Crosstalk between oncogenic MYC and noncoding RNAs in cancer

Journal Pre-proof Crosstalk between oncogenic MYC and noncoding RNAs in cancer Rongfu Tu, Zhi Chen, Qing Bao, Hudan Liu, Guoliang Qing

PII:

S1044-579X(20)30219-4

DOI:

https://doi.org/10.1016/j.semcancer.2020.10.014

Reference:

YSCBI 1906

To appear in:

Seminars in Cancer Biology

Received Date:

28 July 2020

Revised Date:

9 October 2020

Accepted Date:

24 October 2020

Please cite this article as: Tu R, Chen Z, Bao Q, Liu H, Qing G, Crosstalk between oncogenic MYC and noncoding RNAs in cancer, Seminars in Cancer Biology (2020), doi: https://doi.org/10.1016/j.semcancer.2020.10.014

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Crosstalk between oncogenic MYC and noncoding RNAs in cancer

Rongfu Tu1, 2, #, Zhi Chen1, 2, #, Qing Bao1, 2, #, Hudan Liu1, 2, Guoliang Qing1, 2, *

1

Department of Urology, Zhongnan Hospital of Wuhan University, Wuhan 430071, China

2

Frontier Science Center for Immunology & Metabolism, Medical Research Institute,

Wuhan University, Wuhan 430071, China

These authors contributed equally to this work

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#

[email protected]

Zhi Chen

[email protected]

Qing Bao

[email protected]

Hudan Liu

[email protected]

[email protected]

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Guoliang Qing

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Rongfu Tu

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Running title: MYC and noncoding RNAs in cancer

* Corresponding Author: Guoliang Qing, PhD

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Frontier Science Center for Immunology & Metabolism Medical Research Institute, Wuhan University 185 East Lake Rd, Wuhan, China, 430071

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Tel: +86 027 68750015, Fax: +86 027 68759675

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Abstract The MYC family oncoproteins are deregulated in more than 50% of human cancers through a variety of mechanisms, such as gene amplification or translocation, super-enhancer activation, aberrant upstream signaling, and altered protein stability. As one of the major drivers in tumorigenesis, MYC regulates the expression of a large number of noncoding genes involved in multiple oncogenic processes. Noncoding RNAs, including miRNA, lncRNA, circRNA, rRNA and tRNA, are also deeply involved in the oncogenic MYC network by functioning as MYC regulators/effectors. In this review, we summarize representative studies

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depicting the crosstalk between oncogenic MYC and noncoding RNAs in carcinogenesis with the aim of providing potential implications for both basic science and clinical applications.

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Keywords: MYC, noncoding RNAs, lncRNA, circRNA, tumorigenesis

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Introduction The oncogenic MYC family consists of three members, MYC, MYCN and MYCL, all of which are implicated in tumorigenesis of multiple human cancers [1, 2]. MYC is overexpressed or activated in greater than half of human cancers, and MYC activation is a hallmark of tumor initiation and maintenance [3]. MYCN amplification has been predominantly identified in neural tumors, such as neuroblastoma, but it also occurs in small cell lung cancer and neuroendocrine prostate cancer [4-6], with MYCL deregulation being more restricted in small cell lung cancer [7]. Deregulated expression of the MYC family in transgenic murine tissues of

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many varieties can trigger tumorigenesis, illustrating its transforming activity in vivo and supporting the notion that it is a human oncogene [8, 9]. Numerous studies have shown that enhanced MYC expression is a major driver of tumorigenesis, and elevated MYC levels are

consistently essential for the growth of tumors driven by MYC or other oncoproteins [10-12].

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Inhibition of endogenous MYC triggers rapid regression of incipient and established tumors in a mouse model of Ras-induced lung adenocarcinoma, indicating that MYC inhibition would be

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therapeutically efficacious [12].

MYC family members exhibit high structural homology, including the carboxy terminal

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region of basic-region/helix-loop-helix/leucine-zipper domains (BR/HLH/LZ) and several conserved elements. Functioning as a transcription factor, MYC is required to form a heterodimer with MAX, and the MYC/MAX heterodimer activates or represses gene

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transcription through binding to the E-Box (CACGTG) sequence [13-16]. It has also been reported that in tumor cells expressing high levels of MYC, MYC accumulates in the promoter

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regions of active genes and causes transcriptional amplification, producing increased levels of transcripts within the existing gene expression programs of the cells [17-19]. MYC regulates a

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large number of protein-coding or noncoding genes that are involved in almost all aspects of tumorigenesis [20]. MYC expression in tumors is subject to comprehensive regulation, including chromatin remodeling [21], super-enhancer activation [19, 22], and posttranslational modifications [23, 24]. With the advances of high-throughput technologies, such as next-generation sequencing, we have learned that protein-coding genes represent less than 2% of all human genomes, while the majority of nucleotide sequences transcribe noncoding RNAs that lack 3

protein-coding functions [25]. Generally, noncoding RNAs are divided into short (or small) and long (or large) noncoding RNAs with a threshold of 200 nucleotides [26], and linear long noncoding RNAs (lncRNAs) can be distinguished from circular isoforms by appearance [27]. Noncoding RNAs can also be classified as housekeeping RNAs (such as rRNA and tRNA) and regulatory RNAs (such as microRNA, lncRNA and circular RNA) [28]. Over the past two decades, noncoding RNAs have been shown to regulate cellular processes and pathways in both developmental and pathological contexts, notably in cancers [29]. Noncoding RNAs function as key modulators that regulate many biological processes

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during tumorigenesis. Regulatory noncoding RNAs, including microRNAs (miRNAs), lncRNAs and circular RNAs (circRNAs), are deeply involved in all ten hallmark features of human

malignancies via diverse mechanisms [27]. Moreover, some noncoding RNAs are specifically expressed in certain cancers, which could be potential biomarkers in cancer diagnosis [30].

