RNA interference: A novel Drug target

Abstract: Gene silencing is a natural process occurring in eukaryotes as a defencing mechanism to control RNA viral infections. Expression of various proteins is also regulated by this process. This natural phenomenon is recently being under studies to find if it can be used as new method to cure or diagnosis some diseases where a gene upregulation or downregulation is the cause of that disease. SiRNAs and miRNAs are being studied extensively for their use in treatment of several diseases like Cancers; inflammatory disorders etc.

Present review explains the use of these gene silencers in disease etiology and treatment.

Introduction:

Gene silencing is “switching off” a gene, which usually occurs in Eukaryotic organisms. Gene silencing results in blockade of gene expression so that translation is shunted. It is a self-defence mechanism of eukaryotic cells to overcome the infection by RNA viruses (De Paula, Bentley and Mahato 2007). This gene regulation can occur in transcriptional or post-transcriptional level. Transcriptional gene silencing is due to histone modifications, creating a heterochromatin environment around a gene being expressed and is made inaccessible to RNA polymerase, transcription factors etc. Post-transcriptional gene silencing is the destruction or blockade of mRNA of a particular gene preventing the expression of that gene. This naturally occurring phenomenon can be implemented in selectively switching off genes in some diseased states. It has potential for treating disease such as cancer in which an oncogen is over expressed. Different approaches have been developed in curing or

detecting diseases using gene silencing (Table 1). These include the use of some drugs, oligodeoxynucleotides, Ribozymes, DNA enzymes, and micro managers like siRNA and miRNAs (NCBI) as shown in Figure 1.

Antisense and RNA inhibition are gene knockdown technologies where protein synthesis is lost but the transcription of gene is unaffected. Protein synthesis is affected as the mRNA molecules become unstable or inaccessible.

The main double stranded RNAs involved in gene silencing are small interfering RNAs (siRNAs), micro RNAs (miRNAs), and short hairpin RNA (shRNA). All three types of RNA form RNA-induced silencing complex (RISC) and cleave the double stranded RNA resulting in mRNA degradation and post transcriptional gene silencing (Wang et al. 2010).

RNAi by Small interfering RNA:

SiRNAs are dsRNAs of 19-30 nucleotide length with 3’-overhangs of 2nt on both strands (De Paula, Bentley and Mahato 2007). These are capable of degrading the complementary mRNA (Wang et al. 2010). As illustrated in figure 2. The RNAi by siRNA involves the cleavage of 500-100 nucleotide length dsRNA into small fragments of siRNA by endoribonucleaseDicer. The formed siRNA are then incorporated into RNA-induced silencing complex (RISC), a protein complex. Dicer also helps in helps in entry of siRNA into RISC. Argonaute 2 (Ago-2) protein helps in cleavage and release of single stranded RNA from dsRNA. Formation of single stranded RNA activates RISC and directs complementary base pairing. Ago-2 then cleaves target mRNA and causes mRNA degradation and gene silencing (Wang et al. 2010 cited in Grimm 2009).

RNA interference by miRNA:

Micro RNAs (miRNAs) are small, noncoding sets of 19 to 24 nucleotides. These can regulate the expression of transcription factors, oncogenes, and tumour suppressor genes. The genes that encode miRNA are transcribed from DNA but are not translated. Bio genesis of miRNA involves the formation of primary transcripts (pri-miRNAs) by RNA polymerase II. Pri-miRNAs are then converted to pre-RNAs by RNase enzyme. Dicer cleaves the pre-miRNA into miRNA duplex (Ross, Carlson and Brock 2007)

RNAi by miRNA is obtained by one of three methods

1. Complementary binding and direct cleavage of the target mRNA.

2. Cleavage-independent mRNA degradation.

3. Direct or indirect blockade of protein synthesis.

Table 2: Comparison of siRNA and miRNA (modified from Ross, Carlson and Brock 2007)

SiRNA and miRNA in medicine

SiRNA: Implications in Asthma therapy:

Chemokines are proteins secreted by different types of cells during any invasion by micro-organisms. These proteins have the ability to induce chemotaxis in cells. These proteinsguide the lymphocytes to the site of infection and also help in wound healing. Based on the amino-terminal cysteine residue arrangement, these are divided into four groups. CC and CXC are two largest groups of Chemokines having adjacent cysteine amino acids or cysteine residues separated by a single amino acid moiety, respectively. In addition to their role in immune system, these are also used in pathogenesis of certain diseases including atherosclerosis, rheumatoid arthritis, chronic obstructive pulmonary disease (COPD),

rheumatoid arthritis, allergic rhinitis and asthma (Errahali et al. 2009 cited in Charo and Ransohoff 2006). Studies have shown that there exists a strong correlation between eosinophil (EOS) numbers in asthmatics. EOS selective Chemokines are involved in guiding the EOS towards the air passages in asthma. CCL11, CCL12, and CCl26 are eotaxins involved mainly in EOS trafficking through CC receptor three (CCR3). This receptor is highly expresses in human EOS. Of all the three eotaxins CCL26 plays a predominant role in eosinophilic pulmonary inflammation. It was found that Interleukin-4 (IL-4) upregulates CCR3 and expression and release of CCl24 and CCl26. CCl26 was believed to be involved in auto regulatory mechanism where in presence of IL-4, CCl24 and CCl26 protein expression was increased. Taka and others showed that multiple receptor/ligand pathways are involved in CCl26 sustained and increased secretion from cells during inflammatory conditions and CCl26 may trigger leukocyte trafficking to elicit disease symptoms.

