MicroRNA expression and its association with DNA repair in preimplantation embryos

Abstract

Active DNA repair pathways are crucial for preserving genomic integrity and are likely among the complex mechanisms involved in the normal development of preimplantation embryos. MicroRNAs (miRNA), short non-coding RNAs, are key regulators of gene expression through the post-transcriptional and post-translational modification of mRNA. The association of miRNA expression with infertility or polycystic ovarian syndrome has been widely investigated; however, there are limited data regarding the importance of miRNA regulation in DNA repair during preimplantation embryo development. In this article, we review normal miRNA biogenesis and consequences of aberrant miRNA expression in the regulation of DNA repair in gametes and preimplantation embryos.

Mammalian preimplantation embryo development follows a series of critical events. These events begin at gametogenesis and continue until parturition. Male and female gametes are derived from primordial germ cells during spermatogenesis and oogenesis, respectively. Following fertilization, oocyte and sperm nuclei fuse, resulting in syngamy. In preimplantation embryos, because the cell cycle is short, the risk of genetic errors during replication or segregation is increased [1]. Once DNA damage is detected, cells may undergo apoptosis or activate different DNA repair mechanisms, such as base excision repair (BER), nucleotide excision repair (NER), double strand break repair (DSBR) and mismatch repair (MMR) [2,3,4, 5]. Poor activation of DNA repair may affect embryo implantation, because apoptosis of even a single cell at the cleavage stage is likely to delay embryo development into the blastocyst.

In the early stages of preimplantation embryo development, maternal mRNAs direct DNA repair. In mammals, during the cleavage stage divisions programming of maternal and paternal chromosomes occurs to create the embryonic genome (embryonic genome activation, EGA) and to begin preimplantation embryo development. Upon EGA, remarkable reprogramming of expression occurs. In mammals, these reprogramming events are controlled by transcription, translation, and microRNA (miRNA) regulation [6]. miRNAs form a large family of short non-coding RNAs between 17–25 nucleotides (nts) in length that have been shown to be expressed in preimplantation embryos [7,8,9].

The biological significance of miRNAs and the roles of these small non-coding RNAs in gametes and preimplantation embryos are poorly understood. This review briefly summarizes the biogenesis of miRNAs and their expression in gametes and preimplantation embryos, as well as their role in regulating DNA repair.

MicroRNA Biogenesis

The biogenesis of a small group of miRNAs is induced in an ATM-dependent manner. These miRNAs are associated with KH-type splicing regulatory protein, an AU-rich binding protein involved in Drosha and Dicer processing and in mRNA decay [10,11,12]. Generally, miRNAs are transcribed into long RNAs with a stem-loop structure by RNA polymerase II [13] (Fig. 1). Intergenic miRNAs, which contain their own promoters and regulatory units, are transcribed into pri-miRNA by RNA polymerase II, whereas intronic miRNAs are co-transcribed using host genes from a common promoter [14, 15]. The intergenic pri-miRNAs are processed by Drosha, a 130–160-kDa protein with one dsRNA-binding and two catalytic domains [16]. In the presence of Pasha/DiGeorge syndrome critical region gene 8 (DGCR8), both strands of the hairpin are cut, generating a pre-miRNA product of approximately 70 nt in size [17]. Although both Drosha and DGCR8 were found to be essential for the formation of mature miRNA in Drosophila melanogaster and Caenorhabditis elegans, some pri-miRNAs do not involve these processes by Drosha and DGCR8 and they either use other endonucleases or are directly transcribed into short hairpin structures [18,19,20]. Pre-miRNAs are then transported from the nucleus into the cytoplasm by Exportin-5 (Exp5), which is a nucleocytoplasmic transporter in the karyopherin family and has binding sites for pre-miRNAs in the presence of Ras-related nuclear protein and guanosine triphosphate [21, 22]. Pre-miRNAs are further cleaved by the cytoplasmic RNase endonuclease Dicer, forming 21–22-nt double-stranded structures. The mRNA levels of Dicer in oocytes are higher compared to those in other cell types, suggesting that Dicer plays an important role in the female germline [23]. The crucial role of Dicer in the female germline was also supported in a recent study showing that loss of a splice variant of Dicer, DicerO, led to sterility in female rodents [24]. Reduced and disorganized spindles and incorrect chromosome alignment were observed in Dicer mutant mice and C. elegans oocytes, respectively [7, 25]. Both strands of the pre-miRNA can be associated with Argonaut (Ago)-protein-containing complex and mediated by RNA-induced silencing complex/mi-ribonucleoprotein (RISC/miRNP); however, one strand is mostly degraded [16, 26]. There is no discrimination of which miRNA strand is degraded in mammals. However, the strand that regulates and is involved in the loading of the other strand, miR*, on RISC is typically degraded in mammals [27]. Selection of the strand for degradation may also depend on the stability of the 5′ end and the sequence characteristics such as the bias for A and U [28, 29].

