MicroRNAs as Therapeutic Targets
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《新英格兰医药杂志》
New revelations about small RNA molecules continue to rivet molecular biologists. Lurking in cells among the thousands of messenger RNA (mRNA) transcripts that direct the synthesis of proteins, microRNAs function to inactivate specific mRNAs and deplete their corresponding proteins (Figure 1). The microRNAs average only about 22 nucleotides in length and are transcribed from DNA as hairpin precursors. They regulate such major processes as development, apoptosis, cell proliferation, hematopoiesis, and patterning of the nervous system.2,3,4,5 Thus, the identification of the actions of microRNAs adds new layers of complexity to our understanding of human biology.
Figure 1. Silencing MicroRNAs with Antagomirs.
MicroRNAs are transcribed from endogenous DNA and form hairpin structures (called pre-microRNAs) that are processed by an enzyme to form mature microRNA duplexes that are about 21 to 23 nucleotides long. A protein complex called RNA-induced silencing complex (RISC) allows the antisense strand of the microRNA to couple with matching messenger RNA (mRNA) sequences at 3' untranslated regions (the bulge in the microRNA denotes a region found in microRNAs that is not complementary to the mRNA). The binding of the microRNA to mRNA disrupts the translation or processing of the message, thereby disrupting the expression of the protein. In a recent study, Krützfeldt and colleagues1 prepared a cholesterol-linked single-stranded RNA, or antagomir, complementary to miR-122, a microRNA that is highly expressed in the liver. When injected into mice, antagomir-122 caused the depletion of miR-122 and decreased plasma cholesterol levels. Thus, miR-122 may down-regulate a repressor of genes in the cholesterol biosynthetic pathway, and antagomir-122 may enhance the expression of the repressor, which in turn inhibits the transcription of cholesterol-synthesizing enzymes. In other words, antagomir-122 may counter a brake (orange) on the production of a transcriptional repressor protein.
Several studies have shown that the introduction of a small, exogenous inhibitory RNA into animal models of disease abrogates molecular pathology and, in some cases, the disease itself. Now, a study has shown that inhibition of miR-122, a specific microRNA that is highly expressed in the liver, can be therapeutic in mice. Using a pharmacologic approach (Figure 1), Krützfeldt and colleagues1 synthesized single-stranded 23-nucleotide RNA molecules complementary to the targeted miR-122 in such a way as to stabilize the RNA and protect it from degradation. Next, the stabilized 23-mer RNAs were covalently linked to cholesterol molecules, aiding their delivery into liver cells. On injection of these cholesterol-conjugated RNA molecules — termed "antagomirs" — into the tail veins of mice, efficient and specific ablation of the targeted endogenous miR-122 was observed.
How might antagomirs cause the depletion of targeted microRNAs? The data provided by Krützfeldt et al. are not conclusive but provide clues. For example, when high levels of miR-122 and antagomir-122 duplexes were present in liver cells, degradation products of these duplexes could be detected. This finding is in keeping with study results showing that gene-silencing mechanisms are triggered by short double-stranded RNA species — a process known as RNA interference.2,5 Thus, the simplest explanation for the effectiveness of the antagomir is its hypothetical ability to bind and promote rapid degradation of the target microRNAs by nucleases normally present in cells.
Control experiments show that treatment of mice with antagomir-122 selectively removes miR-122 but not other species of microRNA from the liver. Although lower levels have some effect, the highest dose of antagomir-122 — 240 mg per kilogram of body weight — was required for the complete depletion of miR-122. Remarkably, this loss of miR-122 was sustained for as long as 23 days after treatment, indicating that the antagomir has a durable effect. Furthermore, when miR-16, another microRNA species that is ubiquitously expressed, was targeted by the complementary antagomir-16, loss of miR-16 was observed in all tissues except brain. Consistent with this finding was the presence of antagomir-16 in the liver, kidney, lung, heart, fat, skin, colon, and skeletal muscle. Thus, an injection of antagomir into mice leads to broad distribution of the antagomir, which can then effectively silence the targeted microRNA in most tissues.