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Although the detailed mechanisms whereby noncoding RNAs participate in the regulation

of tumorigenesis remain elusive, numerous studies have shown that noncoding RNAs play key

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roles in human cancers, in large part through regulation of the MYC network. Noncoding RNAs not only regulate expression of the MYC family but are also important downstream effectors of

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RNAs in carcinogenesis.

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MYC. In this review, we aim to discuss crosstalk between oncogenic MYC and noncoding

MYC and miRNAs

MiRNAs are single-stranded RNAs of ~ 22 nt in length that are generated from

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endogenous hairpin-shaped transcripts. Most miRNA transcription is mediated by RNA pol II, though a minor group of miRNAs are transcribed by RNA pol III, and the primary transcripts

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(pri-miRNAs) are usually several kilobases long and contain local stem-loop structures [31, 32]. Pri-miRNAs are subsequently cleaved into small hairpins, which are termed pre-miRNAs by the RNase protein Drosha in the nucleus, and pre-miRNAs are then exported to the cytoplasm, where they are cleaved near the terminal loop into miRNA duplexes by Dicer. MiRNAs primarily function as guide molecules in posttranscriptional gene regulation by base-pairing with target mRNAs, usually in the 3’-untranslated region (3’-UTR) [33]. As predicted, miRNAs regulate expression of ~90% of human genes [34]. Dozens of miRNAs have been identified to 4

participate in regulation of the MYC network, and some representative miRNAs are listed in Table 1.

1. MYC-regulated miRNAs The first identified miRNA cluster directly activated by both MYC and MYCN was miR-17~92, which encodes six distinct miRNAs, including miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR-92a-1 [35, 36]. The MYC-stimulated miR-17~92 cluster has been shown to target multiple tumor suppressors, such as PTEN, p21 and Bim [37, 38]. In agreement with its oncogenic activity, the miR-17~92 cluster is frequently activated in

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numerous solid tumors, including MYCN-amplified neuroblastomas and B-cell lymphomas [35, 39, 40]. Conditional knockout of the miR-17~92 cluster reduces the progression of

MYC-induced B-cell lymphoma in the Eμ-myc model, indicating that the miR-17~92 cluster

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represents an important mediator of MYC oncogenic functions [41]. As an additional critical target of MYC, miR-378 achieves its oncogenic effects in part by targeting and inhibiting the

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anti-proliferative BTG family member TOB2, a tumor suppressor that transcriptionally represses the proto-oncogene CCND1 (encodes cyclin D1) [42]. In MYCN-amplified

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neuroblastomas, MYCN induces the expression of miR-421, which suppresses the ATM-dependent DNA damage response by targeting the 3′-UTR of ATM mRNA [43] (Fig. 1A). Interestingly, a large body of evidence indicates that widespread miRNA repression, but

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not activation, by MYC contributes to tumorigenesis. Analysis of the miRNA expression profile in P493-6 cells and mouse bone marrow with MYC overexpression showed that the

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predominant consequence of MYC activation is widespread repression of miRNA expression, and enforced expression of the repressed miRNAs diminishes the tumorigenic potential of

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lymphoma cells. Chromatin immunoprecipitation (ChIP) reveals that much of this repression is likely due to the direct binding of MYC to these miRNA promoters [44]. Similarly, MYCN is also predominantly a suppressor of miRNAs [45]. Indeed, miRNAs are widely downregulated in MYCN-amplified neuroblastoma compared to non-MYCN-amplified neuroblastoma [46, 47]. Notably, many miRNAs directly inhibited by MYC mediate its oncogenic roles. For example, the miR-15a/16 cluster is located in an intronic region of the DLEU2 gene, which is directly repressed by MYC. By directly targeting several oncogenic factors, such as cyclin D1, 5

WNT3A, MCL1 and BCL2, miR-15a/16 induces apoptosis and cell cycle arrest, thereby exerting its tumor-suppressive functions [48-50]. By repressing miR-23a and miR-23b, MYC increases expression of GLS1, a key enzyme responsible for the conversion of glutamine to glutamate, which serves as a substrate in the TCA cycle [51]. MYCN contributes to tumorigenesis in part by repressing miR-184, leading to increased levels of AKT2. As such, two important genes with positive effects on cell growth and survival, MYCN and AKT2, can be linked into a common genetic pathway through miR-184 [52] (Fig. 1B). Let-7 is a tumor suppressor family of miRNAs implicated in numerous cancers that is repressed by both MYC

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and MYCN in an unconventional fashion. MYC directly activates the expression of the RNA binding protein LIN28b, which binds the let-7 precursor stem loop and thereby prevents processing by Drosha and Dicer [53, 54].

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2. MiRNAs targeting MYC

Dozens of miRNAs have been described to inhibit MYC or MYCN expression by directly

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binding to their mRNAs [45, 55]. MiR-145, a tumor suppressor induced by p53, mediates MYC mRNA degradation by binding to its 3′-UTR. Specific silencing of MYC by miR-145 accounts at

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least in part for miR-145-mediated inhibition of tumor cell growth [56]. MiR-24 suppresses MYC expression by directly binding to two partially complementary sites in the MYC 3’-UTR

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[57] (Fig. 1C). MiR-184, miR-320 and miR-744 have been reported to inhibit MYC expression through direct binding to its open reading frame (ORF) [58-60]. The MYCN 3’-UTR is also directly targeted by several miRNAs, including miR-19, miR-29, miR-101, miR-449 and

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miR-202. These miRNAs are further shown to decrease MYCN protein expression when overexpressed in MYCN-amplified neuroblastoma cells [61]. Interestingly, miR-375 has been

