The role of microRNAs in tumorigenesis
Calin
et al. first made the connection between microRNAs and cancer by showing that
miR-15 and
miR-16 are located at chromosome 13q14, a region deleted in more than half of B-cell chronic lymphocytic leukemia (CLL). Detailed deletion and expression analysis showed that
miR-15 and
miR-16 are located within a 30 kb region of loss in CLL, and that both genes were deleted or downregulated in approximately 68% of CLL cases [
8]. They further showed that miR genes were frequently located in cancer-associated genomic regions or in fragile sites. The full complement of miRNAs in a genome might be extensively involved in cancers [
9]. Bottoni
et al. found that
miR-15a and
miR-16-1 were expressed at lower levels in pituitary adenomas as compared to normal pituitary tissue. Their expression was inversely correlated with tumor diameter and with arginyl-tRNA synthetase expression, but was directly correlated with
p43 secretion, suggesting that these miRNAs might influence tumor growth [
10]. Cimmino
et al. then demonstrated that
miR-15a and
miR-16-1 expressions were inversely correlated to
Bcl2 expression in CLL and that both miRNAs negatively regulated
Bcl2 at a posttranscriptional level.
Bcl2 repression by these miRNAs induced apoptosis in a leukemic cell line model. Therefore
miR-15 and
miR-16 were natural antisense
Bcl2 interactors that could be used for therapy of
Bcl2-overexpressing tumors [
11]. Recently, Garzon
et al. showed that all-trans retinoic acid (ATRA) downregulation of
Bcl2 and
Ras was correlated with the activation of
miR-15a/miR-16-1 [
12].
Amplification and overexpression of the
miR-17-92, which comprised 7 miRNAs and resided in intron 3 of the
C13orf25 gene at 13q31.3, has been reported with pointers to functional involvement in the development of lymphoma and lung cancer. He
et al. compared B-cell lymphoma samples and cell lines to normal tissues, and found that the levels of the primary or mature miRNAs derived from the
miR-17-92 locus were often substantially increased in cancer cells. Their studies indicated that miRNAs could modulate tumor formation and function as oncogenes, implicating the
miR-17-92 cluster as a potential human oncomicroRNAs (oncomiRs) [
13]. O'Donnell
et al. demonstrated that c-Myc activated expression of a set of 6 miRNAs on human chromosome 13 that was tied to the development of human lymphoma. It was found that the expression of E2F1 was negatively regulated by
miR-17-5p and
miR-20a in HeLa cells. Their findings revealed a mechanism through which the c-Myc protein simultaneously activated E2F1 transcription and limited its translation, allowing a tightly controlled proliferative signal [
14]. Woods
et al. proposed a model whereby
miR-17-92 promoted cell proliferation by shifting the E2F transcriptional balance away from the pro-apoptotic E2F1 and toward the proliferative E2F3 transcriptional network [
15].
On the other hand, Hayashita
et al. found that
miR-17-92 was markedly overexpressed in lung cancer, especially with small-cell lung cancer. Their findings suggested that marked overexpression of the
miR-17-92 cluster with occasional gene amplification might play a role in the development of lung cancer and that the
C13orf25 gene might well be serving as a vehicle in this regard [
16]. Matsubara
et al. showed that inhibition of
miR-17-5p and
miR-20a with antisense oligonucleotides could induce apoptosis selectively in lung cancer cells overexpressing
miR-17-92, suggesting the possibility of oncomiR addiction to expression of these miRNAs in a subset of lung cancers. Their discoveries contributed towards better understanding of the oncogenic roles of
miR-17-92, which might ultimately lead to the future translation into clinical applications [
17].
Iorio
et al. showed that miRNAs were aberrantly expressed in human breast cancer compared with normal breast tissue, with the most significantly downregulated miRNAs being
miR-10b,
miR-125b and
miR-145, whereas the most significantly upregulated miRNAs being
miR-21 and
miR-155. The miRNA expression was also found to be correlated with specific breast cancer biopathologic features, such as estrogen and progesterone receptor expression, tumor stage, vascular invasion or proliferation index [
18]. Si
et al. found that
miR-21 was highly overexpressed in breast tumors compared to the matched normal breast tissues. They found that the anti-
miR-21-mediated cell growth inhibition was associated with increased apoptosis and decreased cell proliferation. Their results suggested that
miR-21 functioned as an oncogene and modulated tumorigenesis through regulation of genes such as
Bcl2 and it might serve as a novel therapeutic target [
19]. Zhu
et al. performed two-dimensional differentiation in-gel electrophoresis of tumors treated with anti-
miR-21 and identified the tumor suppressor tropomyosin 1 as a potential
miR-21 target. Downregulation of tropomyosin 1 in breast cancer by
miR-21 might explain why suppression of
miR-21 could inhibit tumor growth, further supporting the notion that
miR-21 functions as an oncogene [
20]. Meng
et al. identified
miR-21 was also highly overexpressed in malignant cholangiocytes. Inhibition of
miR-21 increased sensitivity to gemcitabine, it modulated gemcitabine-induced apoptosis by phosphatase and tensin homolog deleted on chromosome 10-dependent activation of PI 3-kinase signaling [
21]. Tran
et al. found that
miR-21 was highly expressed in the head and neck cancer cell lines. Several tumor suppressor genes were identified to be potential targets of miRNAs, including kinesin family member 1B isoform alpha, hypermethylated in cancer 2 and pleomorphic adenoma gene 1 [
22].
