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Abstract

  1. Top of page
  2. Abstract
  3. Distribution of Micro RNA Genes and Their Related CpG Islands in the Human Genome
  4. Intergenic Tumor-Suppressive Micro RNA Genes Silenced by CpG Island Hypermethylation in Cancer Cells
  5. Intragenic Tumor-Suppressive Micro RNA Genes and Their Host Genes Silenced by CpG Island Hypermethylation in Cancer Cells
  6. Future Perspectives on Tumor-Suppressive Micro RNA Silenced by Tumor-Specific DNA Hypermethylation in Cancer Research
  7. Concluding Remarks
  8. Acknowledgments
  9. Disclosure Statement
  10. References

MicroRNA (miRNA) genes, located in intergenic or intragenic non-coding regions of the genome, are transcribed and processed to small non-protein-coding RNA of approximately 22 nucleotides negatively regulating gene expression. Some miRNA have already been reported for their genetic alterations, aberrant expression and oncogenic or tumor-suppressive functions. After 2008, there has been a striking increase in the number of publications reporting tumor-suppressive miRNA (TS-miRNA) silenced epigenetically in various types of cancers, suggesting important clinical applications for miRNA-based molecular diagnosis and therapy for cancers. Here, we introduce a correlation of the gene silencing of TS-miRNA through CpG island hypermethylation with the genomic distances between intergenic and intragenic miRNA genes or protein-coding host genes and CpG islands located around these genes. Furthermore, we also discuss the potential of miRNA replacement therapy for cancers using double-stranded RNA mimicking TS-miRNA. (Cancer Sci 2012; 103: 837–845)

MicroRNA (miRNA) are small, non-coding and single-strand RNA of 19–22 nucleotides with a primary role in post-transcriptional silencing generally through imperfect pairing with the 3′-UTR of protein-coding transcripts.[1] Approximately 98% of the human genome is known to be non-coding DNA harboring a large number of intergenic and intronic miRNA genes.[1-3] Intergenic miRNA genes are located in the non-coding regions between genes, while intragenic miRNA genes, or intronic miRNA genes, are harbored within introns of their protein-coding host genes. In normal cells, some of these endogenous RNA play crucial roles in many processes, such as proliferation, development, differentiation and apoptosis.[4-7] In cancer cells, several studies demonstrate the deregulation of miRNA expression and the genetic aberration of a few miRNA genes within amplified or deleted regions,[8-10] showing that miRNA can contribute to the multistep processes of carcinogenesis as oncogenes or tumor suppressor genes (TSG).[6, 11-13] Recently, several miRNA genes have also been demonstrated to have copy number variations (CNV), although whether CNV affect miRNA genes in human cancers remains unclear.[14]

DNA hypermethylation of CpG sites within CpG islands is known as an epigenetic aberration leading to the inactivation of tumor-suppressive miRNA (TS-miRNA) in cancer cells,[15] in the same manner as that of many classical TSG.[16] In fact, the expression of several miRNA is generally downregulated in malignant tissues compared with corresponding non-malignant tissues. Recent studies, including our own, clearly demonstrate DNA methylation-mediated downregulation of TS-miRNA gene expression in various types of cancers.[17-24] Although the genomic distances between the 5′-end of intergenic miRNA genes or host genes harboring intronic miRNA and their proximal CpG islands vary, these distances might provide more important information for the understanding of silencing of TS-miRNA genes through DNA hypermethylation. However, few studies have focused on these genomic distances.

The many achievements in the field of TS-miRNA discovery and in vitro/in vivo delivery technology may offer the possibility of new therapeutic approaches for cancer. Because one miRNA can target many messenger RNA (mRNA) of protein-coding genes, the in vivo applications of miRNA for cancer therapies are considered better than those of short interfering RNA (siRNA). In addition, among miRNA-based in vivo delivery approaches, including the use of DNA plasmids or viral vectors, miRNA replacement therapy using double-stranded RNA (dsRNA) mimicking TS-miRNA is one of the most promising, offering hope for new cancer therapies.[25]

We recently evaluated the genomic distribution of 1523 miRNA genes and their CpG islands, and then determined the genomic distance between these genes and CpG islands located within 10 kb upstream of miRNA gene using the miRBase database (Release 18: November 2011) and the UCSC Genome Browser on Human February 2009 Assembly (hg19). In this review, based on information from these databases, we provide insights into the relationship of the gene silencing of TS-miRNA through aberrant DNA hypermethylation with the genomic distances between TS-miRNA genes or protein-coding host genes harboring intragenic miRNA and related CpG islands. We also discuss the potential of these TS-miRNA as therapeutic agents for cancer.

