MicroRNAs as Oncogenes and Tumor Suppressors
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《新英格兰医药杂志》
Recent discoveries related to microRNAs, RNA interference, small interfering RNAs (siRNAs), and small modulatory RNAs have revealed a new class of mechanisms of gene regulation that are mediated by small, noncoding RNAs. Among these small RNAs are the microRNAs. These are thought to control gene expression at the post-transcriptional level by degrading or repressing target messenger RNAs (mRNAs). Surprisingly simple and elegant, microRNA-mediated gene regulation is guided by the base-pairing rules of Watson and Crick.
MicroRNAs are individually encoded by their own set of genes and are an integral component of the genetic program. Some are located in noncoding regions of the genome, whereas others occur in the introns (noncoding regions) of protein-coding genes. Furthermore, as shown in the figure, a complex set of proteins is required for the formation and function of microRNAs. Many of these proteins, as well as the microRNAs themselves, are found in a wide range of animal species and are important for development.
Biogenesis of MicroRNA and MicroRNA-Mediated Gene Regulation in Animal Cells.
Mature functional microRNAs of approximately 22 nucleotides are generated from long primary microRNA (pri-microRNA) transcripts. First, the pri-microRNAs, which usually contain a few hundred to a few thousand base pairs, are processed in the nucleus into stem-loop precursors (pre-microRNA) of approximately 70 nucleotides by the RNase III endonuclease Drosha and its partner Pasha, a homologue of the human DiGeorge syndrome critical region gene 8 (DGCR8). The pre-microRNAs are then actively transported into the cytoplasm by exportin 5 and Ran-GTP and further processed into small RNA duplexes of approximately 22 nucleotides by the Dicer RNase III enzyme and its partner Loqacious (Loqs), a double-stranded RNA-binding–domain protein that is a homologue of the human immunodeficiency virus transactivating response RNA-binding protein (TRBP). The functional strand of the microRNA duplex is then loaded into the RNA-induced silencing complex (RISC). Finally, the microRNA guides the RISC to the cognate messenger RNA (mRNA) target for translational repression or degradation of mRNA.
There is accumulating evidence that microRNA-mediated gene regulation has a broad impact on gene expression. First, microRNA genes constitute about 1 to 5 percent of the predicted genes in worms, mice, and humans — there may be as many as 1000 microRNA genes in the human genome. Second, microRNAs are expressed at high levels in animal cells and are dynamically regulated in response to physiological and developmental cues. Each type of cell is likely to have a specific microRNA milieu — defined by a unique set of microRNAs — that controls gene expression. Lastly, each microRNA has the potential to regulate a large number of target genes. According to recent computational predictions, as many as 200 target genes can be regulated by a single microRNA, which implies that over one third of protein-coding genes in humans are regulated by microRNAs. Thus, the microRNA milieu can modulate levels of protein expression by dampening the translation of thousands of mRNAs. Indeed, microRNA-mediated gene regulation is now considered a fundamental layer of genetic programs that operates at the post-transcriptional level. MicroRNAs play important roles in various biologic processes in worm, fly, fish, mouse, and human cells, where they regulate apoptosis, proliferation, differentiation, development, and metabolism. All these effects may occur by regulating the expression of signaling molecules, such as cytokines, growth factors, transcription factors, and proapoptotic and antiapoptotic genes, to name a few.
Given the global effect of microRNAs on gene expression, it is not surprising that small regulatory RNAs have been implicated in the pathogenesis of cancer. Some microRNA genes are in regions of translocations and deletions that occur in leukemia. The miR-15a–miR-16-1 cluster is located at chromosome 13q14, a region deleted in most cases of chronic lymphocytic leukemia (CLL), mantle-cell lymphoma, multiple myeloma, and prostate cancer; the miR-142 gene was found at the junction of the t(8;17) translocation, which may contribute to the progression of an indolent lymphoma into aggressive B-cell leukemia. In addition, many oncogenes and tumor-suppressor genes are potentially regulated by microRNAs. The expression profiles of microRNAs are frequently altered in tumors, and in some cases, a reduction in the expression of microRNA may cause increased expression of the oncogenic target genes. For example, the RAS oncogene is regulated by the let-7 microRNA; decreased expression of let-7 microRNA in some human lung tumors causes increased expression of the RAS oncogene and thereby may contribute to tumorigenesis.