Costinean
et al. showed that E(mu)-mmu-
miR-155 transgenic mice exhibited preleukemic pre-B-cell proliferation evident in spleen and bone marrow, followed by frank B-cell malignancy. Their findings indicated that
miR-155 could induce polyclonal expansion, favoring the capture of secondary genetic changes for full transformation [
23]. Using miRNA cloning and qRT-PCR of mature miRNAs, Fulci
et al. demonstrated that
miR-21 and
miR-155 were dramatically overexpressed in CLL patients [
24]. Besides, Roldo
et al. showed that the expression of
miR-103 and
miR-107, associating with a lack of
miR-155 expression, could discriminate pancreatic tumors from normal pancreas. Their results suggested that the alteration in miRNA expression was related to endocrine and acinar neoplastic transformation [
25].
Felli
et al. demonstrated that treatment of CD34+ progenitors with
miR-221 and
miR-222 caused impaired proliferation and accelerated differentiation of erythropoietic cells, coupled with the downmodulation of Kit protein. The decline of
miR-221 and
miR-222 during exponential erythropoietic growth unblocked Kit protein production at mRNA level, thus leading to expansion of early erythroblasts [
26]. He
et al. also showed that
miR-146,
miR-221 and
miR-222 distinguished unequivocally between papillary thyroid carcinoma (PTC) and normal thyroid. The upregulation of these miRNAs was strongest associated with a dramatic loss of Kit transcript and its protein. Sequence changes in genes targeted by these miRNAs could contribute to the regulation of Kit involved in PTC pathogenesis [
27]. Analyzing the genome-wide miRNA expression profile in human PTCs using microarray, Pallante
et al. detected a significant increase of
miR-181b,
miR-221 and
miR-222 in the comparison of PTCs with normal thyroid tissue. Further confirmation by Northern blot analysis and qRT-PCR, their results suggested miRNA deregulation as an important event in thyroid cell transformation [
28].
The analysis of both glioblastoma tissues and glioblastoma cell lines allowed Ciafre
et al. to identify a group of miRNAs whose expression was significantly altered in the tumor.
miR-221 was strongly upregulated, whereas
miR-128,
miR-181a,
miR-181b and
miR-181c were downregulated in glioblastoma [
29]. Pekarsky
et al. discovered that the expression levels of
miR-29 and
miR-181 were inversely correlated with
Tcl1 expression in CLL. Their results showed that
miR-29 and
miR-181 might be candidates for therapeutic agents in CLL overexpressing
Tcl1 [
30]. By
in silico analysis, Mott
et al. identified a putative target site in the
Mcl1 mRNA and found that
miR-29b was downregulated in malignant cells, consistent with Mcl1 protein upregulation. Enforced
miR-29b expression reduced Mcl1 protein expression in the KMCH cholangiocarcinoma cells, thus
miR-29 was an endogenous regulator of Mcl1 protein expression [
31].
Examined by RT-PCR, Bandres
et al. identified 13 significantly deregulated mature miRNAs in colorectal cancer, including
miR-31,
miR-96,
miR-133b,
miR-135b,
miR-145 and
miR-183. In addition, the expression level of
miR-31 was correlated with the stage of colorectal cancer [
32]. Akao
et al. found that
miR-143 and
miR-145 expression levels were extremely reduced in the colon cancer cells. The transfection of each precursor miRNA into the cells demonstrated a significant growth inhibition in human colon cancer DLD-1 and SW480 cells, whereas
Erk5 was determined to be the target gene of
miR-143 [
33].
Laneve
et al. showed that
miR-9,
miR-125a and
miR-125b acted in an additive manner by repressing the truncated isoform of the neurotrophin receptor tropomyosin-related kinase C. They found that the downregulation of this isoform was critical for regulating neuroblastoma cell growth [
34].
In vitro functional studies of neuroblastoma cell lines indicated that
miR-184 played a significant role in apoptosis. Chen and Stallings suggested that neuroblastoma derived
Myc myelocytomatosis viral related oncogene might mediate a tumorigenic effect through directly or indirectly regulating the expression of miRNAs that were involved with neural cell differentiation and/or apoptosis [
35].