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identified as a negative regulator of MYCN through directly binding with its 5’-UTR under stress conditions [62] (Fig. 1C). In addition to regulating MYC in a direct fashion, miRNAs can also influence MYC

expression in an indirect manner. Cancerous inhibitor of PP2A (CIP2A), a guardian of oncoprotein MYC, is identified as a candidate miR-375 target. Transient transfection of miR-375 in oral cancer cells reduced the expression of CIP2A, resulting in decreased MYC protein levels, leading to reduced proliferation, colony formation, migration and invasion [63]. 6

MiR-363 destabilizes MYC by directly targeting and inhibiting USP28, which is required for MYC stability [64]. Cancer cell-secreted exosomal miR-105 is recognized as a positive regulator of MYC-dependent metabolic reprogramming of stromal cells. MiR-105 activates MYC through direct downregulation of MXI1, a protein that antagonizes the MYC–MAX dimeric transcriptional factor by forming heterodimers with MAX [65] (Fig. 1C).

3. Feedback loops between MYC and miRNAs In addition to the one-way regulation between MYC and miRNAs as described above,

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more complex feedback loops have been identified between MYC and miRNAs (Fig. 1D). MiR-34a, miR-148 and miR-451 are examples of MYC-repressed miRNAs that can directly repress MYC translation. In hepatocellular carcinoma (HCC), MYC directly binds to the conserved region within the promoter of miR-148 and represses its expression, and miR-148

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can also suppress MYC expression by targeting its 3’-UTR. Inhibition of miR-148 induced hepatocellular tumorigenesis by promoting G1 to S phase progression, whereas activation of

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miR-148 exhibited the opposite effects [64]. MiR-34a, one of the most well-known miRNAs induced by p53, is repressed by MYC binding in the vicinity of the miR-34a promoter [44]. On

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the other hand, miR-34a downregulates MYC and MYCN by targeting their 3’-UTRs [66, 67]. MiR-144/451, a bicistronic miRNA gene encoding miR-144 and miR-451, is suppressed by

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MYC in a promoter-dependent manner. Both miR-144 and miR-451 binding sequences exist in the MYC 3’-UTR, but only miR-451 is able to repress MYC mRNA, forming a positive feedback loop to safeguard against high levels of MYC in B lymphocytes [68]. Let-7a, a member of the

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let-7 family, is repressed by MYC in an unconventional fashion [53, 54]. However, in MYCN-amplified neuroblastomas, let-7a has been reported to inhibit MYCN translation

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through direct binding with its 3’-UTR [69] (Fig. 1D). MiR-17 is a member of the miR-17~92 cluster, which is transcribed by both MYC and

MYCN. A regulatory feedback loop exists between miR-17 and MYC, in which miR-17 suppresses some of the distinguishing features mediated by MYC by directly targeting its 5’-UTR [70]. MiR-17 also downregulates MYCN in MYCN-amplified neuroblastoma cells, generating a negative feedback regulatory loop between MYCN and miR-17 [71]. MYC and E2F1 form a positive feedback circuit by activating the transcription of each other and then 7

activating genes involved in DNA replication and cell cycle control [72]. MYC-induced miR-17 is also reported to suppress E2F1 expression by targeting its 3’-UTR, revealing a mechanism through which MYC simultaneously activates E2F1 transcription and limits its translation, allowing a tightly controlled proliferative signal [73] (Fig. 1D).

MYC and lncRNAs LncRNAs are defined as a subset of noncoding RNAs whose length is >200 nt. LncRNAs with different subcellular locations tend to have diverse biological functions. Nuclear lncRNAs

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can modulate chromatin remodeling, recruit transcription factors and epigenetically regulate gene expression. Cytoplasmic lncRNAs may be involved in the regulation of mRNA stability,

translation, protein stability and/or localization. In general, both nuclear and cytoplasmic lncRNAs are involved in diverse biological processes. In Table 2, we provide a list of lncRNAs

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that regulate MYC or, reciprocally, are regulated by MYC.

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1. MYC-regulated lncRNAs

Numerous MYC-regulated lncRNAs have been identified, indicating that lncRNAs are

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important players in mediating the oncogenic roles of MYC [74, 75]. LncRNAs regulated by MYC promote tumor progression through diverse mechanisms, for instance, acting as molecular sponges for tumor suppressor miRNAs. In HCC, MYC-activated LINC00176 is

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required for cell proliferation. Exploration of the mechanism revealed that LINC00176 acts as a competitive endogenous RNA to bind the tumor suppressor miRNAs miR-9 and miR-185 [76].

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In gastric cancer, highly expressed LINC00346, driven by MYC, is essential for tumor initiation and progression. MYC activates LINC00346 expression by directly binding to its promoter, and

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LINC00346 then functions as a molecular sponge for miR-34a, leading to malignant progression of tumor cells [77] (Fig. 2A). MYC-activated lncRNAs can also promote expression of critical oncogenes by regulating

their mRNA stability. The MYC-activated lncRNA LAST (lncRNA-assisted stabilization of transcripts)

promotes

tumorigenesis

by

upregulating

CCND1

mRNA

stability.

