Researchers have found a new way of distinguishing cancerous from non-cancerous tissues

Researchers from Osaka University find a new way to distinguish individuals with early pancreatic cancer from healthy controls, representing a promising new metric in cancer diagnosis

Levels of molecules associated with genetic function, such as microRNA, can be an important indicator of abnormal activity associated with cancer.

However, little is known about how different molecules are altered in cancerous cells.

Now, researchers from Japan have found a new way of distinguishing cancerous from non-cancerous tissues.

In a study published on August in Nature Communications, researchers from Osaka University revealed that the rate at which microRNA molecules undergo a process called methylation is able to discriminate cancer patients from healthy individuals.

MicroRNAs exhibit abnormal expression in cancer tissues and are stable in body fluids, making them a useful biomarker for cancer.

Although microRNAs are generally measured in terms of RNA expression levels, this technique lacks sensitivity and accuracy.

Particularly, although microRNAs are measured based on the assumption that they recognize and regulate targets regardless of whether or not they are methylated, their action may actually vary according to methylation status.

This is something the researchers at Osaka University aimed to address.

“We found that a small group of mature microRNAs are methylated, which could potentially alter their stability and target recognition,” says Masamitsu Konno, co-lead author of the study.

“Thus, we wanted to investigate whether methylation could be an important indicator of abnormal microRNA function.”

To evaluate the potential of microRNA methylation as a biomarker for early cancer diagnosis, the researchers determined whether levels of methylated RNAs increase or decrease in cancer cells.

To do this, they measured microRNA methylation levels in serum samples from patients with pancreatic cancer and healthy controls.

“While we found methylated microRNA in the samples from pancreatic cancer patients, it was either present in very low levels or absent in the control group,” explains senior author of the study Hideshi Ishii.

“Further, methylation levels in serum samples were able to distinguish early pancreatic cancer patients from healthy controls with extremely high sensitivity and specificity.”

The researchers also found that compared with established biomarkers, microRNA methylation was a more powerful indicator of early-stage pancreatic cancer.

“Our data indicate that levels of methylated microRNA may be more useful than those of microRNA as a biomarker for gastrointestinal cancer,” says Jun Koseki, co-lead author of the study.

“Clarifying the mechanisms by which methylation regulates microRNA function throughout the different stages of cancer may facilitate the development of targeted therapies, leading to improved patient outcomes.”

As early detection and treatment of cancer can have a substantial effect on patient outcome, new ways to screen for cancer could be vitally important.

Given the advantages with respect to existing biomarkers for cancer, it is possible that RNA methylation will be an important component of future systems for early cancer detection.

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Epigenetic mechanisms regulate multiple aspects of chromatin structure and function, including the regulation of transcriptionally repressive and permissive configurations for gene expression. These mechanisms include DNA methylation, histone modifications—including methylation, acetylation, phosphorylation, and ubiquitination—, and regulation by microRNAs (miRNAs).¹Its regulation has recently been established as an emerging mechanism in tumor development and tumor therapy. Epigenetic alterations are often reversible, which has led to the emergence of the promising field of epigenetic therapy. miRNAs are small noncoding RNA transcripts processed in the nucleus and cytoplasm. Mature miRNAs (miRs) are assembled into RNA-induced silencing complex (RISC), enabling posttranscriptional control of gene expression. They are involved in many molecular pathways and in pivotal biologic processes, including cell growth, development, differentiation, proliferation, and cell death, bringing new diagnostic and therapeutic opportunities in cancers.^2,3 DNA methylation is the main epigenetic feature of DNA with a key function in gene transcriptional regulation and preserving genome stability, and its alterations impair transcriptional levels and contribute to pathological conditions including cancer.^4,5

