Study On Micrornas In Angiogenesis Biology Essay
MicroRNAs are small RNA molecules consist of about 22 nucleotides. They are non-coding RNAs that play an important role to function as negative regulators in gene expression. In early time, they are only regards as some tiny detritus in cells. But soon they become our research target since some studies have revealed that microRNAs are evolutionarily conserved across species . Till the end of 2010, Scientists have found and validated about 721 miRNAs in the human genome . However, the actual number of miRNAs should be greater than the above number. According to a prediction by computer programme, the number of miRNAs should be more than 1000, which may only be encompassing about 2.5% of the total number of genes . It is believed that about one third of human genes are regulated by these miRNAs . With the development of novel tools in identifying miRNAs, the number of miRNAs will definitely increase significantly . But still, more efforts are need in studying miRNAs since we know little about the miRNA regulatory system. Many identified miRNAs were discovered to be located commonly in cancer associated regions . Some researchers obtain clinical samples to further investigate, and they found out that clusters of miRNAs are expressed in cancerous cells . Recent studies have shown that tumor suppressor genes expression will be lowered when there is an upregulation of oncogenic miRNAs, on the other hand, oncogene translation will be increased when miRNAs downregulation increases. Therefore, a fully understanding on miRNAs will provide a novel way in cancer treatment and diagnosis.
Fig.2. Biogenesis of microRNA.
Nature Cell Biology 11, 228 - 234 (2009)
The above figure illustrates the biogenesis of miRNAs. First of all, the primary miRNA transcripts (pri-miRNA) are produced by RNA polymerase II or III. Then, the pri-miRNAs are cleaved by the Drosha–DGCR8 (Pasha) in the cell nucleus. The pre-miRNAs formed, are exported into the cell cytoplasm by Exportin-5–Ran-GTP. In cell cytoplasm, the RNase Dicer combines to the double-stranded RNA-binding protein (TRBP) and cleaves the pre-miRNA hairpin to form mature form. The functional strand of the mature miRNA is associated together with Argonaute (Ago2) proteins forming the RNA-induced silencing complex (RISC). Then, miRNA directs RISC to suppress target mRNAs by mRNA cleavage, translational repression or deadenylation.
Inhibition mechanism of translation by microRNAs
We know that there are three steps involved in translational inhibition: Initiation of ribosome, elongation and termination. Recent studies [5,6], have shown that the same mRNA distribution pattern across poly-ribosomes was maintained by miRNA-inhibited mRNAs compared with non-suppressed mRNAs. Models of post-initiation inhibition of mRNA translation have been established base on the above results. 
Studies have provided a way to distinguish between initiation and post-initiation inhibition. The method bases on the result of whether (IRES)-containing mRNAs were opposed to miRNA-mediated inhibition . However, the results obtained from different groups were incompatible.
In some researches, they showed that [8,15] IRES-initiated translation was influenced by miRNA-mediated repression, and thus eliminating 4E-cap recognition factor as a possible target for miRNA function.
Some studies [10,17] stated that the inhibitory step of miRNAs to their specific target mRNAs locates at the initiation. IRES-mediated translation was shown to have a less efficiency than m7G-capped mRNAs. Therefore the translational kinetics will be affected, which led to miRNA-initiated mRNA translational inhibition . Consequently, the origin of IRES sequence will become a factor affecting the protein requirements of host for IRES mediated translation initiation.
Some studies found supporting evidences on miRNA-mediated translational inhibition by examined reconstituted cell-free extracts extracted from rabbit reticulocytes , Drosophila  and mammalian cell lines [14,21]. The results indicated that repression requires the classic m7G 5' cap. This finding reveals that miRNA-initiated translational repression happened at the initiation step.
Moreover, researchers further investigate miRNA - initiated repression by supplying excess of the eIF4F complex, and they found out that miRNA - initiated repression was moderated  which further prove that miRNA - initiated translational repression happened at the initiation step.