There is also evidence that microRNAs can act as oncogenes or tumor suppressors. He et al. found that the miR-17-92 cluster of microRNAs, which are highly expressed in many malignant lymphomas containing amplified 13q31–32 chromosomal fragments, can augment the oncogenic effect of the c-Myc gene in mice when coexpressed with c-Myc in hematopoietic stem cells.1 Interestingly, O'Donnell et al. found that this cluster, when up-regulated by the expression of c-Myc, down-regulates the expression of the E2F1 protein — one of the transcriptional targets of c-Myc — at the translational level.2 These findings suggest that the miR-17-92 cluster acts as an oncogene under one set of conditions and as a tumor suppressor under another.1,2
A number of studies have demonstrated the power of analyses of microRNA expression profiles in the classification and diagnosis of cancer. Lu et al. developed a sensitive method of microRNA-expression profiling and demonstrated that such profiles can be highly effective in classifying cancers in humans and predicting the developmental origins of cancers.3 They demonstrated that microRNA microarrays are more effective in cancer classification than mRNA microarrays containing more than 16,000 protein-coding genes.
In this issue of the Journal, Calin and colleagues (pages 1793–1801) report their finding that mutations in microRNA genes are frequent in CLL. Intriguingly, many mutations were located in the flanking sequences of the pre-microRNA regions, and a cell-culture assay indicated that a point mutation of the miR-16-1 precursor abolishes the expression of the mature miR-16. These observations suggest that a loss of expression of microRNA contributes to the pathogenesis CLL. Calin et al. also performed analyses of microRNA expression profiles in 94 samples of CLL cells and discovered a microRNA signature might be associated with ZAP-70 expression and the mutation status of the immunoglobulin heavy-chain (IgVH) gene, both of which are known prognostic factors in CLL. All these studies suggest that microRNAs are important players in tumorigenesis and, of greater consequence, point to the crucial role of post-transcriptional genetic mechanisms that are mediated by microRNAs. These new insights concerning the function of microRNAs may lead to advances in the diagnosis and treatment of leukemia.
The systematic analysis of microRNA gene-expression profiles through the use of comprehensive microRNA microarrays will allow us to establish a microRNA taxonomy of cancer. The use of DNA microarrays to monitor the RNA profiles of tumor specimens has been tremendously successful in building a molecular taxonomy of cancer that can be used to identify new drugs, make a prognosis, and predict patterns of drug resistance. However, cellular behavior is also governed by translational and post-translational control mechanisms that are not reflected in mRNA profiles of tumor specimens. Since microRNAs regulate gene expression at the post-transcriptional level, the availability of a comprehensive microRNA expression profile could provide information that is complementary to that derived from mRNA transcriptional profiling.
MicroRNAs are evolutionarily selected gene regulatory molecules. Therefore, microRNA expression profiles may be particularly rich in gene regulatory information. In contrast, only a small percentage of the 16,000 genes on the mRNA-expression arrays are regulatory molecules. This difference may explain why Lu et al. found that microRNA-expression arrays are more efficient in classifying cancer than mRNA-expression arrays.3 However, a cautionary note is indicated. The sensitivity and specificity of current microRNA microarray techniques are limited, and the microRNA probes used in these analyses account for only a fraction of the microRNA genes in the genome — a substantial number of microRNAs have yet to be identified by cloning and sequencing. MicroRNAs that are rarely expressed but are functionally important in stem or progenitor cells may be underrepresented in the database because most of the microRNA cloning analyses used tissues or cultured cells.
Nevertheless, the identification of microRNAs that are potential tumor suppressors (tumor-suppressor microRNAs) or oncogenes (oncogenic microRNAs) may lead to the discovery of a new class of oncogenes and tumor suppressors. For example, a loss of expression of tumor-suppressor microRNA may lead to elevated levels of the protein products of target oncogenes. Conversely, overexpression of oncogenic microRNAs may reduce the levels of protein products of target tumor-suppressor genes. This scenario suggests that the regulatory mRNA target genes of the tumor-suppressor microRNAs and oncogenic microRNAs may be a new class of tumor suppressors and oncogenes, respectively, that are regulated by a post-transcriptional mechanism. If this proposition is correct, the identification and validation of the target genes of these microRNAs could reveal new targets for cancer diagnosis and therapy.