In this review, we discuss relevant dysregulation of miRs and DNA methylation in human cancers, and highlight recent advances, particularly in our understanding of the mutual regulation between miRs and DNA methylation. Finally, we will discuss the potential application of both miRs and DNA methylation as novel therapeutic approaches for treating cancer patients.Go to:

miRNA dysregulation

The processing machinery of miRs and related miR dysregulation in cancers

The primary miR (pri-miR) is transcribed from miR-coding DNA sequences by RNA polymerase II, and processed into a precursor miR (pre-miR) by RNase III endonuclease Drosha in the nucleus.⁶ RNase III nuclease Dicer1 carries out the maturation of the pre-miR into a final 22 nucleotide-long double-stranded RNA in the cytoplasm. The RISC complex, made up of the mature miR (called guide strand), Dicer, TAR RNA binding protein (TRBP), protein activator of PKR (PACT), and Argonaute protein, enables silencing of mRNAs.⁷ miR expression can be affected at any step of the miR processing. A significant number of miRs are located within cancer-associated genomic regions or in fragile sites.^8,9 A study showed that a general decrease in miRs, caused by knockdown of Dicer and Drosha, promoted tumorigenesis, indicating that the dysregulation of targeted proteins participating in miR processing was involved in human malignancies.^10,11 Colorectal cancer cells with defects on Dicer1 showed enrichment of tumor stemness features and an epithelial-mesenchymal transition (EMT), leading to the downregulation of miR-34a, miR-126, and miR-200 family and the acquisition of a greater capacity for tumor initiation and metastasis.¹² These findings indicate that miRNA machinery genes contribute to cancer development and progression.

miR dysregulation in tumor initiation and development

The first report on miR deregulation in cancer was the deletion or downregulation of the miR-15a-miR-16–1 cluster in B cells of patients with chronic lymphocytic leukemia (CLL).¹³ Ectopic expression in leukemic cells showed that miR-15a-16–1 cluster overexpression resulted in apoptosis of the leukemic cells. This cluster could target BCL-2, an anti-apoptotic gene overexpressed in patients with CLL. More importantly, miR-15a-16–1 inhibited BCL-2 translation via direct binding to its 3′UTR; there was a negative correlation between miR-15a-16–1 (downregulation) and BCL-2 (upregulation) in CLL patients. Klein et al. reported that miR-15a-16–1 cluster knockdown mice developed CLL-like disease and lymphomas, supporting a tumor suppressor role of these miRs in CLL.¹⁴ Later on, several reports demonstrated that miRs expression was dysregulated in cancer. miR-362–3p and miR-329 were downregulated in human breast cancer and performed a tumor-suppressor function by inhibiting cellular proliferation.¹⁵ miR-148a was downregulated in non-small cell lung cancer (NSCLC) and functioned as a tumor suppressor gene by modulating matrix metalloproteinase 15 (MMP15) and Rho-associated kinase 1 (ROCK1).¹⁶ By contrast, miR-224 was upregulated in NSCLC tissues and promoted cell proliferation by targeting TNF α-induced protein 1 (TNFAIP1) and SMAD directly, indicating a function as a potent oncogenic miR.¹⁷ Higher expression of miR-126 was associated with a negative outcome in acute myeloid leukemia (AML) patients, indicating its oncogene effect.¹⁸ miR-590 promoted cell proliferation and invasion by increasing G1/S transition and inhibiting MMP-9 in T-cell acute lymphoblastic leukemia.¹⁹ The above findings suggest a role for miRs in human malignancies either as oncogenes or tumor suppressors. Nevertheless, the same miRs might function drastically differently in different cancer types. miR-9 promotes the proliferation of human gastric cancer cells through the targeting of CDX2 (a nuclear homeobox transcription factor), while its overexpression suppresses the proliferation of ovarian carcinoma cells, partly by downregulating NF-κB1.^20,21 This may be due to the same miRNAs having different targets in different cancers.

miR dysregulation in cancer metastasis

miR-10a was the first miR discovered to be highly expressed in metastatic breast cancer, positively regulating cell migration and invasion. miR-10a promotes breast cancer cell invasion and metastasis by targeting HOXD10, thus leading to the expression of the pro-metastatic gene RHOC.³⁵ However, miR-10b inhibition could suppress metastasis in a mouse breast cancer model.³⁶ Another study showed that high levels of miR-107 in breast cancer cells correlated with metastasis and poor outcome. miR-107 expression increases breast cancer metastasis via inhibition of let-7.³⁷ Furthermore, miR-107 was correlated with gastric cancer metastasis and Dicer was its direct target.³⁸ Restoration of Dicer expression impaired miR-107-induced gastric cancer cell invasion and metastasis, indicating that Dicer has been proven to act as a suppressor of metastasis in gastric cancer cells. miR-221/222 is a well-known miRNA cluster that has been shown to influence cancer metastasis by positively regulating tumor invasion and EMT in breast and colorectal cancer.^39,40 Those studies suggest a therapeutic application of targeting metastasis-associated miRNAs.