In order to study miRNA and mRNA interactions, some novel methods were used in some researches, such as tethering Ago proteins to a 3' untranslated reading part without associating with miRNA. Translational repression was still occurred . It is important to further investigate the interaction between the Agos or Ago-associated proteins and translational machinery. There is a notion in some studies that Ago2 and eIF4E compete for binding site of the target mRNA cap structure and eventually inhibit translation initiation. This notion was supported by the finding that Ago2 in mammal can bind directly to the m7G-cap.
The above results led to the establishment of a model which is compatible with the results derived from the study of cell reconstitution. Therefore, it becomes an important evidence to support the idea that miRNAs block translational initiation.
Angiogenesis is the process in which new blood vessels form from pre-existing vessels. It is an essential natural process in the organisms involved in many physiological processes such as reproduction and healing. A precise balance of growth and inhibitory factors control the angiogenic process. Under unbalance condition, the result is either over-stimulation or under-stimulation on angiogenesis.
The body controls angiogenesis via a series of "on" and "off" switches. When the amount or concentration of angiogenic growth factors exceeds angiogenesis inhibitors, angiogenesis will be turned on. When inhibitors are produced in excess of stimulators, angiogenesis will be switched off. Under normal, angiogenic growth factors and angiogenesis inhibitors will be maintained in a balance condition. Normally, angiogenesis will be inhibited since there are more inhibitors released than stimulators.
In angiogenic process, angiogenic growth factors are released in injured or diseased tissues. The released angiogenic growth factors will then migrate to ECs and bind to the specific receptors present on them. ECS are then activate by them. Signals are generated and transmitted to the cell nucleus. Small holes are produced in the basement membrane surrounding blood vessels by the production of enzymes. The endothelial cells start to differentiate and migrate towards the diseased tissue. The new blood vessel sprout are pulled forward with the help of specialized adhesion molecules and the tissue around the vessel tip are dissolved by Matrix metalloproteinases. The tissue is rearranged around the newly formed vessel along the extending vessel. Afterwards, the newly arranged ECs are rolled up to form a new blood vessel tubes. Continuous blood vessel loops are then formed by connecting each vessel tube segments. Finally, the continuous blood vessel network formed and allow blood circulation.
Role of miRNAs in angiogenesis
MicroRNAs are believed to be involved in vascular development and the study of Dicer on animal model provides the first evidence on it. Dicer is an enzyme involved in the maturation process of microRNA. Researches artificially impair the Dicers in mice and the mice died at around 13 days after gestation . Mice with Dicer deficiency in endothelial cells shows a diminished response in angiogenesis after injecting vascular endothelial growth factor-A (VEGF-A). In the study of human endothelial cells, the ECs with with Dicer deficiency using silencing RNA showed a decrease in proliferation and endothelial sprouts formation. However, some pro-angiogenic factors expression increased after Dicer silencing. The reason may be due to the accompnying up-regulation of potent anti-angiogenic factors or Dicer-independent miRNA biogenesis pathway was involved. However, the above results or data only give an indirect evidence to show that miRNAs are involved in angiogenesis. But they do not show the specific miRNAs that is involved in and responsible for the observed results.
Studies found out that over-expression of miR-18a, miR-17-5p, and miR-20a restored EC proliferation in endothelial cells with Dicer deficiency [33,34].With the usage of zebrafishes and mice models, the role of individual miRNA in vascular angiogenesis is being revealed by miRNA genetic analysis. miR-126 was discovered to be involved in vascular integrity and hemorrhage during embryonic development by knocking down miR-126 in zebrafish . On the other hand, intersegmental vessel formation defection was induced in zebrafish with overexpressed miR-92a. In some studies, knowdown of miR-126 in ECs caused an 50% lethal rate in embryonic mice. Also, survivals showed a defection in postnatal retinal vascularization, which implies that miR-126 plays an important role in angiogenesis .