It is important to note that molecules that are regulated at the post-transcriptional level are likely to be missed by standard transcriptional profiling analyses involving mRNAs. Moreover, because the base-pairing of microRNAs with their target genes is imperfect, they represent a challenge to the computational and experimental analyses of microRNA target genes. Much remains to be learned about the molecular principles guiding the microRNA and target-gene interactions before we can confidently identify microRNA target genes and elucidate the molecular mechanisms through which the oncogenic and tumor-suppressor microRNAs regulate normal development and tumorigenesis.
Are microRNAs the magic cure for the diagnosis and treatment of cancer? We certainly hope so. But the struggle to combat cancer — including the discovery of oncogenes, tumor suppressors, telomerase, and cancer stem cells over the past quarter century — has revealed the complex nature of cancer in humans. The molecular definition of cancer seems to be constantly evolving as we learn more about the genetic components of cell growth, apoptosis, senescence, metastasis, and angiogenesis. The discovery of microRNA-mediated gene regulation as a fundamental genetic program at the post-transcriptional level adds yet another layer of complexity to cancer genetics. Nevertheless, understanding the molecular mechanisms by which microRNAs regulate normal development and tumorigenesis may lead to novel concepts in the diagnosis and treatment of cancer in humans.
Source Information
Dr. Chen is an assistant professor in the Department of Microbiology and Immunology, Baxter Laboratory of Genetic Pharmacology, Institute for Cancer/Stem Cell Biology and Medicine, Stanford University School of Medicine, Stanford, Calif.
References
He L, Thomson JM, Hemann MT, et al. A microRNA polycistron as a potential human oncogene. Nature 2005;435:828-833.
O'Donnell KA, Wentzel EA, Zeller KI, Dang CV, Mendell JT. c-Myc-regulated microRNAs modulate E2F1 expression. Nature 2005;435:839-843.
Lu J, Getz G, Miska EA, et al. MicroRNA expression profiles classify human cancers. Nature 2005;435:834-838.(Chang-Zheng Chen, Ph.D.)
MicroRNAs are individually encoded by their own set of genes and are an integral component of the genetic program. Some are located in noncoding regions of the genome, whereas others occur in the introns (noncoding regions) of protein-coding genes. Furthermore, as shown in the figure, a complex set of proteins is required for the formation and function of microRNAs. Many of these proteins, as well as the microRNAs themselves, are found in a wide range of animal species and are important for development.
Biogenesis of MicroRNA and MicroRNA-Mediated Gene Regulation in Animal Cells.
Mature functional microRNAs of approximately 22 nucleotides are generated from long primary microRNA (pri-microRNA) transcripts. First, the pri-microRNAs, which usually contain a few hundred to a few thousand base pairs, are processed in the nucleus into stem-loop precursors (pre-microRNA) of approximately 70 nucleotides by the RNase III endonuclease Drosha and its partner Pasha, a homologue of the human DiGeorge syndrome critical region gene 8 (DGCR8). The pre-microRNAs are then actively transported into the cytoplasm by exportin 5 and Ran-GTP and further processed into small RNA duplexes of approximately 22 nucleotides by the Dicer RNase III enzyme and its partner Loqacious (Loqs), a double-stranded RNA-binding–domain protein that is a homologue of the human immunodeficiency virus transactivating response RNA-binding protein (TRBP). The functional strand of the microRNA duplex is then loaded into the RNA-induced silencing complex (RISC). Finally, the microRNA guides the RISC to the cognate messenger RNA (mRNA) target for translational repression or degradation of mRNA.