Fig. 1.

Schematic diagram of miRNA biogenesis. Pri-miRNAs are cleaved by the Drosha/DGCR8 complex producing pre-miRNA. Exp5 transports pre-miRNAs into the cytoplasm that are then processed by Dicer and Ago2/RISC complex to form mature miRNA. Adapted from [13]. Copyright adapted with permission from “Development”, via DOI:- http://dev.biologists.org/content/138/9/1653.long/.

Gametogenesis and miRNA Expression

miRNA expression has been observed as early as oogenesis and spermatogenesis in mouse, bovine, and human (Supplementary Table 1: online only). Two percent of the miRNAs analyzed (15/722) in human oocytes showed different expression levels between GV and MII oocytes, such that four miRNAs, including hsa-miR-602, hsa-miR-193a-5p, hsa-miR-297, and hsa-miR-625, were up-regulated in oocytes matured in vitro compared to immature GV oocytes, whereas 11 miRNAs, including hsa-miR-888, hsa-miR-212, hsa-miR-662, hsa-miR-299-5p, hsa-miR- hsa-miR-339-5pm hsa-miR-20a, hsa-miR-486-5p, hsa-miR-141, hsa-miR-768-5p, hsa-miR-376a, and hsa-miR-15a, were down-regulated in mature oocytes [30]. Ago2-deficient oocytes were matured with abnormal spindles, the chromosomes could not unite properly, and showed reduced expression levels of miRNAs [31].

Similar to the oocyte, sperm carries a range of miRNAs. Approximately 20% of these miRNAs are located in the nuclear or perinuclear region of the sperm, indicating that these miRNAs are transferred to the zygote at the time of fertilization. However, the role of these sperm-borne miRNAs is not clear since they are already present in the mature oocyte (meiosis II) [32].

Aberrant expression of miRNA biogenesis genes causes defects in both oogenesis and spermatogenesis. Complete loss of Dicer in somatic cells in the mouse reproductive tract not only showed reduced expression of miRNAs, but also caused the female mice to become infertile with compromised oocytes and embryo integrity [7, 9, 33,34,35]. Loss of Dicer in the germ-line of male mice (homozygote Dicer) led to decreased fertility resulting from abnormal spermatogenesis. In these mice, the number of germ cells was reduced with abnormal spermatids, abnormal phenotype of spermatocytes with condensed nucleus, abnormal sperm motility, and mutant testes with Sertoli tubules [36]. However, it has been suggested that maternal cytoplasmic Dicer transfer disguised the early abnormal phenotypes [25, 37]. Deletion of Dicer led to the loss of sperm, which may be related to the reduced levels of miRNA production [7, 38,39,40].

Preimplantation Embryos and miRNA Expression

Similar miRNA expression profiles in mature mouse oocytes and early developing embryos indicate that the zygote contains maternally inherited miRNAs [7]. As mouse and bovine embryos undergo cleavage divisions, variation in the expression levels of some miRNAs were observed (Supplementary Table 1). In murine embryos, miRNA expression is reduced by as much as 60% between the 1- and 2-cell stages. However, at the end of the 4-cell stage, the expression of miRNAs were doubled in mouse embryos compared to the 2-cell stage embryo, supporting that EGA begins between the 1-cell and 4-cell stages (Supplementary Table 1) [7]. Although the synthesis and degradation of miRNAs co-occur during mouse preimplantation embryo development, overall miRNA expression increases towards the blastocyst stage [41]. Despite numerous studies in mice, only two studies have analyzed the expression of miRNAs in human blastocysts [9, 42].