On the contrary, metastasis-suppressor miRNAs, miR-126, miR-335, and miR-206, are negative regulators of tumor invasion and metastasis.^41,42 In addition, let-7 family has been demonstrated to play an important role in tumor metastasis. The metastasis suppressive effect of let-7 in breast cancer allegedly occurs through targeting of HMGA2 and the oncogene HRAS.⁴³ let-7g was also proved to inhibit breast cancer metastasis by the regulation of Grb2-associated binding protein 2 (GAB2) and fibronectin 1 (FN1), consequent activation of p44/42 MAPK, and specific matrix metalloproteinases.⁴⁴ In an in vivo melanoma model, let-7b inhibited Basigin, a protein involved in tumor progression and decreased metastases.⁴⁵ Inhibition of the matrix metalloproteinase MMP11 and PBX3 by let-7c has been shown to suppress colorectal cancer metastasis in vitroand in vivo.⁴⁶ Targeting of myosin IIA (MYH9) by let-7f showed inhibition of gastric cancer metastasis in vivo.⁴⁷

Due to the existence of different targets for the same miRNA, miRNAs may function as either oncogenes or tumor suppressor genes. Forced expression of miR-200 inhibited EMT, invasion, and metastasis of lung adenocarcinoma cells.⁴⁸ However, another study showed that overexpression of miR-200 in breast cancer promoted metastasis in a mouse model through direct targeting of Sec 23a, which acts on the secretion of metastasis-suppressive proteins.⁴⁹ This phenomenon was also observed again on miR-9. miR-9 overexpression in non-metastatic breast cancer tumor cells led to the formation of pulmonary micrometastases in mice.⁵⁰ While other studies have on the contrary revealed an anti-metastatic action for miR-9. Zhang et al. showed that miR-9, through targeting of MMP-14, regulates VEGF in neuroblastoma cells.⁵¹ In uveal melanoma, miR-9 suppresses migration and invasion of highly invasive cells by modulating the NF-κB pathway, including, notably, MMP-2, MMP-9, and angiogenesis-related protein VEGF-A.⁵² Prospero homeobox 1 (PROX1), a tumor suppressor, was proved to bind the miR-9–2 promoter and trigger its expression to suppress E-cadherin in colon cancer cells.⁵³ Those reports showed the crucial and complex role of miRNAs in cancer metastasis.Go to:

DNA methylation dysregulation

The DNA methylation machinery and its dysregulation in cancers

DNA methyltransferases (DNMTs), including DNMT1, DNMT3a, and DNMT3b, are essential during the process of DNA methylation. DNMT1 is called maintenance methylase, as it preferentially methylates hemimethylated substrates, whereas DNMT3a and DNMT3b are characterized as de novo methylases for their ability to methylate unmethylated DNA. The level of expression and activity of all 3 DNMTs has been reported to be significantly higher in hepatoma compared with the normal liver.⁵⁴

Significant increase of DNMT1 expression has been associated with tumorigenesis in colon cancer. Among other epigenetic changes, aberrant de novo hypermethylation of many tumor suppressor genes has been shown to play a crucial role in many malignancies including, lung and ovarian cancer^55,56.

DNA methylation dysregulation in tumor initiation and development

Global DNA hypomethylation is a well-known characteristic of human cancers

In human cancers, global patterns of DNA methylation are usually altered. The forced reduction in global DNA methylation could result in tumor induction or inhibition, depending on the tumor stage and target organ.⁵⁷ Decreasing global DNA methylation in some cell types promotes tumorigenesis by promoting chromosomal instability. However, global DNA hypomethylation suppresses tumor development in tongue, esophagus, stomach, small intestine, colon, and pancreas using hypomethylated-DNA mice in conjunction with various cancer models.^58-60 Hatano et al. also reported that DNA demethylation exerts a tumor suppressor effect in the colon by inducing tumor cell differentiation.⁶¹ Thus, global DNA methylation regulates gene expression in a highly context-dependent manner.⁶²

