**3. Functions of miRNAs**

The general function of miRNA is oriented towards gene silencing [3, 4]. miRNAs specifically recognize mRNA and downregulate gene expression by one of the two posttranscriptional mechanisms: (1) translational repression and (2) mRNA cleavage. The determinant of the regulatory mechanism process is mainly dependent on the degree of

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**Figure 1.**

*Therapeutic Implication of miRNA in Human Disease DOI: http://dx.doi.org/10.5772/intechopen.82738*

miRNA–mRNA complementarity. If there is a high degree of complementarity between the miRNA and mRNA, it will enable the Ago-catalyzed degradation of target mRNA sequences through the mRNA cleavage mechanism process. However, if there is a low degree of degree of miRNA–mRNA complementarity, a central mismatch will omit

*miRNA biogenesis, function, and strategies for miRNA-based therapies. miRNA is transcribed from miRNA gene via RNA polymerase II as pri-miRNA and cleaved by Drosha complex in the nucleus. The resulting precursor miRNA (pre-miRNA) is exported to the cytoplasm via exportin 5 complex. In the cytoplasm, Dicer complex cleaves pre-miRNA to form mature miRNA duplex. The strand is separated and the functional strand is loaded into the RISC complex. The function of miRNA is depending on the complementarity of the seed region of mature miRNA to the 3'UTR of the target mRNA gene, either undergoing mRNA cleavage or translational repression. Strategies for miRNA-based therapies: improving miRNA in disease can be achieved by the following approaches: (a) Small molecule miRNA inhibitors can regulate miRNA expression at the transcriptional level. (b) Antisense oligonucleotides can bind to the target miRNA and induce degradation effect. (c) The miR-mask oligonucleotides are synthetic oligonucleotides complementary to the 3' UTR target mRNA that compete with endogenous miRNA for its target. (d) The miRNA sponges are oligonucleotide constructs with multiple complementary miRNA binding sites to the target miRNA. (e) The miRNA mimics are synthetic miRNAs which can restored the downregulated miRNA expression. (f) The AAV miRNA vectors are a group of adenovirus-associated vectors that have been inserted genes coding for miRNAs and they are used for restoring downregulated miRNA expression.*

The exact mechanism for translational repression by miRNA is still not fully understood. However, recent studies suggest that, as the miRNA is incorporated into a RISC [3, 4], the associated protein silencing complex can either repress translational mechanisms typically associated with ribosomal translation, or induce deadenylation of the 3′ poly-A protective posttranscriptional mRNA modification, thought to be involved in repression via the mRNA 5′ terminal cap. The mechanism for mRNA degradation is mainly involved in endonucleolytic cleavage, which is facilitated by Argonaute cleavage proteins. It has been shown that when miRNAs have a high degree of sequence complementarity, then target mRNA degradation processes are facilitated through Ago protein slicer activity [2, 3]. miRNA typically binds to the 3′ untranslated region (3' UTR) in mRNA that follows the translation termination codon. The mechanism of miRNA translation inhibition requires partial sequence match, whereas the mechanism of miRNA-mediated mRNA degrada-

degradation and promotes the translational repression mechanism.

tion requires a near-perfect complementary match (see **Figure 1**).

#### **Figure 1.**

*Antisense Therapy*

involving miRNAs.

**2. miRNA biochemical synthesis**

about 70–100 nucleotides, called pre-miRNA.

eventually efficacy studies of modified inhibitors of miRNAs in primates in 2010 illustrates the explosion of research surrounding miRNAs in just 5 years. There are now over 2000 miRNAs that have been discovered in humans and it is believed that they collectively regulate one third of the genes in the genome [2]. miRNAs have been linked to many human diseases and are being pursued as clinical diagnostics and as therapeutic targets, showing promise in many fields, ranging from cancer therapy to cardiac disease, to even suggestions as a potential biomarker for numerous diseases and treatment responses. This chapter will briefly discuss the miRNAs biogenesis, their function, regulation, and implication in disease, then discuss the miRNA-based therapeutic strategies, their therapeutic implication in diseases, and some of the current clinical trials

miRNAs are encoded in the genomes (inter or intragenic) and are transcribed from genes located in nuclear DNA; however, such genes are not eventually translated into protein [3]. These transcribed genes are typically longer than the eventual gene product miRNA and undergo much post-transcriptional modification between initial transcription and the functional miRNA end-product. After initial transcription of the DNA sequence, the miRNA sequence contains a reverse-complement base pair segment that forms a double stranded RNA hairpin loop. The entire DNA transcript, including the double stranded RNA loop, constitute the primary miRNA structure (called pri-miRNA). Pri-miRNA is usually several kilobases long and has local stem loop structures. The primary transcripts undergo further processing in the nucleus. The ribonucleases Drosha and DiGeorge syndrome critical region gene 8 (DGCR8) complex are mainly involved in the pri-miRNA processing, which is cleaved at the stem of the hairpin structure and generates a hairpin intermediate of