Abnormalities in Preimplantation Embryos Resulting from Aberrant miRNA Biogenesis

The correct biogenesis and expression of miRNAs is important in preimplantation embryos, as any changes in the expression of DNA repair genes may cause defective DNA repair in the embryos. The 5′ and 3′ nucleotides of the mature miRNAs are determined by Drosha cleavage, and defects in Drosha cleavage may lead to changes in the seed sequence of miRNAs. Exp5 is fundamental for miRNA expression in human and Drosophila, as the knock-down of Exp5 reduces miRNA and tRNA levels in human and Drosophila, respectively [43]. Exp5 was also suggested to stabilize pre-miRNAs. In the absence of Exp5, no pre-miRNA accumulation was detected in the nucleus [22], while when Exp5 was over-expressed, endogenous and exogenous miRNA levels were increased [44]. Knockout of Ago2 in mouse embryonic fibroblasts and hematopoietic cells decreased the levels of mature miRNAs [38, 45, 46]. Any changes to the miRNA sequence or miRNA expression level will likely alter the regulation of mRNAs, which may cause changes in crucial pathways in preimplantation embryos [47] (Table 1).

Table 1. Functions of miRNA processing genes and defects caused by abnormal functioning of these genes.

Genes	Functions	Defects caused by abnormal functioning of genes
Drosha	• Vital for the formation of mature miRNA [47]	• 5′ and 3′ nucleotides of the mature miRNA are determined by Drosha cleavage and any defects in Drosha cleavage may lead to changes in the seed sequence of miRNAs [47]
	• miRNA processing by cutting both strands of miRNA forming pre-miRNA product in the presence of DGCR8 [16, 127]
Exp5	• Stabilization of pre-miRNAs [44]	• Knockdown of Exp5 was shown to reduce miRNA expression [43]
	• Transport of pre-miRNAs from the nucleus into the cytoplasm in the presence of Ran and GTP [44]
Dicer	• Maturation of miRNA [16, 26]	• Cell proliferation defects [36]
		• Lack of Dicer in Drosophila germ line stem cells postponed the G1/S phase transition
		• Dicer deletion in hippocampal, mouse and zebrafish initiated problems in nervous system and led to inability of forming mature miRNAs that resulted in variations of brain morphogenesis and differentiation of neurons [128, 129]
		• Complete loss of Dicer1 caused reduced expression of miRNAs and infertile female mice [7, 9]
		• Homozygote Dicer1 germ-line mutant male mice caused decreased male fertility [36]
		• Dicer deficiency led to embryo death in mouse around embryonic day 7.5 [7, 25, 56] and in zebrafish [58]
Ago2	• Component of miRISC [16, 26]	• In the absence of Ago2, oocytes developed to the mature oocytes but with abnormal spindles, and chromosomes were not able to unite properly with reduced miRNA expression levels [57]
	• miRNAs associated with Ago2/RISC complex target mRNAs [13]

The early differentiation of the maternal to zygotic transition was normal in Dicer mutant zebrafish and mouse embryos; however, defects were triggered in somatogenesis, morphogenesis that affected gastrulation, and heart development, as well as led to apoptosis in the limb mesoderm [25, 48,49,50,51]. Injection of miR-430, which is expressed at the time of EGA and increases the rate of deadenylation and degradation of maternal mRNAs [49], partially repaired the gastrulation, retinal development, and somatogenesis in zebrafish and C. elegans [25].

Deletion of DGCR8 caused cell proliferation defects; however, injection of miR-19, miR-20a, miR-20b, miR-294, and miR-295 prevented these aberrations in mouse embryonic stem cells [52]. A lack of Dicer in Drosophila germ line stem cells postponed the G1/S phase transition [53], suggesting that miRNAs may be vital for stem cells to bypass this checkpoint. Additional studies showed that deletion of Dicer in the developing animals caused aberrations [54, 55]. Dicer deficiency and loss of Ago2 function led to embryo death in mice around embryonic day 7.5 [7, 25, 56] and 9.5 [57], respectively, and in zebrafish [58].

Regulation of mRNAs by miRNAs

miRNAs are either transcribed as separate genes or as miRNA clusters from precursor transcripts, whereas intronic miRNAs are encoded by introns of protein-coding genes, and are formed following pre-mRNA splicing. Approximately 30% of genes in the human genome are estimated to be targeted by miRNAs [59]. Until recently, all studies reported that miRNAs down-regulated their target mRNAs; however, in the past few years, miRNAs were suggested to stabilize their targets [60,61,62,63,64,65,66,67]. This stabilization of mRNAs is not well understood; however, several possible mechanisms have been proposed. miRNA regulation through binding to AU-rich elements of specific proteins [68, 69], miRNA regulation between repression and activation of genes with repression more active in proliferating cells and activation in G1/G0 arrest [70], a computational hypothesis of differential mRNA regulation by miRNAs [61, 62], and competition of pseudogenes with their legitimate genes for the same miRNAs, reducing the down-regulatory effect of miRNAs on their target genes [67], are the main proposed stabilization pathways. Therefore, pseudogenes, non-coding genes, and circular RNAs can function as endogenous decoys for miRNAs [64,65,66,67, 71,72,73]. From a similar perspective, it was also suggested that if an miRNA has multiple mRNA targets, the down-regulatory effect of the miRNAs is reduced [63]. Based on these observations, the competitive endogenous RNA (ceRNA) hypothesis was proposed, in which coding and non-coding RNAs can regulate the mRNA/miRNA association by competing for miRNA binding sites (miRNA response elements) [60]. Similarly to the ceRNA hypothesis, it is also possible that miRNA/miRNA interactions positively regulate mRNAs, such that one miRNA down-regulates another miRNA, leading to increased expression of its target gene [69].