Hypermethylation of tumor suppressor gene/gene family promoters is generally observed in cancers

Epigenetic silencing of tumor suppressor genes (TSGs) is often associated with the tumorigenic process.⁶³ There are several known tumor suppressor genes that are silenced via promoter methylation in different tumors, such as Rb, p16INK4a BRCA-1, VHL, E-cadherin, and MLH1.⁶⁴In Hodgkin lymphoma patients, the promoter of IGSF4 was hypermethylated resulting in protection from cell apoptosis.⁶⁵ Genome-wide analysis of DNA methylation has shown that the epigenetic process can also occur in chromosome regions such as contiguous CpG islands and gene families.^66–68 Hypermethylation of HOXA gene clusters was reported in breast and lung cancer.^69,70 Lastly, the protocadherin (PCDH) gene cluster was found to be hypermethylated in Wilms tumor and the region across chromosome 2q14.2 in colorectal cancer.^71,72

Hypomethylation of oncogene promoters is frequently seen in human cancers

Epigenetic modulation of oncogenes is involved in tumorigenesis. LIM-only protein 3 (LMO3) gene, whose overexpression was correlated with a poor prognosis in glioma patients, is hypomethylated and overexpressed in glioma cells and tissues.⁷³ In astrocytoma patients, PR domain containing 16 (PRDM16) gene is hypomethylated and the hypomethylation status of the PRDM16 promoter can predict poor prognoses for astrocytoma patients.⁷⁴ The Villalba group recently reported that high TMPRSS4 expression—a type II transmembrane serine protease (TTSP) implicated in tumor development and progression—is an independent prognostic factor in squamous cell carcinomas (SCC), and aberrant TMPRSS4 hypomethylation, which correlates with high TMPRSS4 expression, is an independent prognostic factor in SCC.⁷⁵ These findings indicate the crucial role of oncogene DNA methylation in the human cancers.

DNA methylation dysregulation in cancer metastasis

Both DNA hypomethylation and hypermethylation of promoter regions are frequently detected in human cancers. What is their role in human cancer metastasis, respectively?

Hypomethylation and the correlated activation of gene expression are crucial for metastasis progression in human cancers. For example, S100 calcium binding protein A4 (S100A4) induces EMT and promotes metastasis and was reported to be hypomethylated and upregulated in LMP2A-positive NPC tissues.⁷⁶ Another study reported that the synergistic hypomethylation of Jagged1 and Notch1 genes was involved in the metastasis of breast cancer.⁷⁷ In addition, Iroquois homeobox 1 (IRX1) hypomethylation is a potential molecular marker for lung metastasis.⁷⁸ On the other hand, hypermethylation and correlated inactivation of genes also play important roles in human cancer metastasis. Semaphorin-3F (SEMA3F), a candidate tumor suppressor in human cancers, was hypermethylated in colorectal cancer, consequently leading to cancer migration and invasion.⁷⁹ Breast cancer metastasis suppressor 1 (BRMS1), a metastasis suppressor gene in several solid tumors, showed hypermethylation in its promoter, leading to its downregulation and breast cancer cell invasion.⁸⁰ Hyaluronan (HA), as a major component of extracellular matrix known to play a critical role in tumor metastasis, was associated with DNA hypermethylation.⁸¹ In addition, one study showed that the IL-8/AKT1 pathway regulated E-cadherin expression by stabilizing DNMT1 protein, thus enhancing hypermethylation of the E-cadherin promoter.⁸² These data suggest that both hypomethylation and hypermethylation of related genes are critical for the modulation of the process of cancer metastasis.

Overall, these findings highlight the mechanisms by which aberrant DNA methylation contributes to tumorigenesis (Fig. 1) and tumor metastasis. Alterations of both genome-wide and gene-specific DNA methylation are critical events in tumor initiation and progression. Hypomethylation of genome-wide CpGs or oncogenes promoters could promote or inhibit tumorigenesis, and genes involved in tumorigenesis and tumor metastasis could be hypermethylated or hypomethylated in their promoters.

Mutual regulation of miRNAs and DNA methylation

Both miRNAs and DNA methylation play important roles in cancer initiation, progression, and metastasis. A mutual regulation loop between miRNAs and DNA methylation has been observed in human cancers [Table 2].

Table 1.

