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5.2 Potential oncomir candidates
We combined the miRNA expression profile to identified human cancer-related miRNAs which are highly tissue-specifically expressed or differentially expressed in particular tissues (Table 5.4). Those miRNAs may play essential roles to maintain stability of human cellular mechanism in particular tissues.
Table 5.4 Human cancer-related miRNAs which are differentially expressed in certain tissues.
To understand the regulatory mechanism of miRNAs in cancer study, we identified complex interactions among transcription factors, miRNAs and their target genes. All the cis-regulatory elements and miRNA target genes had been annotated their molecular function by using Gene Ontology (59). Related information of gene function in different cancer are referred to the literatures.
Cancer miRNA Up/down Ref
Hepatocellular carcinoma hsa-mir-122 Down Kutay et al., 2006 Hepatocellular carcinoma hsa-mir-30e* Down Budhu et al., 2008 Hepatocellular carcinoma hsa-miR-92a-1 Down Meng et al., 2007 Hepatocellular carcinoma hsa-mir-148a Down Budhu et al., 2008
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5.3 Results
We identified 20 oncomirs of eight cancer types from 205 cancer-related miRNAs. Those oncomirs are most important than other cancer-related miRNAs because their features related to tissue specificity and can directly regulate downstream genes to result oncogenesis.
The regulatory networks were constructed by predicting the cis-regulatory elements and miRNA targets. Some experimental validations can support our predictions. The dotted red lines are our predictions in the networks and solid red lines indicate that putative regulatory relationships are verified by experimental data in published references. The solid black lines are the accomplished works in other studies and we integrated them in our networks.
5.3.1 Hepatocellular carcinoma
Four oncomirs: miR-122, miR-148a, miR-30e* and miR-92 are downregulated in hepatocellular carcinoma (HCC). Figure 5.3 shows the regulatory networks of those oncomirs. Several cis-regulatory elements of oncomirs are down-expressed (T1 stage>
T3 stage) and may result their targets dowregulated. The gene NR3C1 is a receptor for glucocorticoids that can act as both a transcription factor and as a regulator of other transcription factors. The glucocorticoids can attenuate the response of estrogen which is a risk factor for breast cancer (90). Besides, it controls all the oncomiRs in HCC in our prediction and the dowregulated of NR3C1 may infect the expression of oncomiRs.
Recent study indicates TCF7L2 which had been thought to boost malignant cell growth instead as a transcriptional repressor that restricts colorectal cancer (CRC) cell growth (13). It is one of putative target of transcription factor and tumor suppressor TP53 in TRANSFAC database.
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The overexpression of transcription factors and nuclear factor of activated T-cells c1 (NFATC1) will activate its target gene autotaxin (ATX). ATX is known to stimulate migration of tumor cells (91).
One of the miR-122 targets in our prediction, ADAM17 (a disintegrin and metalloprotease 17) have been experimentally verified, miR-122 can silence ADAM17 and resulted reduction of migration, invasion of tumors in the livers of nude mice (88).
Three oncomirs: miR-122, miR-148 and miR-30e*, can silence transcription factor SOX4 and miR-122 can also silence NRP1 which is one of SOX4 targets and plays an important role in tumor metastasis of mouse livers (88).
Figure 5.3 Oncomir regulatory network in HCC, all the oncomiRs and their cis-regulatory elements are downregulated and oncomir target genes are overexpressed in microarray data.
5.3.2 Brain cancer
Figure 5.4 shows the regulatory network in brain cancer, miR-181a-1 and
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miR-181a-2 are down regulated and miR-221 is upregulated in this study. The human GSTP1 gene is frequently overexpressed in many human cancers and the expression increases with tumor progression, the presence of a cAMP response element (CRE) in the 5'-region of the human GSTP1 gene, raising the possibility that the cAMP signaling pathway to active CRE (Ser133) binding protein-1 (CREB1) (92). CREB1 is one of the cis-regulatory elements of miR-221 in our prediction. Overexpression of miR-221 was shown to cause downregulation of cyclin-dependent kinase (CDK) inhibitors CDKN1C/p57 and promoted cell cycle progression (93).
hsa-mir-181a functions as tumor suppressors in human glioma cells (94).