Welch
et al. showed that
miR-34a on chromosome 1p36.23 was generally expressed at lower levels in unfavorable primary neuroblastoma tumors relative to normal tissue.
miR-34a directly targeted the mRNA encoding E2F3 and significantly reduced E2F3 protein levels. Their results suggested that
miR-34a acted as a suppressor of neuroblastoma tumorigenesis [
36]. Chang
et al. showed that
miR-34a was frequently absent in pancreatic cancer cells. They demonstrated that this miRNA was directly transactivated by
p53.
miR-34a-responsive genes were highly enriched for those that regulated cell-cycle progression, apoptosis, DNA repair and angiogenesis. It was likely that an important function of
miR-34a was the modulation and fine-tuning of the gene expression program initiated by
p53 [
37].
Nervi
et al. found that the expression level of
miR-223 was correlated with the differentiation fate of myeloid precursors. The activation of both pathways of transcriptional regulation by the myeloid lineage-specific transcription factor CCAAT/enhancer-binding protein-α (C/EBPa) and posttranscriptional regulation by
miR-223 appeared essential for granulocytic differentiation and clinical response of acute promyelocytic leukemia blasted to ATRA [
38]. Using miRNA microarray platform and qRT-PCR, Garzon
et al. reported the expression of miRNAs in acute promyelocytic leukemia patients and cell lines during ATRA treatment. They found upregulation of
miR-107 targeted nuclear factor 1-A, a gene involving
miR-223 and C/EBPa in a regulatory loop during granulocytic differentiation. Besides, ATRA downregulation of
Ras and
Bcl2 was shown to correlate with the activation of
let-7a miRNA [
12].
Johnson
et al. showed that the
let-7 miRNA family negatively regulated
let-60/Ras. Loss of
let-60/Ras suppressed
let-7 miRNA complementary sites, restricting reporter gene expression in a
let-7-dependent manner.
let-7 miRNA expression was lower in lung tumors than in normal lung tissue, while Ras protein was significantly higher in lung tumors, providing a possible mechanism for
let-7 miRNA in cancer [
39]. Akao
et al. found that the levels of Ras and c-Myc proteins in
let-7 miRNA low-expressing DLD-1 human colon cancer cells were lowered after the transfection with
let-7a-1 precursor miRNA, whereas the levels of both of their mRNAs remained almost unchanged. Their findings suggested the involvement of
let-7 miRNA in the growth of colon cancer cells [
40]. Meng
et al. demonstrated that
let-7a miRNA was upregulated and contributed to the survival effects of enforced Interleukin-6 activity in malignant human cholangiocytes. It contributed to the constitutively increased phosphorylation of the signal transducers and activators of transcription-3 factors by a mechanism involving neurofibromatosis 2 [
41]. Brueckner
et al. showed that the human
let-7a-3 precursor miRNA on chromosome 22q13.31 was associated with a CpG island. They identified
let-7a-3 precursor miRNA as an epigenetically regulated miRNA gene in lung adenocarcinomas with oncogenic function and suggested that aberrant miRNA gene methylation might contribute to the human cancer epigenome [
42].
Mayr
et al. reported that the chromosomal translocations associating with human tumors disrupted the repression of High mobility group A2 (
Hmga2) by
let-7 miRNA. They found that the loss of miRNA-directed repression of an oncogene provided a mechanism for tumorigenesis, and disrupting a single miRNA-target interaction could produce an observable phenotype in mammalian cells [
43]. Lee and Dutta also demonstrated that ectopic expression of
let-7 miRNA reduced
Hmga2 and cell proliferation in lung cancer cell. Their results suggested that some tumors activated the oncogene through chromosomal translocations that eliminated the oncogene's 3' untranslated region with the
let-7 miRNA target sites [
44]. Hebert
et al. reported that
Hmga2 expression in head and neck squamous cell carcinoma cells was regulated in part by
miR-98. They showed that the transfection of pre-
miR-98 during normoxia diminished
Hmga2 and potentiate resistance to doxorubicin and cisplatin. Their findings implicated the role of
miR-98 as a key element in modulating tumors during hypoxia [
45].
Stem cells have the ability to escape cell cycle stop signals, which are similar to cancer cells. On the basis of cell cycle markers and genetic interactions, Harfield
et al. reported that dicer-1 mutant germline stem cells were delayed in the G1 to S transition, which was dependent on the cyclin-dependent kinase inhibitor dacapo, suggesting that miRNAs were required for stem cells to bypass the normal G1/S checkpoint. The miRNA pathway might be part of a mechanism that made stem cells insensitive to environmental signals that normally stop the cell cycle at the G1/S transition [
46].