Samples of different targets by the same miR in human cancers.

miRNA	Targets	Human Cancer	Mechanistically	Oncogene/Suppressor	Ref.
miR-9	CDX2	GCa	promotion of cell proliferation	oncogene	20
	NF-κB1	OCb	suppression of cell proliferation	suppressor	21
miR-155	P85α	HCCc	promotion of EMTd	oncogene	22
	ErbB2	BCe	inhibition of malignant transformation	suppressor	23
miR-24	metallothionein 1M	HCCc	enhance of cell growth	oncogene	23
	Jab1/Csn5	NPCf	suppression of cell growth	suppressor	24
miR-146a	NUMB	melanoma	promotion of melanoma initiation and progression	oncogene	25
	CCND1&CCND2	LCg	inhibition of cell proliferation and cell cycle	suppressor	26
	RhoA	BCe	inhibition of cell migration and invasion	suppressor	26
miR-205	CDK2AP1	LSCCh	promotion of cell proliferation and invasion	oncogene	27
	PTEN	ECi	inhibition of cell apoptosis	oncogene	28
	ZEB1	GCa	suppression of cell invasion and EMTd	suppressor	29
	TGF-α	OSj	inhibition of cell proliferation, invasion and migration	suppressor	30
miR-9	E-cadherin & SOCS5	PCk	promotion of cell invasion and migration	oncogene	31
	LASS2	BLCl	cell proliferation promotion and cell apoptosis inhibition	suppressor	32
	TM4SF1	CRCm	inhibition of cell invasion and migration	suppressor	33
	TAZ	HCCc	inhibition of cell proliferation	suppressor	34

Open in a separate window^aGC: gastric cancer;^bOC: ovarian carcinoma;^cHCC: hepatocellular carcinoma;^dEMT: epithelial-mesenchymal transition;^eBC: breast cancer;^fNPC: nasopharyngeal carcinoma;^gLC: lung cancer;^hLSCC: laryngeal squamous cell carcinoma;ⁱ_EC: endometrial cancer;^jOS: osteosarcoma;^kPC: prostate cancer;^lBLC: bladder cancer;^mCRC: colorectal carcinoma.

Table 2.

Samples of mutual regulation of miRNAs and DNA methylation.

miRNA	DNA methylation	Mechanistically	Human Cancer	Ref.
miR-29	DNMTa (3a and 3b)	miR-29 directly targeted DNMT	LCb	83
miR-148a	DNMTa (1)	miR-148a directly targeted DNMT	LSCCc	84
miR-124 and −506	DNMTa (3b and 1) targeted DNMT1 indirectly	targeted DNMT3b directly and	CRCd	85
miR-221	DNMTa (3b)	miR-221 directly targeted DNMT	BCe	86
miR-212	MeCP2	miR-29 directly targeted MeCP2	GCf	87
miR-373	MBD2	miR-373 directly targeted MBD2	HCg	88
miR-373	hypermethylation	promoter-associated CpG islands of miR-373 was hypermethylated (MBD2 was required)		89
miR-296	hypermethylation	miR-296–5p was repressed by hypermethylation	GBh	90
miR-34a	hypermethylation	miR-34a promoter was hypermethylated	LSCCc	91
miR-106a	hypomethylation	miR-106a promoter was hypomethylated	GCf	92
miR-196b	hypomethylation	promoter CpG islands of miR-196b was hypomethylated	OSCCi	93
miR-145	hypomethylation	CpG island promoter of miR-145 was hypomethylated	PCj	94
miR-145	DNMT (3b)	3′UTR of DNMT3b was directly targeted by miR-145	PCj	94

Open in a separate window^aDNMT: DNA methylationtransferase;^bLC: lung cancer;^cLSCC: laryngeal squamous cell carcinoma;^dCRC: colorectal caner;^eBC: breast cancer;^fGC: gastric cancer;^gHC: hilar cholangiocarcinoma;^hGB: glioblastoma;ⁱOSCC: oral squamous cell carcinoma;^jPC: prostate cancer.

How do miRNAs regulate DNA methylation?