Regulation of Genes Involved in the Cell Cycle by miRNAs Expressed in Preimplantation Embryos

Several miRNAs targeting mRNAs function at cell cycle checkpoints and in DNA repair mechanisms. Proper coordination of the cell cycle is crucial in the response to DNA damage. Cell cycle arrest occurs during replication and at G1/S (first gap phase/DNA synthesis phase) or G2/M (second gap phase/mitosis) checkpoints to activate the correct repair pathway [74]. If the repair mechanisms are unable to repair the damage, which can be caused by inactive DNA repair mechanisms, apoptosis of an embryonic cell can be detrimental to the early developing embryo. Therefore, the correct activation of genes and proteins is critical for fully functioning DNA repair pathways.

Many miRNAs expressed in preimplantation embryos regulate or are regulated by genes functioning at cell cycle checkpoints [75,76,77]. As described above, DNA replication and cell proliferation are shorter in preimplantation embryos. Therefore, the correct regulation of mRNA function during the cell cycle is crucial for avoiding genetic errors, cell cycle arrest, or apoptosis [78].

The tumor suppressor p53 is a DNA damage-induced transcription factor. Recent studies showed that the miR-125b [79], miRNA-380-5p, miR-34, and miR-200 families inhibit the expression of p53 and its related family member p63 in cancer cells [80]. miRNAs expressed in preimplantation embryos, such as miR-16-1, miR-143, and miR-145, were up-regulated by p53- and p68/p72-dependent pathways upon DNA damage in human colon cancer cell lines [81]. Additionally, p53 transcriptionally regulates the miR-192, miR-194, miR-215, and miR-17-92 clusters, and the miR-34 family that targets genes involved in G1/S and G2/M checkpoints [82,83,84,85,86]. Ectopic expression of these miRNAs results in cell cycle arrest in cancer cells and cell lines [79, 82, 83, 87,88,89]. Over-expression of miR-34c, a member of the miR-34 family, suppressed c-Myc expression, which regulates the G1/S cell cycle transition and prevents the replication of damaged DNA in cell lines, whereas inhibition of miR-34c prevents DNA damage-induced S-phase arrest [89]. RB1, another important gene functioning at the G1/S cell cycle transition, has recently been shown to have a direct expression association, indicating a possible stabilization effect of this miRNA on its target mRNAs. miR-21, which is involved in the epithelial-mesenchymal transition and represses tumor suppressors [90], negatively regulates the G1/S phase in laryngeal carcinoma tissues [91]. Members of miR-17-92 in embryonic stem cells [92] and miR-371 and miR-302 clusters in humans [93] were also suggested to control the G1/S transition, and this cluster along with the signaling processes by Oct4, Sox2, and c-Myc may be vital for embryonic cell pluripotency and self-renewal [92]. Additionally, ectopic expression of miR-421 influences the efficiency of the S-phase cell-cycle checkpoint by down-regulating ataxia-telangiectasia mutated (ATM) kinase and increases the sensitivity to ionizing radiation in cell lines [94]. This effect was reversed by blocking the miR-421 and ATM 3′UTR interaction [95]. Similarly, over-expression of miR-18a in colorectal cancer cells suppressed ATM expression [96]. Additionally, over-expression of N-Myc, frequently amplified in neuroblastoma, is capable of inducing miR-421 expression, leading to ATM down-regulation. This suggests that N-Myc-mediated oncogenesis may be associated with the miRNAs in DNA damage response and repair. Up-regulation of miR-101 in the plasmid constructs reduces ATM and DNA-dependent protein kinase protein levels [97]. M059J glioblastoma DNA-PK-deficient cells expressed high levels of miR-100, leading to low expression levels of ATM [98]. Over-expression of miR-143 protects cells from DNA damage-induced death, leading to G2 checkpoint arrest by targeting fragile histidine triad (FHIT) [99]. Figure 2 summarizes the association between miRNAs expressed in mouse, bovine, or human preimplantation embryos and the cell cycle genes and proteins.