There is accumulating evidence that microRNA-mediated gene regulation has a broad impact on gene expression. First, microRNA genes constitute about 1 to 5 percent of the predicted genes in worms, mice, and humans — there may be as many as 1000 microRNA genes in the human genome. Second, microRNAs are expressed at high levels in animal cells and are dynamically regulated in response to physiological and developmental cues. Each type of cell is likely to have a specific microRNA milieu — defined by a unique set of microRNAs — that controls gene expression. Lastly, each microRNA has the potential to regulate a large number of target genes. According to recent computational predictions, as many as 200 target genes can be regulated by a single microRNA, which implies that over one third of protein-coding genes in humans are regulated by microRNAs. Thus, the microRNA milieu can modulate levels of protein expression by dampening the translation of thousands of mRNAs. Indeed, microRNA-mediated gene regulation is now considered a fundamental layer of genetic programs that operates at the post-transcriptional level. MicroRNAs play important roles in various biologic processes in worm, fly, fish, mouse, and human cells, where they regulate apoptosis, proliferation, differentiation, development, and metabolism. All these effects may occur by regulating the expression of signaling molecules, such as cytokines, growth factors, transcription factors, and proapoptotic and antiapoptotic genes, to name a few.
Given the global effect of microRNAs on gene expression, it is not surprising that small regulatory RNAs have been implicated in the pathogenesis of cancer. Some microRNA genes are in regions of translocations and deletions that occur in leukemia. The miR-15a–miR-16-1 cluster is located at chromosome 13q14, a region deleted in most cases of chronic lymphocytic leukemia (CLL), mantle-cell lymphoma, multiple myeloma, and prostate cancer; the miR-142 gene was found at the junction of the t(8;17) translocation, which may contribute to the progression of an indolent lymphoma into aggressive B-cell leukemia. In addition, many oncogenes and tumor-suppressor genes are potentially regulated by microRNAs. The expression profiles of microRNAs are frequently altered in tumors, and in some cases, a reduction in the expression of microRNA may cause increased expression of the oncogenic target genes. For example, the RAS oncogene is regulated by the let-7 microRNA; decreased expression of let-7 microRNA in some human lung tumors causes increased expression of the RAS oncogene and thereby may contribute to tumorigenesis.
There is also evidence that microRNAs can act as oncogenes or tumor suppressors. He et al. found that the miR-17-92 cluster of microRNAs, which are highly expressed in many malignant lymphomas containing amplified 13q31–32 chromosomal fragments, can augment the oncogenic effect of the c-Myc gene in mice when coexpressed with c-Myc in hematopoietic stem cells.1 Interestingly, O'Donnell et al. found that this cluster, when up-regulated by the expression of c-Myc, down-regulates the expression of the E2F1 protein — one of the transcriptional targets of c-Myc — at the translational level.2 These findings suggest that the miR-17-92 cluster acts as an oncogene under one set of conditions and as a tumor suppressor under another.1,2
A number of studies have demonstrated the power of analyses of microRNA expression profiles in the classification and diagnosis of cancer. Lu et al. developed a sensitive method of microRNA-expression profiling and demonstrated that such profiles can be highly effective in classifying cancers in humans and predicting the developmental origins of cancers.3 They demonstrated that microRNA microarrays are more effective in cancer classification than mRNA microarrays containing more than 16,000 protein-coding genes.
In this issue of the Journal, Calin and colleagues (pages 1793–1801) report their finding that mutations in microRNA genes are frequent in CLL. Intriguingly, many mutations were located in the flanking sequences of the pre-microRNA regions, and a cell-culture assay indicated that a point mutation of the miR-16-1 precursor abolishes the expression of the mature miR-16. These observations suggest that a loss of expression of microRNA contributes to the pathogenesis CLL. Calin et al. also performed analyses of microRNA expression profiles in 94 samples of CLL cells and discovered a microRNA signature might be associated with ZAP-70 expression and the mutation status of the immunoglobulin heavy-chain (IgVH) gene, both of which are known prognostic factors in CLL. All these studies suggest that microRNAs are important players in tumorigenesis and, of greater consequence, point to the crucial role of post-transcriptional genetic mechanisms that are mediated by microRNAs. These new insights concerning the function of microRNAs may lead to advances in the diagnosis and treatment of leukemia.