Distribution of Micro RNA Genes and Their Related CpG Islands in the Human Genome

  1. Top of page
  2. Abstract
  3. Distribution of Micro RNA Genes and Their Related CpG Islands in the Human Genome
  4. Intergenic Tumor-Suppressive Micro RNA Genes Silenced by CpG Island Hypermethylation in Cancer Cells
  5. Intragenic Tumor-Suppressive Micro RNA Genes and Their Host Genes Silenced by CpG Island Hypermethylation in Cancer Cells
  6. Future Perspectives on Tumor-Suppressive Micro RNA Silenced by Tumor-Specific DNA Hypermethylation in Cancer Research
  7. Concluding Remarks
  8. Acknowledgments
  9. Disclosure Statement
  10. References

We examined the genomic distribution of the 1523 genes registered as human miRNA genes in the miRBase database (Release 18: November 2011), (Fig. 1A). Interestingly, 20.0% (304/1523) of these miRNA genes are located on chromosomes 14, 19 and X. Notably, some of these genes are concentrated at 14q32.31, 19q13.42 and Xq27.3, and lie within limited regions of 44, 122 and 33 kb, respectively. In 19q13.42 and Xq27.3, the chromosome 19 miRNA cluster (C19MC) and chromosome X miRNA cluster were revealed as primate-specific.[26] Some C19MC miRNA were described as expressed at a very low level in most human tissues.[27] A correlation between their expression patterns and the methylation status of a distal CpG-rich region located approximately 17.6 kb upstream of the C19MC region has also been demonstrated in gastric cancer cells.[28]

image

Figure 1. Genomic feature of micro RNA (miRNA) genes. (A) Genomic distribution of 1523 human miRNA genes registered in the miRBase database (Release 18: November 2011). (B) Ratio of intergenic and intragenic miRNA genes in 1523 human miRNA genes. (C) Genomic distribution of 878 intergenic (left) and 645 intragenic (right) miRNA genes. These data were obtained from the miRBase database (http://www.mirbase.org/index.shtml) and UCSC Genome Browser on Human February 2009 Assembly (hg19) (http://genome.ucsc.edu/cgi-bin/hgGateway). In our database analyses, miRNA genes, which were located within introns of protein-coding host genes and considered to be transcribed in the same direction as those of their host genes, were analyzed as intragenic miRNA genes.

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Tumor-specific downregulation of subsets of miRNA has been generally observed in various types of human cancer,[11] suggesting that some of these miRNA act as TSG. Because the downregulation of some TS-miRNA has been shown to be tightly linked to CpG island hypermethylation, the aberrant DNA hypermethylation of CpG islands located around TS-miRNA genes, similar to various protein-encoding TSG, has been recognized as one of the main epigenetic alterations in cancer cells.[17-24] The most recent study indicated 11.6% (122/1048) of miRNA to be epigenetically regulated in 23 cancer types,[29] and 19.5% (26/133) of the 133 miRNA genes transcribing these 122 miRNA to have a CpG island within 5 kb upstream. In addition, 14.2% (19/133) of these miRNA genes were also demonstrated to reside within CpG islands. Few studies have examined the relationship of the transcriptional regulation of intergenic, intragenic miRNA genes or host genes harboring intronic miRNA genes through proximal CpG island hypermethylation with genomic distances between these individual genes and their related CpG islands. We discuss these relationships in the following sections.

Intergenic Tumor-Suppressive Micro RNA Genes Silenced by CpG Island Hypermethylation in Cancer Cells

  1. Top of page
  2. Abstract
  3. Distribution of Micro RNA Genes and Their Related CpG Islands in the Human Genome
  4. Intergenic Tumor-Suppressive Micro RNA Genes Silenced by CpG Island Hypermethylation in Cancer Cells
  5. Intragenic Tumor-Suppressive Micro RNA Genes and Their Host Genes Silenced by CpG Island Hypermethylation in Cancer Cells
  6. Future Perspectives on Tumor-Suppressive Micro RNA Silenced by Tumor-Specific DNA Hypermethylation in Cancer Research
  7. Concluding Remarks
  8. Acknowledgments
  9. Disclosure Statement
  10. References

A recent study showed that RNA polymerase II (Pol II) promoters driving miRNA expression contained most of the features of the protein-coding gene promoters and that intergenic and some intragenic miRNA were transcribed by RNA Pol II at a distance that could be as large as 40 kb from the miRNA genes.[30] In addition, a computational approach demonstrated that 81.9% (59/72) of predicted promoters of intergenic miRNA genes (37 miRNA clusters among 46 miRNA clusters) contained or overlapped with at least one CpG island.[31]