Downexpression of hsa-mir-181a may cause upregulation of oncogenes like CKS1B, FOS and ADM.
Figure 5.4 miR-181a-1 and -2 are downregulated and miR-221 is upregulated in brain cancer.
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5.3.3 Colon cancer
The miR-200 family of miRNAs plays a major role in specifying the epithelial phenotype by preventing expression of the transcription repressors, ZEB1(95). ZEB1 is a putative cis-regulatory element of miR-215, miR-194-1, miR-194-2 and miR-192 (Figure 5.5). ZEB1 and their target oncomirs are downregulated in colon cancer.
CD47 and MYLK are common targets of 4 oncomirs in colon cancer. Myosin light chain kinase 2, skeletal muscle (MYLK2) encodes a calcium/calmodulin-dependent serine/threonine kinase. In a recent study, MYLK2 gene was somatically mutated in colorectal carcinomas. The aim of this study was to explore the possibility that other common human carcinomas besides colorectal carcinomas harbored MYLK2 mutations in the kinase domain. (96). MYLK2 is also specifically related to prostate carcinoma (97). This enzyme catalyzes the phosphorylation of a specific serine residue on the 20 kD light chain of myosin II (MCL20), consequently regulating the actin-myosin II interaction. since MLC20 phosphorylation is necessary for cell motility, MYLK inhibition blocks cancer cell invasion and adhesion in vitro. As a result, some reports described the use of MYLK inhibitors as anti-cancer agents since they prevent cancer cells migration (97).
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Figure 5.5 Oncomirs miR-215,mIR-194-1,miR-194-2 and miR-192 are downregulated in colon cancer, the miR-200 family may be involved in their upstream regulation.
5.3.4 Breast cancer
TFAP4 is the cis-regulatory element of miR-205 and miR-126 which are downregulated in breast cancer (Figure 5.6). The interesting is that the oncogene c-FOS or the name FOS in HGNC symbol was downregulated in the network of breast cancer.
It plays a role in a variety of physiological functions including cell proliferation and differentiation, but in recent studies have raised the idea that c-Fos may also have tumour-suppressor activity and might have a function in apoptosis(98).
In recent studies, the team led by Joan Massagué (23) found that miR-335, miR-126, and miR-206 are metastasis-suppressor miRNAs. To identify these miRNAs, they compared miRNA expression of the metastatic nodules versus the unselected breast cancer parental cells. These miRNAs were consistently downregulated in metastatic
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tumors. Moreover, the authors found that restoring the expression of miR-335, miR-126, and miR-106 significantly decreased the number of metastatic foci. The cytometry analysis from Zhang et al. (99) showed that mir-126 targeted IRS-1 and inhibited cell cycle progression from G1/G0 to S. RPS6KB1 is amplified and overexpressed in 10-30% of primary breast cancers and breast cancer cell lines (100-102). This gene is a serine/threonine kinase and plays a crucial role in control of cell cycle, growth and survival.
Figure 5.6 miR-205 and miR-126 are downregulated in breast cancer.
5.3.5 Lung cancer
The oncomir miR-142 was upregulated in lung cancer. The regulatory network is shown in Figure 5.7. TFAP2A and CREB1 can activate miR-142 expression in our prediction. Recent studies have shown that AP-2alpha (TFAP2A) and AP-2gamma (TFAP2C) have tumor-suppressive activity in breast cancer, melanoma, and prostate cancer cells. Decreased expression of TFAP2A or TFAP2A has been found in these
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tumor cells and is associated with disease progression and the metastatic capabilities of the tumors (103). But in lung cancer, upregulated expression of TFAP2A activates human telomerase reverse transcriptase (hTERT) which is hallmark of tumorigenesis in lung cancer (104).
The miR-142 target, LATS2, is one of LATS tumour suppressor family which plays an important role in the control of tumour development and cell cycles (105).
LATS1 and LATS2 are functionally conserved and regulate the cell cycle progression and apoptosis. LATS1 is implicated in the regulation of the cell cycle at the G2/M and LATS2 at the G1/S phases (106).
Figure 5.7 miR-142 is upregulated In lung cancer.
