RB, miR-17-92 promotes carcinogenesis through proliferation control, at least in part by directly suppressing important cell cycle regulators such as p21CIP1 and p57KIP2. According to the references, miR-1792 cooperates with the death of RB’s family members to support RB [18] . (Table 1)
8. Long Non-Coding RNA (lncRNA)
Non-coding RNAs greater than 200 nucleotides, known as long non-coding RNAs (lncRNAs), have a limited capacity to encode proteins. LncRNAs are essential biological molecules linked to cancer, according to growing evidence. They can modulate many biological processes, including cell division, death, migration, invasion, and differentiation [20] . (Table 2)
8.1. IncRNA Biogenesis
RNA polymerase II transcription, splicing, and posttranscriptional alterations, like 5’ capping and polyadenylation, are all critical steps in the complicated process of lncRNA formation [24] . The Xist gene, which causes the inactivation of the X chromosome in female animals, is one well-studied example of lncRNA biogenesis. A persistent RNA molecule known as Xist wraps the whole X chromosome after being extensively spliced and polyadenylated [25] . XIST is translated from the X chromosome. The function of chromatin remodeling complexes, such as the Polycomb Repressive Complex 2 (PRC2), in controlling lncRNA expression is another crucial element of lncRNA biogenesis. LncRNAs serve as scaffolding for the PRC2 recruitment to particular genomic loci and chromatin [26] .
8.2. IncRNA in RB
Recent research has revealed that lncRNAs play a crucial part in the development of RB. In particular, RB patients had increased levels of the lncRNAs Hox transcript antisense intergenic RNA (HOTAIR), differentiation antagonizing non-protein coding RNA (DANCR), colon cancer-associated transcript 1 (CCAT1), and nuclear-enriched abundant transcript 1 (NEAT1), which exacerbated the onset and progression of RB. However, other lncRNAs, such as MT1JP, suppressed tumor growth in RB [5] .
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Table 1. Altered miRNAs in retinoblastoma.
The human genome contains the 2628-nucleotide lncRNA CCAT1, which is adjacent to the gene for the transcription factor c-Myc. In this study, RB tissues had considerably higher levels of lncRNA CCAT1 expression than normal tissues. Moreover, Zhang et al. discovered that lncRNA CCAT1 was markedly higher in RB SO-RB50, Y79, and WERI-RB1 cells than in the normal podiatric retina. Hence, lncRNA CCAT1 requires further investigation to explore its regulatory role in RB [25] .
Increased lncRNA HOX transcript antisense RNA (HOTAIR) expression in RB tissues has been identified by microarray analysis as a factor in accelerated RB development and metastasis. The potential therapeutic target of HOTAIR for RB and the ability of RB cells to proliferate and invade are suppressed when HOTAIR is knocked down [28] .
According to Yang et al., HOTAIR influences the survival, apoptosis, and epithelial-mesenchymal transition of RB cells via sponging miR-613. It is significant to note that overexpression of miR-20b-5p has been shown to inhibit RB cell growth and promote apoptosis. However, it has not yet been determined if HOTAIR can act as the ceRNA of miR-20b-5p to control RB development. The authors examined the ceRNA mechanism of the lncRNA HOTAIR in RB development, looking for further RB diagnosis and treatment. There are numerous malignant tumors, including non-small-cell lung carcinoma (NSCLC), colorectal cancer, and colon cancer, where the well-known lncRNA KCNQ1OT1, which functions as an oncogene, was substantially expressed. KCNQ1OT1 served as a sponge for miR-217, miR-212-3, and miR-504 and was involved in developing tumors, their spread, and their resistance to treatment. miR-124 was found to be down-regulated in RB tissues and cell lines, according to a prior study. Moreover, the miR-124/STAT3 axis partially inhibited the effects of the lncRNAXIST on cell proliferation, cell cycle arrest, and cell death in RB cells [30] . Nonetheless, further research needs to be done on KCNQ1OT1’s expression and biological impact in RB [29] .