In our database analyses, intergenic and intragenic miRNA genes made up 57.6% (878/1523) and 42.4% (645/1523), respectively, of human miRNA genes (Fig. 1B,C). Among intergenic miRNA genes examined in our database analyses, 15.3% (134/878) were located within 500 bp downstream of CpG islands (Fig. 2A). These 134 intergenic miRNA genes included 40 genes whose name contained a number lower than 700. Several research groups, including ours, have been investigating some of intergenic miRNA since the early days of miRNA study, and 47.5% (19/40) of these genes are well-known TS-miRNA (Table 1). Moreover, gene silencing of these known TS-miRNA has already been reported to be related with DNA hypermethylation in several types of cancers.

image

Figure 2. Genomic distances between intergenic and intragenic micro RNA (miRNA) genes or protein-coding host genes and their related CpG islands. Each map indicates the relationship between miRNA genes or host genes and CpG islands located around these genes on the genome. All intragenic miRNA genes examined in our database analyses were considered to be transcribed in the same direction as those of their protein-coding host genes. TSS, transcription start site. Each pie graph shows results of our database analyses for genomic distances between CpG islands and the 5′-end of intergenic (A) or intragenic miRNA genes (B and D) or protein-coding host genes (C). These data were obtained from the miRBase database (Release 18: November 2011) (http://www.mirbase.org/index.shtml) and UCSC Genome Browser on Human February 2009 Assembly (hg19) (http://genome.ucsc.edu/cgi-bin/hgGateway).

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Table 1. Intergenic and intragenic tumor-suppressive micro RNA (TS-miRNA) genes (miRNA number in a name < 700): These intergenic TS-miRNA genes and protein-coding host genes harboring intragenic TS-miRNA genes are located within 500 bp downstream of CpG islands
miRNA genes (ID < 700)LociDistances from 5′ end of miRNA genes to 3′ end of their CpG islands (bp)TargetsReferences indicating tumor suppressive functions of miRNAHost geneDistances from TSS of host genes to 3′ end of their CpG islands (bp)Distances from 5′ end of miRNA genes to 3′ end of CpG islands around TSS of host genes (bp)
  1. TSS, transcription start site. These data were obtained from the miRBase database (Release 18: November 2011) (http://www.mirbase.org/index.shtml), UCSC Genome Browser on Human February 2009 Assembly (hg19) (http://genome.ucsc.edu/cgi-bin/hgGateway) and PubMed (http://www.ncbi.nlm.nih.gov/pubmed). All intragenic miRNA genes, indicated in this table, are transcribed in the same direction as those of their host genes.