Mechanistically, LAST cooperates with cellular nucleic acid-binding protein (CNBP) to bind to the 5’-UTR of CCND1 mRNA to protect against nuclease degradation [78]. LncRNA MYU is a 8

direct target of MYC and is upregulated in most colon cancers. MYU associates with the RNA binding protein hnRNP K to stabilize CDK6, thereby promoting G1/S transition of the cell cycle [79]. Similarly, lncRNA E2F1 mRNA stabilizing factor (EMS) associates with RNA-binding protein RBP associated with lethal yellow mutation (RALY) to promote E2F1 mRNA stability and thereby increases E2F1 expression, resulting in enhanced G1/S cell cycle progression [80] (Fig. 2A). MYC-regulated lncRNAs can also promote malignant transformation through regulation of target protein activities. LncRNA cohesion regulator noncoding RNA (CONCR) is

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transcriptionally activated by MYC and upregulated in multiple cancer types. CONCR interacts with DEAD/H-box helicase 11 (DDX11) and enhances its DNA-dependent ATPase and helicase activity. As such, CONCR deficiency results in severe defects in sister chromatid

cohesion and suppresses tumor growth in vivo [81]. In addition to MYC-activated lncRNAs,

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those repressed by MYC also mediate its oncogenic roles. The lncRNA IDH1-AS1, which is

transcriptionally repressed by MYC, enables MYC to collaborate with HIF-1α to activate

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aerobic glycolysis in tumor cells. Conversely, overexpression of IDH1-AS1 promotes homodimerization of IDH1 and enhanced its enzymatic activity (Fig. 2A), resulting in increased (α-KG)

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decreased

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α-ketoglutarate

production

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subsequent

HIF-1α

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downregulation, leading to attenuation of glycolysis [82].

2. LncRNAs regulating MYC

2.1 LncRNAs regulating MYC transcription

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LncRNAs can regulate MYC transcription by influencing its chromatin structure. LncRNA colorectal cancer-associated transcript 1 (CCAT1-L) is specifically expressed in human

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colorectal cancers and located 515 kb upstream of the MYC locus. Repression of CCAT1-L reduces while overexpression of CCAT1-L enhances MYC expression in cis. As such, depletion of CCAT1-L reduces chromatin loop formation between the MYC promoter and its enhancers [83]. LncRNAs also regulate MYC transcription through recruitment of transcription factors. LncRNA MYC-modulating lncRNA (MYMLR), which resides in close proximity (2 kb upstream) to the MYC locus, has been shown to affect MYC transcriptional activity and lung cancer cell 9

cycle progression. Interestingly, MYMLR regulates MYC expression by binding to the enhancer region of the MYC promoter in cooperation with poly-C-binding protein 2 (PCBP2) [84]. LncCMPK2 is dysregulated in CRC (colorectal cancer) tissues. Overexpression of lncCMPK2 accelerates CRC cell proliferation and cell cycle progression, while lncCMPK2 knockdown impairs cell proliferation both in vitro and in vivo. LncCMPK2 interacts with far upstream element binding protein 3 (FUBP3) and guides FUBP3 to bind to the far upstream element (FUSE) of the MYC gene, leading to enhanced MYC transcription [85]. Similarly, lncRNA LINC00346 interacts with CCCTC-binding factor (CTCF) and prevents it from binding

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to the MYC promoter, abolishing CTCF-mediated repression of MYC transcription [86] (Fig. 2B).

2.2 LncRNAs controlling MYC mRNA stability or translation

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Some lncRNAs ensure high MYC levels by sequestering miRNAs targeting MYC mRNA.

LncRNA cell-cycle regulated lncRNA 492 (CCR492) contains four recognition elements of the

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let-7 family. Mechanistically, CCR492 functions as a competing endogenous RNA to antagonize the function of the let-7 family, leading to derepression of MYC [87]. LncRNA small

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nucleolar RNA host gene 3 (SNHG3) is aberrantly upregulated in CRC. Ectopic expression of SNHG3 increases, whereas its inhibition decreases, expression of MYC. Mechanistic

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investigation demonstrated that SNHG3 functions as a decoy to sponge miR-182, leading to reduced MYC mRNA degradation [88].

LncRNAs also regulate MYC mRNA abundance by affecting the interaction between MYC

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mRNA and its RNA binding proteins. Linc-RoR interacts with hnRNP I, which is essential for hnRNP I binding to MYC mRNA. In addition, linc-RoR interacts with AUF1 to prevent it from

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binding to MYC mRNA. Both molecular events enhance MYC mRNA stability and contribute to malignant transformation [89]. However, the interaction between AUF1 and MYC mRNA may have the opposite effects on MYC mRNA stability. FoxO-induced long noncoding RNA 1 (FILNC1) is an energy stress-induced lncRNA. FILNC1 interacts with AUF1 and prevents AUF1 from binding to MYC mRNA, leading to downregulation of MYC protein [90]. Why sequestering AUF1 from binding MYC mRNA has the opposite effects on MYC expression in different cell contexts remains unknown. 10

Moreover, MYC mRNA is positively regulated by the insulin-like growth factor 2 mRNA binding protein (IGF2BP) family, which acts as an m6A reader that guards m6A-modified mRNAs from decay [91]. LncRNA long intergenic noncoding RNA for IGF2BP2 stability (LINRIS) blocks K139 ubiquitination of IGF2BP2 and protects IGF2BP2 from degradation. LINRIS deficiency significantly decreases MYC levels and partially abolishes the downstream effects of IGF2BP2, especially MYC-mediated aerobic glycolysis [92]. Instead, Linc00266-1 encodes a 71-amino acid peptide that interacts with IGF2BP1, which promotes MYC mRNA m6A recognition by IGF2BP1. This enhances MYC mRNA stability, upregulates MYC

2.3 LncRNAs affecting MYC protein stability or activity

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expression and promotes tumorigenesis [93] (Fig. 2B).

LncRNA PVT1 has been found coamplified with MYC in >97% of tumors with

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chromosome 8q24 amplification. Interestingly, PVT1 knockdown reduces MYC protein levels

and inhibits tumor formation. Mechanistically, PVT1 stabilizes the MYC protein by reducing its

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phosphorylation at threonine 58 (Thr58) and protecting it from proteasome-dependent degradation [94]. LINC01638 protects MYC from SPOP-mediated polyubiquitination and

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degradation by directly interacting with MYC [95]. LncRNA glycolysis-associated lncRNA of colorectal cancer (GLCC1) is highly induced by glucose starvation and promotes cell survival

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by enhancing glycolysis. GLCC1 directly interacts with HSP90 and mediates the interaction between HSP90 and USP22, which further promotes the deubiquitination and stabilization of MYC [96].