The X-inactive specific transcript (XIST), a long non-coding RNA (lncRNA), plays a significant role in the inactivation of the X chromosome. In particular, XIST functions as a tumor suppressor or a tumor-promoting lncRNA in various human cancers. Moreover, XIST expression is increased in RB tissues and cell lines, and when XIST is knocked down, RB cell proliferation is inhibited, and apoptosis is induced. As a competing endogenous RNA (ceRNA) of miR-101, XIST raises the expression levels of ZEB1 and ZEB2, promoting the progression of RB cells and their epithelial-to-mesenchymal transformation [31] . It was determined that XIST was an oncogenic lncRNA in RB in 2021. In cervical cancer, XIST enhanced cell proliferation and inhibited cell apoptosis via miR-140-5p, according to a prior study. Furthermore, miR-140-5p has been shown to induce cell death in RB while inhibiting cell proliferation, migration, and invasion [32] .
Wang et al. demonstrated that differentiation-antagonizing non-protein-coding RNA (DANCR) was overexpressed in RB cell lines and tissues and that by stimulating proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) by acting as a competing endogenous RNA (ceRNA) of the miRNAs miR-34c and miR-613, DANCR worsened disease-free survival (DFS) (p = 0.0084) and OS (p = 0.0056). The lncRNA colon cancer-associated transcript 1 (CCAT1) induced similar changes in RB samples, in this case, through the miR-218-5p/MTF2 axis [33] .
Furthermore, increased levels of the promoter of CDKN1A antisense DNA damage-activated RNA (PANDAR), a newly identified lncRNA, were reported to be associated with unfavorable clinicopathological characteristics such as optic nerve invasion and advanced International Intraocular Retinoblastoma Classification (IIRC) stage; this was partly due to PANDAR inhibiting apoptosis when interacting with the Bcl-2/caspase-3 pathway [34] . Similarly, evidence has been reported for the clinical relevance of testis-associated highly conserved oncogenic long non-coding RNA (THOR), whose overexpression enhances the malignant phenotype of RB in association with the oncogene c-Myc and the gene encoding insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1). Lastly, small nucleolar RNA host gene 14 (SNHG14) and plasmacytoma variant translocation 1 (PVT1), two other lncRNAs that promote tumor aggressiveness and lower OS (p < 0.001 and p < 0.032), respectively, were reported to act by sponging, respectively, the miRNAs miR-124 and miR-488-3p [35] .
Taurine upregulated gene 1 (TUG1) is a 7.1-kb lncRNA encoded on chromosome 11A1 that was first identified as an essentialregulator of retinal and normal photoreceptor development that is expressed in retinal tissues. More recent work has highlighted roles for TUG1 as a modulator of key oncogenic processes such as invasion, metastasis, cell cycle progression, proliferation, and apoptosis. TUG1 has also been linked to radioresistance and angiogenesis in the context of hepatoblastoma [36] .
TUG1 was upregulated in RB cells, and the absence of TUG1 repressed cell proliferation, whereas accelerated cell apoptosis in RB. In brief, TUG1 is an oncogenic gene in RB, and sponge of miR-516b-5p and upregulated H6PD to promote cell growth in RB, possibly inspiring future studies to find an effective target for RB treatment [37] .
PROX1-antisense RNA1 (PROX1-AS1) has been reported to participate in the progression of various cancers, such as ovarian and prostate cancer. Moreover, PROX1-AS1 can promote gastric cancer and thyroid proliferation and metastasis. In RB, reducingPROX1-AS1 could upregulate miR-519d-3p to restrain the malignant behaviors and drug resistance of RB cells via silencing SOX2 [38] .
Metastasis associated with lung adenocarcinoma transcript 1 (MALAT1) is a highly conserved mRNA-like lncRNA and was first identified with high expression in metastatic non-small cell lung cancer. According to previous studies, MALAT1 is overexpressed in many other human malignancies, including breast, pancreas, colon, prostate, and liver cancers. Functional studies of MALAT1 demonstrated that its deregulation influences multiple cancer cells’ proliferation, invasion, and/or metastasis. With these findings, it was concluded that MALAT1 is critical for cancer development. However, the mechanism of MALAT1 in tumor cell growth regulation, especially in RB, is still unclear. Liu and Col. identified the role of lncRNA MALAT1 and miR-124 in RB. The study found that MALAT1 silence upregulated miR-124 expression in RB cells, which reversed the inhibitory effect of miR-124 on tumor growth of RB cells. MALAT1 expression could be regulated in RB cells treated with miR-124 overexpression. Also, miR-124 increases the expression of E-cadherin, considered a hallmark of epithelial cells and a repressor of cell invasion and metastasis. These findings suggest that miR-124 is essential in RB’s invasive and/or metastatic potential [39] .