The most striking finding of this study is the physiological effect caused by the silencing of miR-122 through antagomir-122 in living mice: a 44 percent decrease in plasma cholesterol levels. The authors observed that miR-122 silencing results in an increase in expression of several hundred genes, including those that are normally repressed in hepatocytes — which indicates that miR-122 may help maintain the adult-liver phenotype by suppressing "nonliver" genes. As expected, many of these genes contain an miR-122 recognition sequence in their 3' untranslated regions and therefore can be bound directly by miR-122 and disabled (Figure 1). Conversely, about 300 genes were down-regulated in response to antagomir-122. The mechanism of this down-regulation is unknown but could reflect the suppression by miR-122 of a hypothetical transcriptional repressor. (In this case, antagomir-122 would enhance the expression of the repressor, which in turn would turn off the expression of certain genes.) At least 11 genes involved in cholesterol biosynthesis, including the rate-limiting enzyme 3-hydroxy-3-methylglutaryl–coenzyme A-reductase, were among the genes that were down-regulated by antagomir-122.
The potential for ultimately designing molecular medicines based on the modulation of microRNAs seems good. Because we know that microRNAs can regulate malignant cells, insulin secretion, viral infection, and the neuronal phenotype, diseases such as cancer,4 diabetes, and hepatitis3 are hypothetical targets of these therapeutic strategies. This potential, however, is countered by formidable practical hurdles. Years of research will be required to identify ways to enhance the potency and stability of therapeutic agents and their delivery to specific tissues in an effort to modulate microRNAs to defeat diseases in humans. In addition, unwanted cellular responses to such therapeutic agents may occur over the long term. If we are lucky, some of our most talented basic and clinical scientists will rise to the challenge of solving these problems.
Dr. Czech reports receiving consulting fees and research support and holding equity in CytRx. No other potential conflict of interest relevant to this article was reported.
Source Information
From the University of Massachusetts Medical School, Worcester.
References
Krützfeldt J, Rajewsky N, Braich R, et al. Silencing of microRNAs in vivo with `antagomirs.' Nature 2005;438:685-689.
Zamore PD, Haley B. Ribo-gnome: the big world of small RNAs. Science 2005;309:1519-1524.
Sullivan CS, Ganem D. MicroRNAs and viral infection. Mol Cell 2005;20:3-7.
Croce CM, Calin GA. miRNAs, cancer, and stem cell division. Cell 2005;122:6-7.
Eckstein F. Small non-coding RNAs as magic bullets. Trends Biochem Sci 2005;30:445-452.(Michael P. Czech, Ph.D.)
Figure 1. Silencing MicroRNAs with Antagomirs.
MicroRNAs are transcribed from endogenous DNA and form hairpin structures (called pre-microRNAs) that are processed by an enzyme to form mature microRNA duplexes that are about 21 to 23 nucleotides long. A protein complex called RNA-induced silencing complex (RISC) allows the antisense strand of the microRNA to couple with matching messenger RNA (mRNA) sequences at 3' untranslated regions (the bulge in the microRNA denotes a region found in microRNAs that is not complementary to the mRNA). The binding of the microRNA to mRNA disrupts the translation or processing of the message, thereby disrupting the expression of the protein. In a recent study, Krützfeldt and colleagues1 prepared a cholesterol-linked single-stranded RNA, or antagomir, complementary to miR-122, a microRNA that is highly expressed in the liver. When injected into mice, antagomir-122 caused the depletion of miR-122 and decreased plasma cholesterol levels. Thus, miR-122 may down-regulate a repressor of genes in the cholesterol biosynthetic pathway, and antagomir-122 may enhance the expression of the repressor, which in turn inhibits the transcription of cholesterol-synthesizing enzymes. In other words, antagomir-122 may counter a brake (orange) on the production of a transcriptional repressor protein.
Several studies have shown that the introduction of a small, exogenous inhibitory RNA into animal models of disease abrogates molecular pathology and, in some cases, the disease itself. Now, a study has shown that inhibition of miR-122, a specific microRNA that is highly expressed in the liver, can be therapeutic in mice. Using a pharmacologic approach (Figure 1), Krützfeldt and colleagues1 synthesized single-stranded 23-nucleotide RNA molecules complementary to the targeted miR-122 in such a way as to stabilize the RNA and protect it from degradation. Next, the stabilized 23-mer RNAs were covalently linked to cholesterol molecules, aiding their delivery into liver cells. On injection of these cholesterol-conjugated RNA molecules — termed "antagomirs" — into the tail veins of mice, efficient and specific ablation of the targeted endogenous miR-122 was observed.