Intergenic miRNA genes
hsa-mir-9-315q26.10NF-kappaB1, Androgen receptor (AR)Guo, 2009; Ostling, 2011None
hsa-mir-34b11q23.10CDK6, c-MYC, E2F3, MET, CCNE2, CDK4, CAV1, MYB, SFRS2, CREBCorney, 2007t; Kozaki et al.[19]; Toyota et al.[20]None
hsa-mir-34c11q23.1272CDK6, c-MYC, E2F3, MET, CCNE2, CDK4, CAV1, MYB, SFRS2, Androgen receptor (AR)Corney, 2007; Toyota et al.[20]; Ostling, 2011None
hsa-mir-92b1q220PRMT5Pal, 2007None
hsa-mir-124-18p23.10CDK6, C/EBPα, SMYD3, VIM, IQGAP1, IGFBP7Silber, 2008; Furuta, 2010None
hsa-mir-124-320q13.330CDK6, C/EBPα, SMYD3, VIM, IQGAP1, IGFBP7Silber, 2008; Furuta, 2010None
hsa-mir-12714q32.20BCL6Saito et al.[17]None
hsa-mir-129-211p11.20SOX4Dyrskjot, 2009; Huang, 2009None
hsa-mir-1371p21.30CDK6, MITF, Cdc42Kozaki et al.[19]; Bemis, 2008; Liu et al.[65]None
hsa-mir-148a7p15.2406DNMT-1, TGIF2Braconi et al.[60]None
hsa-mir-193a17q11.20E2F6, c-kitKozaki et al.[19]; Gao, 2011None
hsa-mir-196b7p15.20c-mycBhatia, 2010None
hsa-mir-20314q32.330ABCE1, ABLKozaki et al.[19]; Bueno, 2008; Furuta, 2010None
hsa-mir-21011p15.50FGFRL1Camps, 2008; Tsuchiya, 2011None
hsa-mir-21217p13.30MeCP2, PEDIncoronato, 2010; Wada, 2010None
hsa-mir-3752q350PDK1, 14-3-3zeta, RASD1, YAPTsukamoto, 2010; Liu, 2010; de Souza Rocha Simonini, 2010None
hsa-mir-40914q32.310RDXZheng, 2011None
hsa-mir-424Xq26.30PLAG1Pallasch, 2009None
hsa-mir-66320p11.10JunB, JunD, TGFβ1, p21/Waf1/Cip1Pan, 2010; Tili, 2010; Tili, 2010None
Intragenic miRNA genes
hsa-mir-1-120q13.333HDAC4, FoxP1, METDatta et al.[44]C20orf16603726
hsa-mir-15b3q25.333498BCL2Cimmino, 2005; Xia, 2008SMC403498
hsa-mir-16-23q25.333655BCL2Cimmino, 2005; Xia, 2008SMC403655
hsa-mir-23b9q22.32554GLS, uPA, c-MetGao, 2009; Salvi, 2009C9orf30358 318
hsa-mir-26a-13p22.224 730EZH2, cyclins D2, cyclin E2Lu et al.[48]; Sander, 2008; Kota et al.[47]CTDSPL0106 856
hsa-mir-26a-212q14.120 365EZH2, cyclins D2, cyclin E2Sander, 2008; Kota et al.[47]CTDSP2020 365
hsa-mir-26b2q351813SLC7A11Ma, 2010 #133; Liu et al.[65]CTDSP101813
hsa-mir-27b9q22.32317CYP1B1Tsuchiya, 2006C9orf30358 555
hsa-mir-30c-11p34.126 046BCL2-like 11 (BIM)Garofalo, 2011NFYC064 981
hsa-mir-33a22q13.28601Pim-1Thomas, 2011SREBF2066 964
hsa-mir-954p16.165 175SNX1Huang, 2011ABLIM20152 620
hsa-mir-101-29p24.145 517EZH2, COX-2, Mcl-1Varambally, 2008; Strillacci, 2009; Su, 2009RCL1056 744
hsa-mir-10710q23.3151 653CDK6, PLAG1, HIF-1β, PKCɛLee, 2009; Pallasch, 2009; Yamakuchi, 2010; Datta, 2011PANK128151 653
hsa-mir-1269q34.30IRS-1, SLC7A5, SOX2, VEGFA, PIK3R2Tavazoie et al.[34]; Zhang et al.[35]EGFL704049
hsa-mir-128-12q21.376 097E2F3a, Bmi-1, NTRK3Zhang, 2009; Godlewski, 2008; Guidi, 2010R3HDM10133 252
hsa-mir-133a-220q13.3310 216FSCN1Chiyomaru et al.[55]; Kano, 2010C20orf166014 332
hsa-mir-13911q13.424 361ROCK2Wong, 2011PDE2A027 048
hsa-mir-1492q37.30Akt1, E2F1Lin et al.[27]GPC1019 098
hsa-mir-15217q21.320DNMT1, E2F3, MET, RictorHuang et al.[61]; Das, 2010; Tsuruta et al.[24]COPZ2930
hsa-mir-153-12q35516Bcl-2, Mcl-1Xu, 2010PTPRN1214 948
hsa-mir-153-27q36.32006Bcl-2, Mcl-1Xu, 2010PTPRN201 012 214
hsa-mir-18522q11.2111 562Six1, RhoA, Cdc42, Androgen receptor (AR)Imam, 2010; Liu et al.[65]; Ostling, 2011C22orf25011 562
hsa-mir-1861p31.113 087P2X7Zhou, 2008ZRANB218913 087
hsa-mir-1983q13.3345 984c-METTan, 2011FSTL1054 626
hsa-mir-218-14p15.31171 810ECOP, IKK-b, LASP1, PXN, Robo1, BIRC5, GJA1, RictorMartinez, 2008; Wu et al.[56]; Alajez et al.[49]; Uesugi et al.[23]SLIT20273 030
hsa-mir-218-25q34188 233ECOP, IKK-b, LASP1, PXN, Robo1, BIRC5, GJA1, RictorMartinez, 2008; Wu et al.[56]; Alajez et al.[49]; Uesugi et al.[23]SLIT30532 169
hsa-mir-32611q13.415 994Notch, MRP-1/ABCC1Kefas, 2009; Liang, 2010ARRB1015 994
hsa-mir-3357q32.22841SOX4Tavazoie et al.[34]MEST02841
hsa-mir-33817q25.32999SMOHuang, 2011AATK039 752
hsa-mir-3405q35.355 894MITF, c-MetGoswami, 2010; Wu, 2011RNF130055 894
hsa-mir-34614q32.21172Androgen receptor (AR)Grady et al.[43]GRID1098 379
hsa-mir-449a5q11.22333HDAC-1, E2F1, Androgen receptor (AR)Noonan, 2009; Yang, 2009; Lize, 2009; Ostling, 2011CDC20B02333
hsa-mir-449b5q11.22213E2F1, Androgen receptor (AR)Yang, 2009; Lize, 2009; Ostling, 2011CDC20B02213
hsa-mir-4868p11.216311OLFM4Oh, 2011ANK10235 314
hsa-mir-4881q25.2134 811Androgen receptor (AR)Sikand, 2010ASTN1178134 811
hsa-mir-4897q21.390 754PTPN11Kikkawa, 2010CALCR4390 754
hsa-mir-548d-18q24.1347 722ERBB2Chen, 2009; Heyn, 2011ATAD2047 722
hsa-mir-5592p217558ERBB2Chen, 2009EPCAM3437558
hsa-mir-5622q37.1178 223EYA1Drake, 2009DIS3L20210 527
hsa-mir-5917q21.3101 711Shohet, 2011SLC25A130101 711
hsa-mir-5937q32.122 182PLK1Ito, 2010SND10429 362
hsa-mir-63417q24.247 907Androgen receptor (AR)Ostling, 2011PRKCA0483 573