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LncRNAs are also involved in modulating MYC transcriptional activity. Prostate cancer gene expression marker 1 (PCGEM1), a prostate-specific lncRNA induced by androgens,

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promotes MYC transactivation activity through direct interaction with MYC, which then promotes its binding to target promoters [97]. Similarly, lncRNA epigenetically induced lncRNA1 (EPIC1) physically interacts with MYC and increases its binding to the promoter of a subset of MYC target genes involved in cell cycle progression [98]. LncRNA HOTAIR acts as a key scaffold molecule in the HBXIP/HOTAIR/LSD1 complex, which serves as a critical regulator of MYC transcriptional activity. Depletion of HOTAIR blocks the interaction of LSD1 with HBXIP/MYC, leading to increased H3K methylation and decreased MYC occupancy to 11

the target gene promoters [99] (Fig. 2B).

3. Feedback loops between MYC and lncRNAs Many lncRNAs regulated by MYC form positive or negative feedback regulatory loops with MYC. LncRNA-MYC inhibitory factor (MIF) is induced by MYC at the transcriptional level and acts as a competitive endogenous RNA to antagonize miR-586, which abolishes the inhibitory effect of miR-586 on FBXW7, an E3 ligase essential for MYC degradation [100]. LncRNA LINC01123 is a direct transcriptional target of MYC and acts as a molecular decoy for

lncRNA

MYC–regulating

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miR-199a, resulting in MYC mRNA stabilization [101]. The physical interaction between adenocarcinoma-amplified

lncRNA

(OVAAL)

and

serine/threonine-protein kinase 3 (STK3) enhances binding between STK3 and RAF1, which

subsequently activates the RAF/MEK/ERK signaling pathway and upregulates MYC protein

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levels. In turn, MYC directly induces OVAAL transcription, creating a positive feedback loop between MYC and OVAAL [102]

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Although numerous lncRNAs are transcriptionally activated by MYC, there are two cases of lncRNAs transcriptionally repressed by MYC that also affect MYC expression. LncRNA

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GLS-AS is repressed by MYC during nutrient stress. Depletion of GLS-AS promotes proliferation and invasion of pancreatic cancer cells both in vitro and in xenograft tumors. As

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such, GLS-AS ablates MYC expression by impairing glutaminase-mediated stabilization of MYC [103]. FGF13-AS1, a lncRNA transcriptionally inhibited by MYC, physically interacts with IGF2BP1 and disrupts the interaction between IGF2BP1 and MYC mRNA, which in turn

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causes MYC mRNA degradation [104] (Fig. 2C).

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MYC and circRNAs

CircRNAs comprise a large class of single-stranded circular transcripts that are produced

by a noncanonical splicing event called back-splicing, during which a downstream splice-donor site is covalently linked to an upstream splice-acceptor site [105]. Most circRNAs lack the ability to encode proteins, although a few of them can be translated into proteins [106]. CircRNAs are primarily located in the cytoplasm and are quite stable, possibly due to their resistance to the cellular linear RNA decay machinery [107]. Emerging evidence demonstrates 12

that dysregulated circRNAs play potent roles in cancer initiation and progression through regulation of many cellular functions, including sustaining proliferative signaling, promotion of cell migration and invasion, resistance to apoptosis, and induction of angiogenesis [108]. CircRNAs affect cellular processes through diverse mechanisms, including functioning as miRNA sponges, interacting with RNA binding proteins, and acting as transcription and translation regulators [109]. Recently, circRNAs have been identified to function as oncogenic drivers or suppressors of tumorigenesis by regulating the MYC network (Table 3).

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1. MYC-regulated circRNAs Gene expression profile analysis of P493 cells with or without MYC inhibition revealed that MYC regulates expression of multiple circRNAs. Bioinformatics analysis of MYC-regulated circRNAs revealed that these circRNAs are intimately involved in different oncogenic signaling

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pathways, indicating that circRNAs may drive the MYC tumorigenic programs [110]. Of particular interest, MYC directly activates the expression of circSOX4, which promotes

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tumor-initiating cell proliferation and migration in part through activation of the Wnt signaling pathway by binding to β-catenin and subsequently promoting its nuclear translocation [111].

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MYC also activates expression of circMYC through direct activation of the splicing factor SRSF1, which promotes circMYC processing. In turn, activated circMYC, functioning as the

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miR1236 sponge, promotes LDHA mRNA stability and tumor cell glycolysis [112] (Fig. 3A).

2. CircRNAs regulating MYC

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CircRNAs regulate MYC through multiple ways. CircCTIC1 was shown to promote MYC transcription through direct binding to bromodomain PHD finger transcription factor (BPTF), a

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core component of nuclear remodeling factor (NURF) complex, recruiting NURF complex onto the MYC promoter and finally driving the transcriptional initiation of MYC [113]. Instead, circLMTK2 increases MYC mRNA abundance by sponging the repressive miR-150 function and promotes tumor cell migration in vitro and tumor metastasis in vivo [114] (Fig. 3B). In addition, some circRNAs could regulate MYC protein levels and nuclear translocation. In breast cancer, circ-Amotl1 increases the retention of nuclear MYC through direct interaction, promoting MYC stability, and increasing the affinity of MYC binding to a number of promoters 13

[115]. In contrast, circ-FBXW7 encodes a novel 21-kDa protein termed FBXW7-185, which reduces the half-life of MYC by antagonizing USP28-induced MYC stabilization, thus inhibiting tumor cell proliferation and cell cycle acceleration. Of note, the lower expression of circ-FBXW7 associates with higher MYC levels in glioblastoma [116] (Fig. 3B).