The pre-miRNA is then transported out of the nucleus to the cytoplasm for further processing to become mature miRNA. There are nuclear pore complexes in the nuclear membrane where the pre-miRNA can be transported out of the nucleus by means of the RanGTP-dependent nuclear transport receptor exportin 5. In the cytoplasm, the pre-miRNA is processed by another ribonuclease, Dicer to create a mature miRNA. The mature miRNA is a double-stranded miRNA of variable length (~18–25 nucleotides). After the generation of mature miRNA duplex by Dicer, the miRNA duplex is incorporated into an Ago family protein complex, which generates an effector complex. Then one strand of the miRNA is degraded, whereas the other strand remains bound to Ago as mature miRNA (guide strand). After strand separation, the guide strand or mature miRNA is incorporated into an RNA-induced silencing complex (RISC). After loading, the miRNA promotes the RISC to its target mRNA and induces mRNA degradation or translational repres-

The general function of miRNA is oriented towards gene silencing [3, 4]. miRNAs specifically recognize mRNA and downregulate gene expression by one of the two posttranscriptional mechanisms: (1) translational repression and (2) mRNA cleavage. The determinant of the regulatory mechanism process is mainly dependent on the degree of

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sion (see **Figure 1**).

**3. Functions of miRNAs**

*miRNA biogenesis, function, and strategies for miRNA-based therapies. miRNA is transcribed from miRNA gene via RNA polymerase II as pri-miRNA and cleaved by Drosha complex in the nucleus. The resulting precursor miRNA (pre-miRNA) is exported to the cytoplasm via exportin 5 complex. In the cytoplasm, Dicer complex cleaves pre-miRNA to form mature miRNA duplex. The strand is separated and the functional strand is loaded into the RISC complex. The function of miRNA is depending on the complementarity of the seed region of mature miRNA to the 3'UTR of the target mRNA gene, either undergoing mRNA cleavage or translational repression. Strategies for miRNA-based therapies: improving miRNA in disease can be achieved by the following approaches: (a) Small molecule miRNA inhibitors can regulate miRNA expression at the transcriptional level. (b) Antisense oligonucleotides can bind to the target miRNA and induce degradation effect. (c) The miR-mask oligonucleotides are synthetic oligonucleotides complementary to the 3' UTR target mRNA that compete with endogenous miRNA for its target. (d) The miRNA sponges are oligonucleotide constructs with multiple complementary miRNA binding sites to the target miRNA. (e) The miRNA mimics are synthetic miRNAs which can restored the downregulated miRNA expression. (f) The AAV miRNA vectors are a group of adenovirus-associated vectors that have been inserted genes coding for miRNAs and they are used for restoring downregulated miRNA expression.*

miRNA–mRNA complementarity. If there is a high degree of complementarity between the miRNA and mRNA, it will enable the Ago-catalyzed degradation of target mRNA sequences through the mRNA cleavage mechanism process. However, if there is a low degree of degree of miRNA–mRNA complementarity, a central mismatch will omit degradation and promotes the translational repression mechanism.

The exact mechanism for translational repression by miRNA is still not fully understood. However, recent studies suggest that, as the miRNA is incorporated into a RISC [3, 4], the associated protein silencing complex can either repress translational mechanisms typically associated with ribosomal translation, or induce deadenylation of the 3′ poly-A protective posttranscriptional mRNA modification, thought to be involved in repression via the mRNA 5′ terminal cap. The mechanism for mRNA degradation is mainly involved in endonucleolytic cleavage, which is facilitated by Argonaute cleavage proteins. It has been shown that when miRNAs have a high degree of sequence complementarity, then target mRNA degradation processes are facilitated through Ago protein slicer activity [2, 3]. miRNA typically binds to the 3′ untranslated region (3' UTR) in mRNA that follows the translation termination codon. The mechanism of miRNA translation inhibition requires partial sequence match, whereas the mechanism of miRNA-mediated mRNA degradation requires a near-perfect complementary match (see **Figure 1**).