Pioneer studies of these intergenic TS-miRNA demonstrated that miR-127 was decreased by aberrant DNA methylation and histone modification in bladder cancer cells[17] and that miR-124 was inactivated by CpG island hypermethylation in several types of cancers.[18] These studies suggest DNA hypermethylation to be an important molecular mechanism for downregulation of miRNA expression in cancers. Previously, we identified four intergenic TS-miRNA silenced through DNA hypermethylation of CpG islands located within 500 bp upstream in oral squamous cell carcinoma (OSCC) and hepatocellular carcinoma (HCC), and also reported their targets (Table 1). We first identified miR-137 and miR-193a as an intergenic TS-miRNA frequently silenced by tumor-specific DNA hypermethylation in OSCC using expression-based screening with a series of sequential analyses of expression profiles of 148 miRNA, DNA hypermethylation status of selected candidates, and their tumor-suppressive activities in a panel of 18 OSCC cell lines and 11 primary tumors of OSCC with paired normal oral mucosa.[19] Our study also revealed that miR-137 and miR-193 might induce cell cycle arrest at the G1-S checkpoint and apoptosis, respectively, through direct binding to their target mRNA, CDK6 and E2F6, respectively, in OSCC cell lines. We performed methylation-based screening, comparing methylation and expression status for 39 miRNA located at 43 loci containing CpG islands within 500 bp upstream of these miRNA genes in a panel of 19 HCC cell lines and 41 primary HCC tumors with corresponding non-tumorous tissue, resulting in the discovery of hypermethylation-mediated silencing of miR-124 and miR-203 genes as a relatively frequent molecular event in HCC.[21] In this study, miR-124 and miR-203 were elucidated to exert cell growth-inhibitory effects on HCC cell lines through the induction of cell cycle arrest at the G1-S checkpoint and apoptosis, respectively, with the downregulation of protein expression of their targets, CDK6,VIM,SMYD3 and IQGAP1 or ABCE1, respectively. Based on these observations, we believe that the tumor-specific DNA hypermethylation of CpG islands located immediately 5′-upstream of intergenic miRNA genes is a useful landmark to explore novel TS-miRNA silenced epigenetically in cancer cells, similar to classical TSG.

Intragenic Tumor-Suppressive Micro RNA Genes and Their Host Genes Silenced by CpG Island Hypermethylation in Cancer Cells

  1. Top of page
  2. Abstract
  3. Distribution of Micro RNA Genes and Their Related CpG Islands in the Human Genome
  4. Intergenic Tumor-Suppressive Micro RNA Genes Silenced by CpG Island Hypermethylation in Cancer Cells
  5. Intragenic Tumor-Suppressive Micro RNA Genes and Their Host Genes Silenced by CpG Island Hypermethylation in Cancer Cells
  6. Future Perspectives on Tumor-Suppressive Micro RNA Silenced by Tumor-Specific DNA Hypermethylation in Cancer Research
  7. Concluding Remarks
  8. Acknowledgments
  9. Disclosure Statement
  10. References