A single miRNA can regulate multiple target genes, and its alteration may be used as a critical biomarker to improve diagnosis and prognosis in cancer patients.⁹⁵ There are 2 main mechanisms by which miRNAs regulate DNA methylation:

miRNAs regulate DNA methylation by modulating DNMTs

DNA methylation is performed by DNMTs, including DNMT1 (for maintenance methylation) and DNMT3a and DNMT3b (for de novo DNA methylation), as mentioned before. The miR-29 family (29a, 29b, and 29c), the most studied epigenetic miRNA family, regulates DNA methylation by modulating DNMTs. DNMT3a and DNMT3b are frequently upregulated in lung cancer and are associated with poor prognosis, whereas the expression of miR-29 family members is inversely correlated with DNMT3a and DNMT3b in lung cancer tissues, and the miR-29 family directly targets both DNMT3a and DNMT3b.⁸³ In laryngeal squamous cell carcinoma, DNMT1 is a target of miR-148a-3p, which inhibits cellular DNA methylation.⁸⁴ In colorectal cancer, miR-124 and miR-506 are downregulated, and both target DNMT3b directly and DNMT1 indirectly.⁸⁵ In addition, DNMT3a has been identified as a direct target of miR-101 in astrocytoma cells, and miR-101 suppresses PRDM16 expression by targeting DNMT3a at the PRDM16 core promoter.⁷⁴ In lung cancer, ectopic miR-101 expression abolishes DNMT3a 3′-UTR luciferase activity and diminishes endogenous DNMT3a expression, leading to a reduction of global DNA methylation. The loss or suppression of miR-101 function accelerates lung tumorigenesis through DNMT3a-dependent DNA methylation.⁹⁶ These findings provide evidence that miRNAs modulate DNA methylation in cancer by targeting DNMTs.

miRNAs regulate DNA methylation by modulating methylation-related critical proteins

Besides DNMTs, methylation-related proteins, including methyl CpG binding protein 2 (MeCP2) and methyl-CpG binding domain proteins 2 and 4 (MBD2 and MBD4), are also critical to DNA methylation. Therefore, the regulation of those proteins by miRNAs is another main way by which miRNAs influence DNA methylation. For example, miR-212 overexpression represses the expression of the MeCP2 protein but not MeCP2 mRNA in gastric cancer, which suggests that MeCP2 is a direct target of miR-212 in the disease.⁸⁷ In hilar cholangiocarcinoma, precursor miR-373 suppresses the luciferase activity of the 3′UTR of MBD2, whereas miR-373 inhibition increases expression of MBD2, which suggests that miR-373 is a negative regulator of MBD2.⁸⁸Furthermore, another study reported that miR-373 directly targets the 3′UTR of MBD2 and that the promoter-associated CpG islands of miR-373 are hypermethylated and result in miR-373 inhibition. The methylation-mediated inhibition of miR-373 requires the enrichment of MBD2 at the promoter-associated CpG islands; thus, there is a feedback loop between miR-373 methylation and MBD2 activity.⁸⁹

How does DNA methylation regulate miRNAs in human cancers?

Aberrant DNA methylation appears to be a major mechanism by which the normal patterns of miRNA expression are disrupted in human cancers. Many tumor-suppressor miRNAs appear to be downregulated by DNA hypermethylation, and various oncogenic miRNAs (onco-miRs) are known to be upregulated via DNA hypomethylation.

Hypermethylation of gene promoters is a common mechanism of miRNA silencing

The epigenetic silencing of tumor-suppressor miRNAs by promoter-associated CpG island hypermethylation is a hallmark of many human cancers.⁹⁷ Because miRNAs can be found in intergenic regions, intronic regions of protein coding genes, or intronic and exonic regions of noncoding RNAs, the core promoter regions of miRNAs differ accordingly. Despite much effort, miRNA promoters have yet to be identified. One study used 9 histone markers, including H3K4me2, H3K4me3, H3K9Ac, H3K9me2, H3K18Ac, H3K27me1, H3K27me3, H3K36me2, and H3K36me3, to predict the promoters of miRNAs.⁹⁸ Another study showed that upstream promoters at -2 kb to -200 bp upstream the transcription start site are more conserved for independently transcribed miRNAs, whereas core promoters −200 bp to 200 bp from the transcription start site are significantly conserved for miRNAs transcribed with host genes.⁹⁹