5.3.6 Ovarian cancer
FOS (c-Fos) is the putative cis-regulatory element of oncomir miR-125b in ovarian cancer. Mahner found that loss of FOS expression is associated with tumour progression in ovarian carcinoma and that FOS may be a prognostic factor (107). The downregulation of miR-125b may cause upregulation of oncogenes like BCL2, VEGFA
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and MUC1 in our prediction (Figure 5.8).
The 3 potential miR-125b targets are involved oncogenesis. BCL2 (for B cell lymphoma gene-2) proteins are associated with membranes and membrane activity. The BCL2 protein is a part of a complex system of signaling that controls apoptosis. Bufalo et al. shows that overexpression of BCL2 enhances the metastatic potential of a human breast cancer (94). Vascular endothelial growth factor A (VEGFA) is critical to angiogenesis. Evidence shows that VEGFA stimulates angiogenesis in tumor growth and mediates neuroprotection to prevent an apoptotic cell death (108). Mucin core protein 1 (MUC1) is associated with invasive growth of neoplasms (109). It is emphasized in most cases of carcinoma and high expression of MUC1 is closely associated with cancer progression and metastasis (110).
Figure 5.8 miR-125b is downregulated in ovarian cancer.
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5.3.7 Prostate cancer
In prostate cancer, miR34a and miR99a are down regulated and miR-92a is upregulated (Figure 5.9). One of the cis-regulatory element of miR-34a and miR-99a, JUNB is an essential upstream regulator of p16 which is a tumor suppressor protein (111), and contributes to maintain cell senescence that blocks malignant transformation.
Gene expression profiling showed the cis-regulatory element of miR-92a, HSD17B4, to be significantly overexpressed in prostate cancer compared to matched-benign epithelium (112). This gene also overexpressed in metastatic samples compared to primary tumor samples (69).
Gene IGF1R, the type 1 insulin-like growth factor receptor, is the target of both miR-99a and miR34a. IGF1R is overexpressed in prostate cancer, and mediates proliferation, motility, and survival, silencing of the IGF1R gene enhances sensitivity to DNA-damaging agents in both PTEN wild-type and mutant human prostate cancer (113). E2F transcription factors, including miR-34a target gene E2F3, directly modulate expression of EZH2. Recently, overexpression of the EZH2 gene has been implicated in the development of human prostate cancer (114). Another miR-34a target, DDX17 (p72) may affect the transcriptionalactivity of ß-catenin in colon cancer cell and promote
c-Myc, cyclin D1, c-jun, and fra-1, all of which are
proto-oncogenes (19). DUSP5 is a direct target of p53, it represents a novel mechanism by which p53 might negatively regulate cell-cycle progression (115).88
Figure 5.9 miR34a and miR99a are down regulated and miR-92a is upregulated in prostate cancer.
5.3.8 Pancreatic cancer
Figure 5.10 shows the regulatory network in pancreatic cancer, miR-92a is upregulated and miR-375 is downregulated. E2F1 and ETV4 are two cis-regulatory elements of miR-92a. E2F1 is a transcriptional factor that mediates cell cycle progression from G1 to S phase, thereby influencing tumor progression. ETV4 is one of ETS-family factors play major roles in development and cancer, notably as critical targets for extra-cellular signaling pathways, including MAPK-signaling, ETV4 also involves in pancreatic development (116).
Most pancreatic cancers correspond to ductaladenocarcinoma (DAC), which
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develops from epithelium in a multistepprocess(117), the miR-375 target gene MUC4 is a transmembrane mucin expressed in pancreatic ductal adenocarcinoma (DAC) in contrast to normal pancreas, and is an independent predictor of poor prognosis in patients with invasive DAC (118). Recent study shows transcription factor TFAP2A can repress MUC4 (118) and TFAP2A is also cis-regulatory element of miR-375 gene.
The miR-92 target gene FBN2, a large modular extracellular matrix glycoprotein, is known to be a key component of human elastic fiber. A loss of FBN2 expression due to promoter methylation was recently identified in pancreatic cancer (119).
Figure 5.10 Upregulation of miR-92a and downregulation of miR-375 in pancreatic cancer.
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