Sizable regions of genomic DNA are recognized as introns harboring many intragenic miRNA genes, referred to as intronic miRNA genes. Approximately 37% of mammalian miRNA genes appear to be intronic miRNA genes.[32] A recent computational approach demonstrated that 94.2% (49/52) of predicted promoter regions of intronic miRNA genes overlapped with promoters of their host genes.[31] In addition, the majority of mammalian intronic miRNA genes are found to be frequently coexpressed with their protein-coding host genes under the promoter-driven regulation of the host gene, while nearly 26% of intronic miRNA genes are described to be transcribed from their own promoters.[3, 30, 33] In our database analyses using the miRBase database (Release 18: November 2011), intragenic miRNA genes accounted for 42.4% (645/1523) of human miRNA genes (Fig. 1B). Notably, among these 645 intragenic miRNA genes, 13.0% (84/645) were located within 500 bp downstream of CpG islands (Fig. 2B). In contrast, the data on the genomic distance between the 5′-end of protein-coding host genes harboring intragenic miRNA genes and the 3 + +′-end of CpG islands located at the 5′-side of host genes show that 81.2% (477/581) of these host genes, containing 645 intragenic miRNA genes within their introns, were located within 500 bp of CpG islands (Fig. 2C), whereas only 6.4% (41/645) of intragenic miRNA genes were located within 500 bp downstream of these CpG islands (Fig. 2D). In addition, These 477 host genes harbored 534 intragenic miRNA genes, including 176 genes assigned a name with a number below 700. Among these 176 genes, no more than 23.9% (42/176) of intragenic miRNA genes have been identified as TS-miRNA (Table 1).

Previous studies have shown tumor-suppressive activities of miR-126 and miR-335 located within introns of the EGFL7 and MEST genes, respectively, in breast cancer,[34] and report IRS-1,[35]SLC7A5,[36]SOX2,[37]VEGFA,[38, 39] and PIK3R2[39] or SOX4[34] to be their targets, respectively. Coexpression of miR-126 and EGFL7 is reported to be downregulated through histone modification and CpG island hypermethylation in the EGFL7 gene promoter in T24, HeLa and MCF7 cells and primary bladder and prostate tumors.[40] Transcription of miR-335 is also demonstrated to be coregulated with MEST by promoter hypermethylation in breast cancer cells.[41]miR-342 located within intron of the EVL gene is described as acting as an TS-miRNA by targeting DNMT1 in colorectal cancer.[42] CpG island hypermethylation upstream of EVL is indicated to suppress both EVL and miR-342 expression.[43] As regards to miR-1-1 located within the C20orf166 gene, miR-1 is described as a TS-miRNA targeting HDAC4,FOXP1 and MET, and is inactivated by DNA methylation of CpG island in HCC.[44] However, these four host genes have never been examined for tumor-suppressive activities.

Physiological or pathophysiological functions of host genes harboring intragenic TS-miRNA mostly remain unclear, whereas miR-26a and miR-218 are unique intragenic TS-miRNA located within introns of known TSG. miR-26a-1 located within an intron of CTDSPL/SCP3/HYA22/RBSP3 having a tumor-suppressive function[45] is reported to be silenced through CpG island hypermethylation.[46]Cyclin D2 and E2 or EZH2 are described as miR-26a targets in liver cancer[47] and nasopharayngeal carcinoma (NPC),[48] respectively. Regarding miR-218, downregulation of its expression was demonstrated to be associated with DNA methylation of CpG islands at the 5′-ends of SLIT2 and SLIT3 harboring miR-218-1 and miR-218-2, respectively, in NPC.[49] The SLIT2 and SLIT3 genes are indicated to act as TSG through SLIT-Robo signaling in breast cancer cell lines,[50] and to be inactivated by their promoter hypermethylation in several types of cancers[51, 52]. BIRC5,[49]ECOP,[53]GJA1[49], IKK-β,[54]LASP1[55], PXN[56] and Robo1[57] are reported as targets of miR-218. Recently, to explore TS-miRNA having potential for miRNA replacement therapy, we performed function-based screening using OSCC and endometrial cancer (EC) cell lines (Fig. 3A), and identified miR-218 and miR-152 as intragenic TS-miRNA frequently silenced through tumor-specific DNA hypermethylation in OSCC and EC, respectively.[23, 24]COPZ2 is a protein-coding host gene harboring the miR-152 gene and has recently been shown to display no tumor-suppressive activities, but, rather, to protect tumor cells from apoptosis induced by COPZ1-knockdown.[58] Moreover, our studies elucidated that Rictor was a novel direct target of these two TS-miRNA. Rictor, together with mTOR, forms mTOR complex 2 (mTORC2), and the Rictor-mTOR complex directly regulates the phosphorylation of Akt at Ser-473, resulting in cell growth.[59] In our study, miR-218 was clearly demonstrated to act as a suppressor of the TOR-Akt pathway, independently of the PI3K-Akt pathway, in an OSCC cell line without a genetic alteration of EGFR,PIK3CA and PTEN (Fig. 3B).[23] We also identified E2F3 and MET as direct targets of miR-152 other than DNMT1, which had been reported previously.[60, 61] A correlation between aberrant DNA methylation of CpG island of miR-152 and a poor clinical outcome is reported in breast cancer[62] and MLL-rearranged acute lymphoblastic leukemia.[63] These observations strongly support our notion that the tumor-specific DNA hypermethylation of CpG islands located around protein-coding host genes harboring intragenic miRNA genes, as well as intergenic miRNA genes, is a useful landmark to explore novel TS-miRNA silenced epigenetically in cancer cells.