3. Feedback loop between MYC and circRNAs Some circRNAs induced by MYC can also modulate the expression levels of MYC. In oral squamous cell carcinoma (OSCC), MYC indirectly promotes circularization and biogenesis of

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circUHRF1 by activating the expression of epithelial splicing regulatory protein 1 (ESRP1), which functions as a splicing factor that targets the flanking introns of circUHRF1. In turn,

circUHRF1 positively regulates MYC by acting as a molecular sponge of miR-526b. The circUHRF1/miR-526b-5p/MYC/ESRP1 feedback loop promotes proliferation, migration and

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epithelial mesenchymal transformation in OCSCs [117]. Besides, MYC modulates expression

of circ-NOTCH1, a novel circular RNA derived from its host gene NOTCH1, by directly binding

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to the NOTCH1 promoter to regulate gastric cancer progression. Circ-NOTCH1 functions as a molecular sponge of miR-449 to upregulate MYC mRNA, thus promoting metastasis and

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MYC and rRNA/tRNA

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stemness in gastric cancer [118] (Fig. 3C).

Synthesis of rRNA, the first event in ribosome biogenesis, is a fundamental determinant of a cell’s capacity to grow and proliferate [119]. Approximately 50% of nascent RNA is occupied

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by rRNA to participate in the formation of ribosomes [120]. RNA Pol I transcribes 47S pre-rRNA, which undergoes processing into mature 28S, 18S and 5.8S rRNA, together with

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5S rRNA (transcribed by RNA Pol III) to constitute the nucleic acid backbone and catalytic activity of the ribosome [121, 122]. tRNAs are transcribed in the nucleus by RNA polymerase III and undergo many modifications before contributing to cytoplasmic protein synthesis [123].

1. MYC activates rRNA/tRNA biogenesis MYC promotes rRNA and tRNA synthesis via multiple mechanisms. MYC binds to specific consensus elements located in human rDNA, while the presence of MYC coincides with the 14

recruitment of Pol I-specific factor SL1 and increased histone acetylation [124]. MYC also promotes

transcription

of

rRNAs

by

recruiting

the

RNA polymerase

I

cofactor

transformation/transcription domain associated protein (TRRAP) and activating transcription of rDNA [125]. Moreover, MYC and MYCN directly induce expression of two key rRNA processing proteins, nucleolin (NCL) and nucleophosmin 1 (NPM), which promotes the processing of 47S pre-rRNA into mature 28S, 18S and 5.8S rRNA [126, 127]. Similarly, by recruiting RNA pol III cofactors, such as transcription initiation factor TFIIIB (TFIIIB), TRRAP, lysine acetyltransferase 2A (GCN5) and RNA polymerase III subunit D (BN51), MYC promotes

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robust tRNA and 5S rRNA gene transcription [128-130] (Fig. 4).

2. rRNA/tRNA enhances MYC translation

rRNA and tRNA, which promotes ribosome biogenesis or functions as linkers between

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amino acids and the genetic codons, cooperatively enhance global mRNA translational

efficiency. Both MYC and MYCN mRNAs are primarily translated by a cap-dependent

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mechanism that involves the interaction of the 5′ m 7G-cap structure of the mRNA with the cap-binding protein, eukaryotic initiation factor 4E (eIF4E), and the translational initiation

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machinery [131]. In addition, both mRNAs also contain an internal ribosome entry site (IRES), which can directly recruit ribosomes and increase cap-independent MYC/MYCN mRNA [132, 133]. As such, these mechanisms act in concert to increase MYC/MYCN

na

translation

mRNA translation in response to enhanced tRNA/rRNA synthesis, constituting a feedforward

ur

regulatory loop between oncogenic MYC and rRNAs/tRNAs (Fig. 4).

Conclusions and perspectives

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It is now becoming clear that there exists a tight relationship and complicated crosstalk between oncogenic MYC and noncoding RNAs, which play critical roles in tumorigenesis. Noncoding RNAs can function as regulators or effectors of MYC and even form feedback loops with MYC. Although tremendous efforts have been made, there remains a great deal to learn before we have a comprehensive, clear picture regarding this crosstalk with respect to malignant transformation (Fig. 5). A lack of efficient MYC-specific inhibitors has been a persistent clinical problem due to its 15

“undruggable” protein nature. Thus, targeting noncoding RNAs that regulate or are regulated by MYC may uncover new prospects. In principle, depletion or restoration of critical noncoding RNAs within the MYC network may represent an alternative to hijack oncogenic MYC in human cancers. In this regard, there remains an urgent need to systematically identify MYC-related noncoding RNAs and subsequently decipher the pertinent tumor-promoting mechanisms, which will help develop innovative strategies for cancer diagnosis and treatment.

The authors declare that there are no conflicts of interest. Acknowledgments

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Conflict of interest statement

We thank the members of the Qing laboratory for helpful suggestions. This study was

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supported by the National Science Fund for Distinguished Young Scholars (Grant 81725013 to

G.Q.), the National Natural Science Foundation of China (Grant 81830084 to G.Q), and the

re

Medical Science Advancement Program (Basic Medical Sciences) of Wuhan University (Grant

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ur

na

lP

TFJC2018005 to G.Q.).

16

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Liu, P.Y., et al., The long noncoding RNA lncNB1 promotes tumorigenesis by interacting with ribosomal protein RPL35. Nat Commun, 2019. 10(1): p. 5026.

143.