Fig. 2.

miRNAs involved in the regulation of cell cycle checkpoint genes at G0, S, G2, and M phases. a) miRNAs involved in the regulation of cell cycle checkpoint genes at the G1/S transition. b) miRNAs involved in the regulation of cell cycle checkpoint genes at G0/G1 and S/G2 transitions. c) miRNAs involved in the regulation of cell cycle checkpoint genes at G2/M and M/G1 transitions. Main cycles of checkpoint, including G1 (first gap phase), S (synthesis phase), G2 (second gap phase), M (mitosis), and G0 are shown. Cell cycle regulators and effectors, which regulate and/or are regulated by miRNAs expressed in preimplantation embryo development, are grouped according to the cell cycle phase. Positive and negative relationship between miRNAs and mRNAs are shown according to the cell cycle: a) miRNAs involved in the regulation of cell cycle checkpoint genes at G1/S transition. b) miRNAs involved in the regulation of cell cycle checkpoint genes at the G0/G1 and S/G2 transitions c) miRNAs involved in the regulation of cell cycle checkpoint genes at the G2/M and M/G1 transitions [75, 80, 93, 95, 97, 118,119,120,121,122,123,124,125,126].

Cell cycle checkpoint genes play an important role in activating the appropriate DNA repair pathways. It is possible that DNA repair pathways are not fully functional in preimplantation embryos. However, several genes associated with these pathways, including nucleotide excision repair (Fig. 3), base excision repair (Fig. 4), mismatch repair (Fig. 5), and double-strand break repair (Fig. 6), are regulated by miRNAs and these genes expressed during preimplantation embryo development.

Fig. 3.

Schematic diagram of nucleotide excision repair pathway with genes and miRNAs regulating these genes. Genes involved in nucleotide excision repair and miRNAs predicted to regulate these genes are shown (http://www.microrna.org/microrna/home.do, http://www.targetscan.org/, http://mirdb.org/miRDB/). Several miRNAs targeting or suggested to target the initial sensor genes and genes functioning at later stages of nucleotide excision repair pathway.

Fig. 4.

Schematic diagram of base excision repair pathway with genes and miRNAs regulating these genes. Genes involved in base excision repair and miRNAs predicted to regulate these genes are shown (http://www.microrna.org/microrna/home.do, http://www.targetscan.org/, http://mirdb.org/miRDB/). Several miRNAs were shown to target PARP1, which interacts with several genes involved in base excision repair and genes functioning at later stages of the base excision repair pathway. Bioinformatics studies also showed that PCNA is regulated by two miRNAs. Although a direct relationship among miRNAs and base excision repair genes, proteins, and polymerases has not been established, base excision repair components may be indirectly regulated by miRNA-regulated PARP1.

Fig. 5.

Schematic diagram of the double-strand break repair pathways with genes and miRNAs regulating these genes. Genes involved in a) non-homologous end joining and b) homologous recombination pathways, and miRNAs predicted to regulate these genes are shown (http://www.microrna.org/microrna/home.do, http://www.targetscan.org/, http://mirdb.org/miRDB/). Several miRNAs target the initial sensor genes of non-homologous end joining and homologous recombination repair pathways. Several more miRNAs regulate the expression of genes functioning at later stages of both non-homologous end joining and homologous recombination repair pathways.

Fig. 6.

Schematic diagram of mismatch repair pathway with genes and miRNAs regulating these genes. Genes involved in mismatch repair and miRNAs predicted to regulate these genes are shown (http://www.microrna.org/microrna/home.do, http://www.targetscan.org/, http://mirdb.org/miRDB/). Multiple miRNAs have been suggested to regulate the expression of mismatch repair sensor genes (MSH2, MSH3, MSH6, PCNA) and genes functioning at later stages of mismatch repair (MLH1 and PMS2).

DNA Repair Genes and miRNA Expression

DNA repair is a multi-protein repair system and miRNAs regulate the expression of genes involved in different repair mechanisms. Several studies have reported the down-regulatory effects of miRNAs on their target mRNAs, resulting in defective repair. However, many of these studies were performed in tumors, cancer cells, and cell lines. Since there is only one published study describing the possible regulatory roles of miRNAs on repair genes in gametes or embryos [8], these studies in tumors, cells, or cell lines may provide a foundation for understanding the possible relationship between miRNAs and mRNAs during preimplantation development. In this part of the review, we will summarize the studies demonstrating the effects of miRNA expression on DNA repair mechanisms.