Long non-coding RNA (lncRNA) KCNQ1OT1 was reportedly tightly associated with tumorigenesis and the progression of multiple cancers. Emerging research suggested that KCNQ1OT1 was involved in the occurrence, metastasis, and drug resistance of tumors acting as a sponge for miRNAs, such as miR-217, miR-212-3, and miR-504. The function and mechanism of KCNQ1OT1 of RB are proliferation and migration [40] . (Table 2)
9. Circular RNAs (circRNAs)
Circular RNA (circRNA) is a novel class of non-coding RNA that predominantly controls gene activity to regulate biological processes. circRNA is a non-coding RNA, but in some situations, it can act as a translational template for the synthesis of proteins, leading to the production of functional proteins. As circRNAs have many miRNA binding sites, they act like sponges, soaking up miRNAs. By base pairing, circRNA may also engage in interactions with other RNAs. Additionally, circRNA may restrict the function of proteins by interacting with them CircRNAs have recently been shown to regulate and control alternative splicing and host gene expression, interact with RNA-binding proteins that control transcription, and affect cis-transcriptional regulation [47] .
9.1. circRNAs Biogenesis
According to their parts, the circRNAs in eukaryotes can be divided into three subtypes: 1) exonic circRNAs (EcircRNAs), produced by the circularization of one or more exons; 2) exon-intron circRNAs (EIciRNAs), circularized from exons with introns reserved; and 3) intronic circRNAs [(ci)RNAs], made entirely of introns. The sequence of circRNAs is assembled head-to-tail by a 3’, 5’ phosphodiester covalent bond. In contrast to conventional splicing, “back-splicing”, which is how circRNAs are often created, involves connecting an upstream 3’ acceptor and a downstream 5’ donor.
In addition, five groups of circRNAs can be classified according to where they are about nearby coding RNA: 1) and 2) “exonic” and “intronic” formed, respectively, by exons and introns; 3) “antisense”: transcribed into the opposing strand from their gene locus being overlapped by linear isoforms; 4) “sense overlapping”: produced by the same gene locus but does not belong to the “exonic” or “intronic” circRNA; 5) “intergenic”: transcribed from the location that is outside the gene loci [42] .
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Table 2. Altered lncRNAs in retinoblastoma.
9.2. circRNAs in RB
According to preliminary studies, dysregulated circRNAs may play tumor-suppressive and oncogenic roles in the origin and progression of cancer. By controlling the biologically malignant behaviors of cancer cells, such as proliferation, apoptosis, and metastasis, they may affect many cellular processes and aid in the growth of tumors. These genes can change in various ways, such as when new oncogenes appear or old proto-oncogenes become overexpressed. Tumor suppressor genes (TSGs) prevent cell proliferation by reversing the process [42] .
By disrupting the AKT/mTOR signaling pathway, Circ 0001649 limits RB cell proliferation and death, and its low expression level predicts a poor prognosis for patients [48] . Circ ODC1 promotes RB cell proliferation and positively controls SKP2 [49] . Unexpectedly, circ 0000527’s high expression was shown to be strongly correlated with the degree of differentiation and cTNM staging in another investigation, which found that RB tissues and cells had a drastically enhanced level of this gene’s expression. According to these results, circ 0000527 was likely to suggest that RB patients will have a bad prognosis. Moreover, circ 0000527 overexpression markedly reduced cell apoptosis while increasing cell proliferation, motility, and invasion [50] . Circ 0000527, however, had the reverse result after being knocked down. At the same time, miR-646 expression in RB showed a significant decrease, and since miR-646 has several binding sites for circ_0000527, further studies confirmed that circ_0000527 negatively regulates miR-646 expression. In addition, acting as a sponge for miR-1236-3p, which targeted SMAD2 directly, and as a result, miR-1236-3p expression was reduced in RB tissues and cells [51] .
According to reports, Circ 0099; 198 is highly expressed in RB and reduces the expression of miR-1287, which controls the expression of LRP6 [52] .
Circ 0000034 was the unique circRNA studied with a single target, miR-361-3p, whose targets could potentially affect the mechanisms underlying RB and are crucial elements in the initiation and development of this malignancy. MiR-361-3p controls the expression of STX17 by targeting. Because miR-361-binding 3p’s sites are sponging STX17, there is a rise in the expression of circ 0000034. On the other hand, investigations in RB have shown a reduction in miR-361-3p expression [53] . Circ 0000034’s inability to sponge miR-361-3p revealed that circ 0000034’s silencing significantly reduced RB development. By controlling the expression of ADAM19 in this situation, miR-361-3p prevents RB development [54] .