The first evidence that epigenetic mechanisms were involved in silencing miRNAs in cancer came from Saito et al.¹⁰⁰ They showed that miR-127 acted as a tumor suppressor, since it directly targeted the proto-oncogene BCL6, and that the upregulation of miR-127 was associated with its DNA methylation. miR-124a, a tumor-suppressor miRNA, was reported to undergo CpG island hypermethylation associated with its transcriptional inactivation in human tumors from different cell types.¹⁰¹ The promoters of the miR-34 family (miR-34a, −34b, and -34c) were targets of CpG island hypermethylation in multiple malignancies, including esophageal and gastric cancers.^102,103 Also, the CpG islands of the miR-9 family (miR-9–1, -9–2, and -9–3) were usually hypermethylated in ALL and colorectal cancers.^104,105 In glioblastoma, the expression of miR-296–5p was repressed by hypermethylation and contributed to the generation and/or maintenance of cancer stem cells.⁹⁰ Here, miRNAs acted as tumor-suppressor miRNAs that were frequently hypermethylated in their promoters in cancers, leading to tumorigenesis and metastasis. We previously discussed the relationship between miR-200 and EMT, explaining that miR-200 inhibited EMT in lung adenocarcinoma cells and promoted metastasis in breast cancer.^48,49 One study reported that the dynamic methylation regulation of the miR-200 family mediated the EMT process. Both miR-200ba429 and miR-200c141 transcripts underwent dynamic epigenetic regulation related to EMT or MET phenotypes in tumor progression.¹⁰⁶

Essentially, besides miRNAs, other noncoding RNAs (ncRNAs), such as long noncoding RNAs (lncRNAs), undergo promoter hypermethylation silencing in human cancers. Lujambio et al. were the first to report that another class of ncRNAs, transcribed-ultraconserved regions, undergoes DNA methylation-associated inactivation in human cancer cells.¹⁰⁷ TP53TG1, a lncRNA with growth-suppressor features, has been reported to undergo cancer-specific promoter hypermethylation-associated silencing, leading to the resistance of tumor cells to cellular death and thereby linking the lncRNA to classical tumoral pathways.¹⁰⁸ Some lncRNAs affect the production of mature miRNAs; for example, a lncRNA named Uc.283+A could control the processing of precursor miR-195 in cancer.¹⁰⁹

Hypomethylation of onco-miRNAs leads to their activation and tumorigenesis

Several studies have shown that the hypomethylation status of onco-miR promoters is correlated with onco-miRNA activation and promotes tumor progression. Promoter hypomethylation of miR-106a is related to its high expression. Circulating miR-106a upregulated by promoter hypomethylation might be a diagnostic and prognostic indicator for gastric cancer.¹¹⁰ Moreover, the methylation status of the miR-106a promoter region is inversely correlated with the expression of miR-106a in hepatocellular carcinoma (HCC) tissues. The upregulated expression of miR-106a by its promoter hypomethylation contributes to the progression of HCC.¹¹¹ Furthermore, onco-miRNAs, including miR-106b, miR-25, miR-93, miR-23a, and miR-27a, are hypomethylated and upregulated in HCC.¹¹² In CLL, the onco-miRNAs miR-21, miR-34a, and miR-155 are upregulated by their promoter hypomethylation, leading to disease progression.¹¹³ In prostate cancer, a crucial crosstalk between miR-145 and DNMT3b contributes to the disease response to irradiation via a double-negative feedback loop. miR-145 downregulates DNMT3b expression by targeting the 3′UTR of DNMT3b mRNA directly, and knockdown of DNMT3b increases the expression of miR-145 through CpG island promoter hypomethylation.⁹⁴

In summary, there is a feedback loop between miRNAs and DNA methylation in human cancers (Fig. 2). On the one hand, miRNAs regulate DNA methylation by targeting DNMTs or methylation-related proteins; on the other hand, onco-miRs or tumor-suppressor miRNAs can be hypomethylated or hypermethylated, respectively, in cancer. The crosstalk between miRNA and DNA methylation may lead to the discovery of novel therapeutic targets.

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More information: Masamitsu Konno et al. Distinct methylation levels of mature microRNAs in gastrointestinal cancers, Nature Communications(2019). DOI: 10.1038/s41467-019-11826-1

Journal information: Nature Communications
Provided by Osaka University