With the extensive studies on miRNAs recently, specific miRNAs are found out to be enriched in ECs including let-7b, miR-16, miR-21, miR-23a, miR-29, miR-100, miR-221, and miR-222. Moreover, miR-126 is discovered to be enriched in Flk-1+ cells. in a study, 2′-O-methyl oligonucleotide inhibitors was used in the blockage of in vitro angiogenesis, which showed that the highly expressed let-7f and miR-27b exert pro-angiogenic effects as evidenced by with. 
How miRNAs are regulated in in vascular cells? This is the main theme in two recent studies to investigate the above question in response to serum and hypoxia, respectively. miR-130a, a pro-angiogenic microRNA is expressed at low levels in HUVECs and is upregulated in the presence of foetal bovine serum. Growth arrest homeobox (GAX), an anti-angiogenic homeobox proteins and HoxA5 were down-regulated by miR-130a, and the inhibitory effects of GAX on ECs migration and tube formation were functionally antagonized and the inhibitory effects of HoxA5 on tube formation vitro was also inhibited. Studies showed that hypoxia induced the expression of miR-210 in endothelial cells.  Tube formation was enhanced by over-expressed MiR-210. On the other hand, inhibited miR-210 expression decreased cell migration tube formation. A receptor called tyrosine-kinase ligand EphrinA3 is found out to be involved in the regulation of ECs responses to hypoxia. Hypoxia inducible factor (HIF) was revealed in some studies that it induced a subgroup of hypoxia-regulated miRNAs. This finding supports HIF plays an important role as transcription factor for miRNA expression during hypoxia. It is important for us to understand the regulation of miRNAs in ECs.
miR-221 and miR-222 were found to be highly expressed in HUVECS. They exert anti-angiogenic effects. Therefore, cell migration, tube formation, and wound healing of ECs in vitro were inhibited in miR-221 and miR-222 transfected ECs. The down-regulation of the potential target protein c-kit (a receptor for stem cell factor) was involved in the regulation mechanism and without affecting the mRNA level. This indicates a post-transcriptional regulation. Endothelial nitric oxide synthase (eNOS) expression was induced by miR-221 and miR-222 overexpression in Dicer siRNA-transfected cells.  Nitric oxide (NO) plays a key role in the regulation of ECs growth, migration, and angiogenesis, Moreover, eNOS was discovered to be involved in stem and progenitor cells’ mobilization and activity.  The above findings shows that miRNAs targeting eNOS not only regulate angiogenic activities in HUVECS, but also may be involved in vasculogenesis.
Studies revealed that miRNAs may influence other miRNAs since the knockdown of miR-221 and miR-222 changed the HUVECs’ miRNA signature. Experiments found out that there are 9 miRNAs and 23 miRNAs were upregulated or downregulated respectively in response to miR-221/222 deficiency. Among these miRNAs, 1/3 of them were found out to target the same target (c-kit 3′UTR), which suggest a connection between miRNAs that share similar functions. But the underlying regulatory mechanism is not resolved. Some reports suggest that it might be due to the interference with the activity of the miRNA-processing machinery.  In recent studies, they provide data to demonstrate that Myc (a transcription factor) can promote or inhibit different miRNAs.
With the discovery of miRNAs in 1993, people starts to realize that these tiny RNA molecules play an important role in the vascular development, angiogenesis and cancer development. Recent years, there are many studies concerning about miRNAs. Although we have made progress, the role of miRNAs in angiogenesis and their involvement in other complicated biological processes are still needed to investigate. Moreover, clarifying the expression of miRNAs in ECs are important to fully understand the importance of miRNAs involved in different cells and tissues. But such expression pattern needs to be further analyzed, especially, the expression of miRNAs under proangiogenesis and anti-angiogenic conditions
On the other hand, we still haven’t fully understood the miRNAs expression mechanism and the details on their regulation in angiogenesis. In order to analyze the role of miRNAs in vascular development and angiogenesis, it is important identify the genetic and epigenetic components involved in these events. Also, researchers are more will to investigate the application of miRNAs in therapeutic purposes. This may be a breakthrough in the near future.
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