image

Figure 3. Function-based screening of tumor-suppressive micro RNA (TS-miRNA) for miRNA replacement therapy as a cancer treatment. (A) Strategy of our function-based approach to the identification of epigenetically silenced TS-miRNA in cancer cells. Previously, to identify novel TS-miRNA having the great potential for miRNA replacement therapy, we performed function-based screening combined with methylation and expression analyses in oral squamous cell carcinoma (OSCC) and endometrial cancer (EC) cell lines according to this strategy shown in this figure, resulting in identification of novel TS-miRNA,miR-218 and -152, respectively, directly targeting Rictor.[23, 24] (B) A model summarizing the molecular mechanism of miR-218,miR-152 and their direct target Rictor in the TOR-Akt signaling pathway. Our previous studies demonstrated that these TS-miRNA acted as suppressors of the TOR-Akt signaling pathway, independently of the PI3K-Akt signaling pathway and that methylation-mediated silencing of these TS-miRNA might contribute to the pathogenesis of OSCC and EC through the activation of this signaling pathway. (C) Therapeutic effects of dsRNA mimicking miR-152 (dsmiR-152) or control non-specific miRNA (dsNC) on tumor growth in vivo. Left panel: a schema indicating the protocol of in vivo analysis (upper) and bar graph showing effects of dsmiR-152 on tumor growth in three SCID mice (lower). Right panel: photograph shows tumor-bearing SCID mice (upper) and their subcutaneous tumors (lower) at the end of in vivo analysis. These findings strongly support the great potential of dsRNA mimicking miR-152 to be applied to miRNA replacement therapy for cancers.

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Future Perspectives on Tumor-Suppressive Micro RNA Silenced by Tumor-Specific DNA Hypermethylation in Cancer Research

  1. Top of page
  2. Abstract
  3. Distribution of Micro RNA Genes and Their Related CpG Islands in the Human Genome
  4. Intergenic Tumor-Suppressive Micro RNA Genes Silenced by CpG Island Hypermethylation in Cancer Cells
  5. Intragenic Tumor-Suppressive Micro RNA Genes and Their Host Genes Silenced by CpG Island Hypermethylation in Cancer Cells
  6. Future Perspectives on Tumor-Suppressive Micro RNA Silenced by Tumor-Specific DNA Hypermethylation in Cancer Research
  7. Concluding Remarks
  8. Acknowledgments
  9. Disclosure Statement
  10. References

Taking miRNA-induced effects on normal cells into consideration, TS-miRNA, the endogenous expression of which is sufficiently activated in normal cells and remarkably reduced in cancer cells, are assumed to have great potential for miRNA replacement therapy.[25] The concept behind this therapy is a restoration of loss of function in cancer cells by exogenous expression of TS-miRNA. A few TS-miRNA, such as miR-34a[64, 65] and let-7,[66] have already demonstrated therapeutic effects on tumor formation in vivo following the replacement of these miRNA using dsRNA mimicking their mature forms. Because dsRNA are unlike proteins, are substantially smaller than DNA plasmids or viral vectors, and have the ability to enter the cytoplasm of target cells and to be delivered systemically by technologies that have been used for siRNA,[25] a dsRNA-based approach may be better than other approaches for exogenous expression of miRNA in vivo. TS-miRNA silenced through tumor-specific DNA hypermethylation might be better suited as prime candidates for this therapy. Recently, we successfully showed for the first time that dsRNA mimicking miR-152 administered with atelocollagen to SCID mice could suppress the in vivo growth of an EC cell line (Fig. 2C),[24] leading us to consider the possibility of miRNA replacement therapy for cancer using dsRNA mimicking TS-miRNA silenced by tumor-specific DNA hypermethylation.