In regard to CCL26’s role in asthma and its multiple receptor/ligand pathways, Errahali et al. hypothesized that gene silencing of CCl26 in Th2 cytokine-stimulated alveolar type II cells would down regulate CC Chemokines that bind to CCR3 and this would result in decrease in EOSs activation and trafficking. Using CCL26-siRNA-treated A549 alveolar type II cells system, Errahali et al. shown for the first time that siRNA-induced inhibition of CCL26 supressed IL4-stimulated release of CCl24. The results from bioassay illustrate that CCL26 gene silencing by siRNA may be used as a disease target for inflammatory diseases. 

Result: This experiment is carried out to verify the blocking of IL-4 stimulated CCL26 mRNA and protein expression by CCL26 targeted siRNA duplexes. Results demonstrated that siRNA duplexes 6 and 8 had decreased IL-4 stimulated CCL26 mRNA suppression and protein expression as shown in figure. CCR3 downregulation was also illustrated in Figure 3.Along with the CCL24 reduction, it was observed from the results that there is decrease in CCR3 receptor expression and the EOSs migration (Figures 4, 5, 6). Previous studies showed that CCL26 production stimulates the chemotaxis of EOSs, CCL24 production and CCR3 upregulation. The present experiment showed that siRNA can be promising target for Asthma treatment.

MiRNA: Implications in Pancreatic Cancer

High mortality rate is observed in patients with pancreatic cancer even after the treatment with existing therapies (Li et al. 2010). This is due to the ability of invasive behaviour of pancreatic cancer cells. It was shown that pancreatic cancer is associated with overexpression of epidermal growth factor receptor (EGFR) and activation of NF-κB (Li et al. 2010 cited in Xiong and Abbruzzese 2002). Overexpression of EGFR results in large tumour size and decreased

survival in pancreatic cancer (Li et al. 2010 cited in Yamanaka et al. 1993). However, the exact mechanism involved in overexpression of EGFR and NF- κB in pancreatic cancer is not fully elucidated. It was reported that miR-146a was lost in metastatic prostate cancer (Li et al. 2010 cited in Lin et al. 2008). The experiment performed by Li et al. is aimed to check the expression level of miR-146a in prostate cancer cells and to find if reexpression could inhibit the metastatic behavior of pancreatic cancer cells .

Results:

As shown in Figure 7 miRNA array and RT-PCR studies showed that there is higher expression of miR-14a in HPDE cells than that in Colo357 and Panc-1 pancreatic cancer cells, proves that there is lower expression of miR-14a in pancreatic cells than that of normal cells.To find if reexpression of miR-146a led inhibition of EGFR signalling, the Colo357 and Panc-1 cells were infected with pre micro RNAs and cultured for a period of 9 days. It was found that reexpression of miR-146a in the infected cells downregulated EGFR (figure 8 and figure 9). From the experiment conducted by Li et al. it can be understood that miR-146a inhibits invasion and metastasis through EGFR and NF-kB signaling regulation. It was also found from Sanger database that HGFR was a predicted target of miR-146a. Hurgs and colleagues reported that miRNA-146a and miR-146b downregulated the EGFR expression and inhibited breast cacner metastatis (Li et al. 2010 cited in Hurst et al. 2009)

Uses of siRNAs and miRNAs:

As discussed earlier siRNAs can be used to regulate expression of some proteins produced during immune respone. According to Liang et al. (2010) cycE siRNA shows anti-tumor activity via inhibition of DNA replication and promoting apoptosis. As discusses earliermiRNA can be used to control metastatis in pancreatic cancer cells. In carcinomas, cell adhesion proteins like claudins, cadherins, Integrins etc. play a vital role as their loss results in metastatis of of carcinoma. Loss of E-cadherin expression is responsible for invasive behaviour of various cancers of GI, endocrine, pulmonary systems. To date mir-9 was found to regulate the expressionof E-cadherin (Ross, Carlson and Brock 2007). Considering all available research data, we can expect that these gene silencing RNAs can be used to treat diseases and also for disease diagnosis.

Limitations:

Problems involved in RNA interference based therapies incude cellular uptake, off target effects, stability of gene silencers, susceptability to enzymes, pharmacokinetic properties, and immunological factors (De Paula, Bentley and Mahato 2007). Limitations and stategies to overcome these are not explained here as it beyond the scope of this review.

Conclusion:

In conclusion, siRNAs and miRNAs have wide range of applications in pharmaceutical industry. These Gene silencing strategies would be implemented to cure various acquired and chronic diseases.

References:

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