**5.1. Targeting oncogenic miRNAs**

The oncomiRs could be blocked by different approaches such as (i) antisense oligonucleotides, (ii) antagomirs, (iii) locked nucleic acid (LNA) constructs or (iv) sponges [145]. Antisense oligonucleotides work as competitive inhibitors of miRNA, leading to the up-regulation of tumour suppressor proteins, inducing apoptosis and blocking tumour formation in vitro and in vivo. Some of the potential ways to enhance miRNA stability include chemical modifications. In order to improve their effectiveness and stability, antisense oligonucleotides, have been modified on their 50 end, by adding 20-O-methyl and 20-O-methoxyethyl groups [146]. Oligonucleotides with 2′-O-methyl groups have proved to be effective inhibitors of miRNA expression in several cancer cell lines [30,147,148,149]. The utility of anti-miRNA oligonucleotide in vivo through intravenous injection of modified anti-miRNA oligonucleotide O-methyl-modified cholesterol-conjugated single stranded RNA analogues has been studied, with phosphorothioate linkages, an 'antagomirs', to target the liver-specific miR-122. Specific miR-122 silencing for up to 23 days was conferred with only a single injection of 240 mg•kg-1 body weight [150].

On the other hand, the locked-nucleic-acid antisense oligonucleotides exhibit relatively low toxicity and have been optimized by reducing their molecular size which has increased their therapeutic potential [151]. LNA anti-miR' constructs have been used successfully in several in vitro studies to knock down specific miRNA expression [151,152]. Also it has showed that miR-221 and miR-222 knockdown through antisense LNA oligonucleotides increases p27Kip1 in human prostate cancer (PC3) cells and strongly reduces their clonogenicity in vitro [153]. In vivo, the use of LNAs has achieved unexpected success for the treatment of hepatitis C in non-human primates [154]. These finding demonstrate the impressive potential of this strategy to overcome a major hurdle for clinical miRNA therapy.

Moreover, other techniques have emerged as an effective way to repress expression levels of miRNA families. A new form of miRNA inhibitors that can be transiently expressed in cultured mammalian cells, "miRNA sponges", was developed. Sponges are ectopically expressed mRNAs that contain multiple miRNA target sites of miRNA that share the same seed sequence [155]. In contrast to miRNA sponges, Xiao et al. designed alternative strategy called "miRNA masking" which covers up the miRNA-binding site to depress its target mRNA [156].

Although several in vitro and in vivo technologies have been developed to inhibit the oncomiRs, it is still a long and arduous way to go for substantial applications of miRNAs in cancer treatments. To date, many OncomiRs seem to have a role in apoptosis (Table 5), therefore some have been regarded as hallmarks in tumour progressions and hot targets in cancer therapy. Thus, miR-21 is functionally considered oncogenic because it is overexpressed in various tumours [157,158]. In fact, antisense inhibition of miR-21 leads to the induction of programmed cell death in neuroepithelial cells, through activation of caspases [159]. This apoptosis induction was also confirmed in breast cancer, colon cancer, pancreas cancer, lung cancer, liver cancer, prostate cancer, stomach cancer and oral squamous cell carcinoma (OSCC) [31,160,161,162,163]. So far, multiple targets of miR-21 have been identified and mapped to anti-apoptotic signalling pathways which suggest a promising miRNA treatment for cancer [31,164]. Other examples have been reported; in breast cancer cells, the overexpresion of the anti-apoptotic Bcl-2 is restored by the silencing of miR-15a and miR-16 through the use of specific inhibitors [165]. As the anti-apoptotic Bcl-2 is frequently overexpressed in a number of human cancers, such as Hodgkin's lymphoma, cell lymphoma and breast, miR-15 and miR-16, it could be used for therapy of cancer-associated phenotypes.


**Table 5.** Key microRNAs involved in apoptosis

130 Apoptosis and Medicine

them are strongly related to the apoptosis phenomenon. Therefore, manipulation of miRNA expression levels which target genes and pathways that are involved in apoptosis could be a potential therapeutic strategy for developing efficient therapies against cancer. In addition, given that cancer cells often exhibit a distinctive pattern of miRNA expression, unique profiles of altered miRNAs expression could be useful as molecular biomarkers for tumour diagnosis, prognosis of disease-specific outcomes, and evaluation of tumour aggressiveness. Based on this, several anticancer therapies focusing on restoring miRNA activities and repairing gene

There are two strategies of molecular therapy targeted at miRNA, one by the inhibition of oncogenic miRNAs and the other the over-expression of tumour suppressor miRNAs.

The oncomiRs could be blocked by different approaches such as (i) antisense oligonucleotides, (ii) antagomirs, (iii) locked nucleic acid (LNA) constructs or (iv) sponges [145]. Antisense oligonucleotides work as competitive inhibitors of miRNA, leading to the up-regulation of tumour suppressor proteins, inducing apoptosis and blocking tumour formation in vitro and in vivo. Some of the potential ways to enhance miRNA stability include chemical modifications. In order to improve their effectiveness and stability, antisense oligonucleotides, have been modified on their 50 end, by adding 20-O-methyl and 20-O-methoxyethyl groups [146]. Oligonucleotides with 2′-O-methyl groups have proved to be effective inhibitors of miRNA expression in several cancer cell lines [30,147,148,149]. The utility of anti-miRNA oligonucleotide in vivo through intravenous injection of modified anti-miRNA oligonucleotide O-methyl-modified cholesterol-conjugated single stranded RNA analogues has been studied, with phosphorothioate linkages, an 'antagomirs', to target the liver-specific miR-122. Specific miR-122 silencing for up to 23 days was conferred with

On the other hand, the locked-nucleic-acid antisense oligonucleotides exhibit relatively low toxicity and have been optimized by reducing their molecular size which has increased their therapeutic potential [151]. LNA anti-miR' constructs have been used successfully in several in vitro studies to knock down specific miRNA expression [151,152]. Also it has showed that miR-221 and miR-222 knockdown through antisense LNA oligonucleotides increases p27Kip1 in human prostate cancer (PC3) cells and strongly reduces their clonogenicity in vitro [153]. In vivo, the use of LNAs has achieved unexpected success for the treatment of hepatitis C in non-human primates [154]. These finding demonstrate the impressive

Moreover, other techniques have emerged as an effective way to repress expression levels of miRNA families. A new form of miRNA inhibitors that can be transiently expressed in cultured mammalian cells, "miRNA sponges", was developed. Sponges are ectopically expressed mRNAs that contain multiple miRNA target sites of miRNA that share the same seed sequence [155]. In contrast to miRNA sponges, Xiao et al. designed alternative strategy called "miRNA

potential of this strategy to overcome a major hurdle for clinical miRNA therapy.

masking" which covers up the miRNA-binding site to depress its target mRNA [156].

regulatory networks or drug sensitivity are being developed.

only a single injection of 240 mg•kg-1 body weight [150].

**5.1. Targeting oncogenic miRNAs** 