While the function-based approach is a powerful tool for exploring the use of dsRNA with tumor-suppressive effects, including TS-miRNA and siRNA, as therapeutic agents for cancers, the tumor-suppressive functions of miRNA eventually identified in our studies have been reexamined using two or three kinds of dsRNA purchased from independent companies to take into consideration the off-target effects associated with dsRNA.[23, 24] Although such effects have been known to complicate the interpretation of phenotypic effects in gene-silencing experiments using siRNA,[67] dsRNA mimicking miRNA might potentially cause these unwanted actions, similar to siRNA. These unpredictable target-independent effects should be addressed during data interpretation in all dsRNA-based studies related to functional genomics, drug target discovery and dsRNA-therapeutics. In addition, these dsRNA have mainly been used at 1.0–50.0 nM in dsRNA-based studies, and overexpression above their physiological concentrations might lead to toxic effects in normal cells with an accumulation of these exogenous dsRNA. In contrast, although atelocollagen[24, 64] or lipid-based delivery agents[65, 66] have been used for in vivo dsRNA delivery, these systems are inadequate for clinical applications to fully restore downregulated miRNA in cancer cells. Therefore, the development of solutions attenuating the nonspecific off-target effects associated with dsRNA mimicking TS-miRNA and the advancement of dsRNA-delivery technology may yield a new field of miRNA-based cancer therapy.

Concluding Remarks

  1. Top of page
  2. Abstract
  3. Distribution of Micro RNA Genes and Their Related CpG Islands in the Human Genome
  4. Intergenic Tumor-Suppressive Micro RNA Genes Silenced by CpG Island Hypermethylation in Cancer Cells
  5. Intragenic Tumor-Suppressive Micro RNA Genes and Their Host Genes Silenced by CpG Island Hypermethylation in Cancer Cells
  6. Future Perspectives on Tumor-Suppressive Micro RNA Silenced by Tumor-Specific DNA Hypermethylation in Cancer Research
  7. Concluding Remarks
  8. Acknowledgments
  9. Disclosure Statement
  10. References

In this review, we focused on genomic distances between intergenic TS-miRNA genes or protein-coding host genes harboring intragenic miRNA genes and their related CpG islands, and illustrated how the tumor-specific DNA hypermethylation of CpG islands located immediately 5′-upstream of intergenic miRNA genes and host genes, as well as classical protein-encoding TSG, might be a useful epigenetic marker for exploration of TS-miRNA, cancer diagnosis and prognosis. Moreover, we also discussed the potential of miRNA replacement therapy for cancers using dsRNA mimicking TS-miRNA silenced epigenetically in cancer cells. Further studies of molecular mechanisms of TS-miRNA and significant advancement of dsRNA-delivery technology are essential for the actualization of TS-miRNA replacement therapy for several types of cancers.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Distribution of Micro RNA Genes and Their Related CpG Islands in the Human Genome
  4. Intergenic Tumor-Suppressive Micro RNA Genes Silenced by CpG Island Hypermethylation in Cancer Cells
  5. Intragenic Tumor-Suppressive Micro RNA Genes and Their Host Genes Silenced by CpG Island Hypermethylation in Cancer Cells
  6. Future Perspectives on Tumor-Suppressive Micro RNA Silenced by Tumor-Specific DNA Hypermethylation in Cancer Research
  7. Concluding Remarks
  8. Acknowledgments
  9. Disclosure Statement
  10. References

Our studies were supported in part by a Grant-in-Aid for Scientific Research (A), (B) and (C) and for Scientific Research on Priority Areas and Innovative Areas, and by the Global Center of Excellence Program for International Research Center for Molecular Science in Tooth and Bone Diseases of the Ministry of Education, Culture, Sports, Science, and Technology, Japan, as well as by a Health and Labour Sciences Research Grant by the Ministry of Health, Labour and Welfare, Japan.

References

  1. Top of page
  2. Abstract
  3. Distribution of Micro RNA Genes and Their Related CpG Islands in the Human Genome
  4. Intergenic Tumor-Suppressive Micro RNA Genes Silenced by CpG Island Hypermethylation in Cancer Cells
  5. Intragenic Tumor-Suppressive Micro RNA Genes and Their Host Genes Silenced by CpG Island Hypermethylation in Cancer Cells
  6. Future Perspectives on Tumor-Suppressive Micro RNA Silenced by Tumor-Specific DNA Hypermethylation in Cancer Research
  7. Concluding Remarks
  8. Acknowledgments
  9. Disclosure Statement
  10. References