How might antagomirs cause the depletion of targeted microRNAs? The data provided by Krützfeldt et al. are not conclusive but provide clues. For example, when high levels of miR-122 and antagomir-122 duplexes were present in liver cells, degradation products of these duplexes could be detected. This finding is in keeping with study results showing that gene-silencing mechanisms are triggered by short double-stranded RNA species — a process known as RNA interference.2,5 Thus, the simplest explanation for the effectiveness of the antagomir is its hypothetical ability to bind and promote rapid degradation of the target microRNAs by nucleases normally present in cells.
Control experiments show that treatment of mice with antagomir-122 selectively removes miR-122 but not other species of microRNA from the liver. Although lower levels have some effect, the highest dose of antagomir-122 — 240 mg per kilogram of body weight — was required for the complete depletion of miR-122. Remarkably, this loss of miR-122 was sustained for as long as 23 days after treatment, indicating that the antagomir has a durable effect. Furthermore, when miR-16, another microRNA species that is ubiquitously expressed, was targeted by the complementary antagomir-16, loss of miR-16 was observed in all tissues except brain. Consistent with this finding was the presence of antagomir-16 in the liver, kidney, lung, heart, fat, skin, colon, and skeletal muscle. Thus, an injection of antagomir into mice leads to broad distribution of the antagomir, which can then effectively silence the targeted microRNA in most tissues.
The most striking finding of this study is the physiological effect caused by the silencing of miR-122 through antagomir-122 in living mice: a 44 percent decrease in plasma cholesterol levels. The authors observed that miR-122 silencing results in an increase in expression of several hundred genes, including those that are normally repressed in hepatocytes — which indicates that miR-122 may help maintain the adult-liver phenotype by suppressing "nonliver" genes. As expected, many of these genes contain an miR-122 recognition sequence in their 3' untranslated regions and therefore can be bound directly by miR-122 and disabled (Figure 1). Conversely, about 300 genes were down-regulated in response to antagomir-122. The mechanism of this down-regulation is unknown but could reflect the suppression by miR-122 of a hypothetical transcriptional repressor. (In this case, antagomir-122 would enhance the expression of the repressor, which in turn would turn off the expression of certain genes.) At least 11 genes involved in cholesterol biosynthesis, including the rate-limiting enzyme 3-hydroxy-3-methylglutaryl–coenzyme A-reductase, were among the genes that were down-regulated by antagomir-122.
The potential for ultimately designing molecular medicines based on the modulation of microRNAs seems good. Because we know that microRNAs can regulate malignant cells, insulin secretion, viral infection, and the neuronal phenotype, diseases such as cancer,4 diabetes, and hepatitis3 are hypothetical targets of these therapeutic strategies. This potential, however, is countered by formidable practical hurdles. Years of research will be required to identify ways to enhance the potency and stability of therapeutic agents and their delivery to specific tissues in an effort to modulate microRNAs to defeat diseases in humans. In addition, unwanted cellular responses to such therapeutic agents may occur over the long term. If we are lucky, some of our most talented basic and clinical scientists will rise to the challenge of solving these problems.
Dr. Czech reports receiving consulting fees and research support and holding equity in CytRx. No other potential conflict of interest relevant to this article was reported.
Source Information
From the University of Massachusetts Medical School, Worcester.
References
Krützfeldt J, Rajewsky N, Braich R, et al. Silencing of microRNAs in vivo with `antagomirs.' Nature 2005;438:685-689.
Zamore PD, Haley B. Ribo-gnome: the big world of small RNAs. Science 2005;309:1519-1524.
Sullivan CS, Ganem D. MicroRNAs and viral infection. Mol Cell 2005;20:3-7.
Croce CM, Calin GA. miRNAs, cancer, and stem cell division. Cell 2005;122:6-7.
Eckstein F. Small non-coding RNAs as magic bullets. Trends Biochem Sci 2005;30:445-452.(Michael P. Czech, Ph.D.)