Zhang, J., et al., RBP EIF2S2 Promotes Tumorigenesis and Progression by Regulating MYC-Mediated Inhibition via FHIT-Related Enhancers. Mol Ther, 2020. 28(4): p. 1105-1118.

144.

Lu, Y., et al., MYC Targeted Long Noncoding RNA DANCR Promotes Cancer in Part by Reducing p21 Levels. Cancer Res, 2018. 78(1): p. 64-74.

145.

Sun, H., et al., Identification of a Novel SYK/c-MYC/MALAT1 Signaling Pathway and Its 23

Potential Therapeutic Value in Ewing Sarcoma. Clin Cancer Res, 2017. 23(15): p. 4376-4387. 146.

Chen, Z., et al., Integrative Analysis of NSCLC Identifies LINC01234 as an Oncogenic lncRNA that Interacts with HNRNPA2B1 and Regulates miR-106b Biogenesis. Mol Ther, 2020. 28(6): p. 1479-1493.

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Pang, Y., et al., Peptide SMIM30 promotes HCC development by inducing SRC/YES1 membrane anchoring and MAPK pathway activation. J Hepatol, 2020.

148.

Zeng, C., et al., The c-Myc-regulated lncRNA NEAT1 and paraspeckles modulate imatinib-induced apoptosis in CML cells. Mol Cancer, 2018. 17(1): p. 130.

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Cai, J., et al., circHECTD1 facilitates glutaminolysis to promote gastric cancer progression by targeting miR-1256 and activating β-catenin/c-Myc signaling. Cell Death Dis, 2019. 10(8): p. 576. Hsiao, K.Y., et al., Noncoding Effects of Circular RNA CCDC66 Promote Colon Cancer Growth

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and Metastasis. Cancer Res, 2017. 77(9): p. 2339-2350.

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151.

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Figure legends Figure 1. Crosstalk between miRNAs and oncogenic MYC (A) MYC represses downstream targets by transcriptional activation of miRNAs. (B) MYC activates downstream targets by transcriptional repression of miRNAs. (C) MiRNAs participate in tumorigenesis by direct or indirect regulation of MYC. (D) MYC and miRNAs form feedback

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regulatory loops.

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Figure 2. Intricate crosstalk between MYC and lncRNAs (A) MYC-regulated lncRNAs mediate MYC’s oncogenic roles. (i) MYC-activated lncRNAs

ur

function as competitive endogenous RNAs to sponge miRNAs; (ii) MYC-activated lncRNAs cooperate with RNA-binding proteins to stabilize target mRNAs; (iii) MYC-regulated lncRNAs

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enhance target protein activity through direct interaction. (B) LncRNAs regulate MYC through diverse processes, including regulation of transcription, mRNA stability and/or translation, protein stability, and transcriptional activity. TF, transcription factor; RBP, RNA-binding protein. (C) Positive or negative feedback regulatory loops between MYC and lncRNAs. See text for more details.

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b

Figure 3. CircRNA and MYC cooperation in tumorigenesis (A) MYC-activated circRNAs mediate MYC’s oncogenic role. (B) CircRNAs regulate expression of MYC. (C) CircRNAs form a positive feedback regulatory loop with MYC. See text for more details.

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Figure 4. Crosstalk between MYC and rRNA/tRNA

Through recruitment of TRRAP and SL1, MYC promotes the transcription of 47S pre-rRNA.

-p

MYC can also promote processing of 47S pre-rRNA into mature 5.8S, 18S and 28S rRNA by

inducing NCL and NPM. Moreover, MYC promotes transcriptional activity of RNA pol III by

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recruiting TRRAP, GCN5, BN51 and TFIIIB, activating transcription of 5S rRNA and tRNA. Increased tRNA/rRNA synthesis promotes mRNA translation, which in turn enhances MYC

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mRNA translation. See text for more details.

Figure 5. Crosstalk between oncogenic MYC and noncoding RNAs in tumor initiation and progression Crosstalk between oncogenic MYC and noncoding RNAs, including microRNA, lncRNA, circRNA, rRNA and tRNA, is involved in regulation of multiple cellular processes, including cell 27

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cycle, migration, metabolism, survival, and DNA repair. See text for more details.

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Targeting

Cellular processes

References

miR-24

MYC 3’-UTR

Cell cycle

[57]

miR-148

MYC 3’-UTR

Cell cycle

[64]

miR-145

MYC 3’-UTR

Cell cycle

[56]

miR-451

MYC 3’-UTR

Cell cycle

[68]

miR-320

MYC ORF

Cell cycle

[59]

miR-744

MYC ORF

Cell cycle

[58]

miR-363

MYC protein stability

Cell cycle

[64]

miR-449

MYC 3’-UTR

Apoptosis

[118]

miR-184

MYC ORF

Apoptosis

[60]

miR-375

MYCN 5’-UTR

Apoptosis

[62]

miR-34a

MYC/MYCN 3’-UTR

Apoptosis

[66, 67]

miR-33b

MYC 3’-UTR

Metastasis

miR-526b

MYC 3’-UTR

Metastasis

miR-17

MYC 3’-UTR

Metastasis

miR-145

MYC 3’-UTR

Metastasis, apoptosis

[135]

miR-1304

MYC 3’-UTR

Metastasis, apoptosis

[135]

miR-150

MYC 3’-UTR

Metastasis

[114]

miR-199a

MYC 3’-UTR

Metabolic reprogramming

[101]

miR-105

MYC activity

Metabolic reprogramming

[65]

miR-586

MYC protein stability

Metabolic reprogramming

[100]

miR-19

MYCN 3’-UTR

Cell proliferation

[61]

miR-29

MYCN 3’-UTR

Cell proliferation

[61]

miR-101

MYCN 3’-UTR

Cell proliferation

[61]

miR-449

MYCN 3’-UTR

Cell proliferation

[61]

miR-202

MYCN 3’-UTR

Cell proliferation

[61]

let-7a

MYCN 3’-UTR

Cell proliferation

[61, 69]