The E2F family of transcription factors has been illustrated as a critical regulator related to various tissues’ proliferation, differentiation, and apoptosis. E2F transcription factor 5 (E2F5), a member of the E2F family, has been characterized as a transcriptional repressor that can regulate cell proliferation through its interaction with the RB protein to inhibit target gene transcription. In addition, it has been documented to play critical roles in cancer development, including RB. The circ_0084811 was discovered to be distinctly overexpressed in RB cells. Through mechanistic studies, it was established that the enrichment of H3K27ac in the circ_0084811 promoter caused the overexpression of circ_0084811 in RB cells. Functionally, inhibition of circ_0084811 diminished RB cell proliferation and stimulated cell apoptosis [55] .
circ_0075804 regulated tumorigenesis in RB, performed an animal study using xenograft mice model. Similar to the former findings in vitro, circ_0075804 silencing notably suppressed xenograft tumor growth. The expression of circ_0075804 and PEG10 protein was reduced, whereas miR-138-5p expression was increased in tumor tissues with the silencing of circ_0075804. Circ_0075804 silencing increased Bax and E-cadherin expression and reduced the level of Bcl-2 in tumor tissues [56] .
CircDHDDS, a miR-361-3p molecular sponge, similarly controlled the expression of WNT3A. The circDHDDS/miR-361-3p/WNT3A axis thus facilitated RB development by controlling RB cell proliferation, cell cycle regulation, migration, and invasion. Circ 0000034 is one of the primary circRNAs in RB, as evidenced by the fact that its expression rises in RB and that silencing it prevents the formation and progression of RB [57] . (Table 3)
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Table 3. Altered circRNAs in retinoblastoma.
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Table 4. Altered ceRNAs in retinoblastoma.
9.3. The ceRNAs Axes in RB
In recent years, increasing numbers of reports have found that circRNAs play a role in the biological functions of a competing endogenous RNAs (ceRNA) network. circRNAs act as ceRNAs to regulate several biological processes as cell proliferation, apoptosis, invasion, and migration. The term’ ceRNA’ was created in 2011 to characterize this additional layer of posttranscriptional control [63] .
The fact that ceRNA interaction networks are multifactorial means that they may be valuable in investigating complicated diseases such as RB in terms of therapeutic targets since the levels of several disease-related RNAs change simultaneously by targeting just one of them.
Most circRNAs examined as ceRNA axes have higher expression levels, although lower levels of the desired ceRNA axis limit circTET1 in RB. In typical circumstances, the ceRNA axes within the cell are in equilibrium. The trend toward abnormal conditions can be accelerated by varying the expression of each axis component.
According to Huang et al. [45] , Circ-E2F3 stimulates the miR-204-5p/ROCK1 pathway, which advances RB. In a study by Li et al. [64] , the miR-497/WEE1 axis allows TRPM2-AS to function as an oncogenic lncRNA in RB. DANCR serves as a ceRNA that targets miR-34c and miR-613 in the RB, as Wang et al. [32] demonstrated, to facilitate MMP-9-mediated progression and metastasis.
Recently, Jie Sun et al., 2023, demonstrated that the hsa_circ_0015965/lncRNA MEG3-has-miR-378a-5p-NOTCH1pathway promotes the malignant progression of RB [64] . (Table 4)
10. Conclusion
The most frequent intraocular tumor in children is Retinoblastoma (RB), and during the past 50 years, the prognosis has improved. Initially, non-coding RNAs (ncRNAs) were believed to be the byproducts of random transcription with no purpose. However, the functional significance of ncRNAs in cellular physiology and pathological processes has recently come to light thanks to new research. Significant interest in and evidence of a close relationship between miRNA, lncRNA, and circRNA and tumor growth have been generated. The competitive endogenous RNA (ceRNA) theory states that mRNAs, lncRNAs, and circRNAs compete for the same miRNA, which affects target mRNAs. A growing body of research has shown that ceRNAs are crucial to the development of RB. In detail, this article examines the miRNAs, lncRNAs, circRNAs, and cecRNAs involved in RB. The results of the summary analysis of the ncRNAs and associated ceRNA networks in RB may offer fresh insights and approaches for creating customized RB therapeutics.