The systematic analysis of microRNA gene-expression profiles through the use of comprehensive microRNA microarrays will allow us to establish a microRNA taxonomy of cancer. The use of DNA microarrays to monitor the RNA profiles of tumor specimens has been tremendously successful in building a molecular taxonomy of cancer that can be used to identify new drugs, make a prognosis, and predict patterns of drug resistance. However, cellular behavior is also governed by translational and post-translational control mechanisms that are not reflected in mRNA profiles of tumor specimens. Since microRNAs regulate gene expression at the post-transcriptional level, the availability of a comprehensive microRNA expression profile could provide information that is complementary to that derived from mRNA transcriptional profiling.
MicroRNAs are evolutionarily selected gene regulatory molecules. Therefore, microRNA expression profiles may be particularly rich in gene regulatory information. In contrast, only a small percentage of the 16,000 genes on the mRNA-expression arrays are regulatory molecules. This difference may explain why Lu et al. found that microRNA-expression arrays are more efficient in classifying cancer than mRNA-expression arrays.3 However, a cautionary note is indicated. The sensitivity and specificity of current microRNA microarray techniques are limited, and the microRNA probes used in these analyses account for only a fraction of the microRNA genes in the genome — a substantial number of microRNAs have yet to be identified by cloning and sequencing. MicroRNAs that are rarely expressed but are functionally important in stem or progenitor cells may be underrepresented in the database because most of the microRNA cloning analyses used tissues or cultured cells.
Nevertheless, the identification of microRNAs that are potential tumor suppressors (tumor-suppressor microRNAs) or oncogenes (oncogenic microRNAs) may lead to the discovery of a new class of oncogenes and tumor suppressors. For example, a loss of expression of tumor-suppressor microRNA may lead to elevated levels of the protein products of target oncogenes. Conversely, overexpression of oncogenic microRNAs may reduce the levels of protein products of target tumor-suppressor genes. This scenario suggests that the regulatory mRNA target genes of the tumor-suppressor microRNAs and oncogenic microRNAs may be a new class of tumor suppressors and oncogenes, respectively, that are regulated by a post-transcriptional mechanism. If this proposition is correct, the identification and validation of the target genes of these microRNAs could reveal new targets for cancer diagnosis and therapy.
It is important to note that molecules that are regulated at the post-transcriptional level are likely to be missed by standard transcriptional profiling analyses involving mRNAs. Moreover, because the base-pairing of microRNAs with their target genes is imperfect, they represent a challenge to the computational and experimental analyses of microRNA target genes. Much remains to be learned about the molecular principles guiding the microRNA and target-gene interactions before we can confidently identify microRNA target genes and elucidate the molecular mechanisms through which the oncogenic and tumor-suppressor microRNAs regulate normal development and tumorigenesis.
Are microRNAs the magic cure for the diagnosis and treatment of cancer? We certainly hope so. But the struggle to combat cancer — including the discovery of oncogenes, tumor suppressors, telomerase, and cancer stem cells over the past quarter century — has revealed the complex nature of cancer in humans. The molecular definition of cancer seems to be constantly evolving as we learn more about the genetic components of cell growth, apoptosis, senescence, metastasis, and angiogenesis. The discovery of microRNA-mediated gene regulation as a fundamental genetic program at the post-transcriptional level adds yet another layer of complexity to cancer genetics. Nevertheless, understanding the molecular mechanisms by which microRNAs regulate normal development and tumorigenesis may lead to novel concepts in the diagnosis and treatment of cancer in humans.
Source Information
Dr. Chen is an assistant professor in the Department of Microbiology and Immunology, Baxter Laboratory of Genetic Pharmacology, Institute for Cancer/Stem Cell Biology and Medicine, Stanford University School of Medicine, Stanford, Calif.
References
He L, Thomson JM, Hemann MT, et al. A microRNA polycistron as a potential human oncogene. Nature 2005;435:828-833.
O'Donnell KA, Wentzel EA, Zeller KI, Dang CV, Mendell JT. c-Myc-regulated microRNAs modulate E2F1 expression. Nature 2005;435:839-843.
Lu J, Getz G, Miska EA, et al. MicroRNA expression profiles classify human cancers. Nature 2005;435:834-838.(Chang-Zheng Chen, Ph.D.)