MYCN ORF

Cell proliferation

[136]

Targeting

Cellular processes

References

TOB2

Cell cycle

[42]

E2F1

Cell cycle

[73]

miR-17~92

PETN, BIM

Cell cycle, apoptosis

[37, 38]

miR-421

ATM

DNA repair

[43]

miR-9

CDH1

Metastasis, angiogenesis

[137]

MYC-repressed

Targeting

Cellular processes

References

miR-148

MYC 3’-UTR

Cell cycle

[64]

miR-451

MYC 3’-UTR

Cell cycle

[68]

miR-15a

CyclinD1, BCL2

Cell cycle, apoptosis

[48-50]

miR-16

CyclinD1, BCL2

Cell cycle, apoptosis

[48-50]

miR-184

AKT2

Apoptosis

[52]

miR-122

BCL2 , E2F1

Apoptosis

[55]

miR-34a

MYC/MYCN 3’-UTR

Apoptosis, DNA synthesis

[66, 67]

miR-571

Geminin

DNA repair

[138]

miR-204 MYC-activated miR-378

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miR-17

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Targeting MYC

na

Table 1：miRNAs of MYC-regulated and targeting MYC

29

[55]

[117, 134] [70]

LDHA

Metabolic reprogramming

[55]

miR-23a/b

GLS1

Metabolic reprogramming

[51]

miR-27

ATG10

Autophagy

[139]

miR-29

CDK6, IGF-1R

Cell proliferation

[140]

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miR-30a

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Table 2：lncRNAs of MYC regulated and targeting MYC Regulation mode

Cellular processes

References

MYMLR

MYC transcription

Cell cycle

[84]

LncCMPK2

MYC transcription

Cell cycle

[85]

CCR492

MYC translation

Cell cycle

[87]

EPIC1

MYC activity

Cell cycle

[98]

LncUSMycN

MYCN transcription

Cell cycle

[141]

LncNB1

MYCN protein stability

Apoptosis

[142]

LINC00346

MYC transcription

Metastasis

[86]

LINC00266-1

MYC mRNA stability

Metastasis

[93]

LINC01600

MYC protein stability

Metastasis

[143]

LINC01638

MYC protein stability

Metastasis

[95]

LINRIS

MYC mRNA stability

Metabolic reprogramming

[92]

FILNC1

MYC translation

Metabolic reprogramming

[90]

GLCC1

MYC protein stability

Metabolic reprogramming

[96]

PCGEM1

MYC activity

Metabolic reprogramming

[97]

CCAT1-L

MYC transcription

Cell proliferation

[83]

Linc-RoR

MYC mRNA stability

Cell proliferation

[89]

SNHG3

MYC translation

Cell proliferation

[88]

PVT1

MYC protein stability

Cell proliferation

[94]

HOTAIR

MYC activity

Cell proliferation

[99]

MYC-activated

Targeting

Cellular processes

References

CONCR

DDX11

Cell cycle

[81]

MYU

CDK6 mRNA

Cell cycle

[79]

LAST

CCND1 mRNA

Cell cycle

[78]

EMS

E2F1 mRNA

Cell cycle

[80]

DANCR

CDKN1A

Cell cycle

[144]

LINC00176

miR-9, miR-185

Cell cycle

[76]

MALAT1

EZH2

Apoptosis

[145]

hnRNPA2B1

Apoptosis

[146]

miR-34a

Metastasis

[77]

SRC, YES1

Metastasis

[147]

LINC00346

-p

re

lP

ur

LINC00998

na

LINC01234

ro of

Targeting MYC

miR-586

Metabolic reprogramming

[100]

LINC01123

miR-199a

Metabolic reprogramming

[101]

OVAAL

STK3

Drug resistance, apoptosis

[102]

MYC-repressed

Targeting

Cellular processes

References

NEAT1

SFPQ

Apoptosis

[148]

IDH1-AS1

IDH1

Metabolic reprogramming

[82]

GLS-AS

GLS1 mRNA

Metabolic reprogramming

[103]

FGF13-AS1

IGF2BP1

Metabolic reprogramming

[104]

Jo

lncRNA-MIF

31

Table 3: circRNAs of MYC-regulated and targeting MYC Direct targeting

Indirect targeting

Cellular processes

References

circ-FBXW7

USP28

MYC stability

Cell cycle

[116]

circPVT1

MYC

/

Cell cycle

[149]

circ-Amot1

MYC location

/

Apoptosis

[115]

circLMTK2

miR-150

MYC 3’-UTR

Metastasis

[114]

circ-PRMT5

miR-145, miR-1304

MYC 3’-UTR

Metastasis

[135]

circ-NOTCH1

miR-449

MYC 3’-UTR

Metastasis

[118]

circUHRF1

miR-526b

MYC 3’-UTR

Metastasis

[117]

circHECTD1

miR-1256

MYC

Metastasis

[150]

CCDC66

miR-93

MYC 3’-UTR

Metastasis

[151]

circ-CTIC1

NURF

MYC transcription

Self-renewal

[113]

circ_0091581

miR-526b

MYC 3’-UTR

Cell proliferation

[134]

MYC-activated

Direct targeting

Indirect targeting

Cellular processes

References

circ-SOX4

β-catenin

/

Metastasis

[111]

circ-NOTCH1

miR-449

MYC 3’-UTR

Metastasis

[118]

circUHRF1

miR-526b

MYC 3’-UTR

Metastasis

[117]

circ_0091581

miR-526b

MYC 3’-UTR

Cell proliferation

[134]

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Targeting MYC

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