In a recently published study reporting the possible regulatory roles of miRNAs on their target repair genes, it was shown that the expression of hsa-miR-23b was inversely associated with the expression of its target nucleotide excision repair gene GTF2H2, indicating a possible down-regulatory role of this miRNA on repair gene [8]. Furthermore, forced expression of another miRNA, miR-192, impaired nucleotide excision repair because increased expression of this miRNA was shown to down-regulate ERCC3 (XPB) and ERCC4 (XPF) in hepatoma HepG2.2.15 cells [100]. Similarly, the down-regulatory effects of miRNAs on genes involved in double-strand break repair, including inverse expression association of hsa-miR-128 with its target genes DCLRE1A and RAD50 in human blastocysts [8], were reported. Over-expression of miR-24 and miR-138 reduced the expression of H2AX, leading to an increased sensitivity to ionizing radiation and reduced repair capacity [88, 101, 102]; over-expression of miR-138 inhibited homologous recombination, leading to an increased sensitivity to DNA damaging agents [102]. Moreover, miR-23a, miR-23b [103], miR-24 [88], and miR-145 [104], target the initial sensor gene H2AX, which is involved in double-strand break repair. In breast cancer cell lines and tumors, miR-18a was over-expressed and the ectopic expression of miR-18a down-regulated ATM by interacting with the 3′UTR of the gene. Over-expression of miR-18a reduced homologous recombination and DNA repair in breast cancer cells and, as expected, inhibition of miR-18a improved the homologous recombination and DNA repair efficiency [105]. miR-182 targets BRCA1; over-expression of this miRNA may be involved in the up-regulation of BRCA1 in sporadic breast tumors and increase the sensitivity to PARP1 inhibition in cultured cells or in xenograft models [106]. miR-99 [107] and siSNF2H, which facilitates homologous recombination and the non-homologous end joining repair pathways [108], affect the localization of BRCA1 and Rad51 to DNA damage sites and therefore influence DNA repair efficiency [109]. Forced expression of miR-210 suppressed RAD52 expression, while over-expression of miR-373 decreased RAD23B and RAD52 [110]. Both of these miRNAs are induced in a hypoxia-inducible factor-1α-dependent manner, indicating an association between the miRNA and DNA repair pathways [110, 111]. Another study showed that the tumor suppressor miR-146, which is expressed in cleavage stage embryos, reduced the expression of BRCA1 in tumor tissues [112]. In silico studies showed that the conserved binding sites for miR-205 within BRCA1 and this miRNA and miR-146b were down-regulated in BRCA1 tumors [107].

Studies suggest that miR-155, which is expressed at different cleavage stages in mouse preimplantation embryos, down-modulates the mismatch repair heterodimer proteins MSH2-MSH6, MLH1, and PMS2 [113]. Mismatch repair recognition protein complex, involving hMSH2 and hMSH6, is down-regulated by increased miR-21 expression in colorectal tumors [114].

Studies suggest that defects in mismatch repair post-replication cause defects in microsatellites, which are short tandem repetitive DNA sequences [115]. Differential expression of miRNAs, including miR-31, miR-625, miR-196b, miR-181c [116], and several members of the miR-17-92 family [117], between tumors with microsatellite instability and/or the absence of protein expression of hMLH1 and tumors with no microsatellite instability and normal protein expression of hMLH1 were reported. Additionally, 39 miRNAs showed differential expression levels between normal colon tissue and tumor specimens. Up-regulation of miRNAs are thought to directly or indirectly lower the tumor suppressor protein expression, leading to improper cell division and tumor formation [116].

Although the expression of several genes involved in repair mechanisms was suggested to be regulated by miRNAs, the possible association between these miRNAs and mRNAs must be further analyzed in gametes and embryos.

Conclusion

Short non-coding RNAs, miRNAs, have gained attention for their regulatory roles of mRNAs and their involvement in many diseases, such as cancers and infertility. Studies of miRNA expression in preimplantation embryos have recently increased to understand their regulatory roles on gene expression. A limited number of articles have been published regarding miRNA expression in gametes and preimplantation embryos. Most of these expressed miRNAs have been associated with DNA repair and aberrant expression of these miRNAs. However, further studies are required to understand the complete contribution of miRNAs in preimplantation embryos. Therefore, the analysis of miRNA expression may identify a regulatory role for these small non-coding RNAs in the expression of DNA repair genes affecting the overall repair activity of the embryo.