Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial hyperplasia and excessive inflammatory cell infiltration in the joints, leading to erosion of the articular cartilage and bone margins, with subsequent destruction of the joint (1). Despite an explosion of studies on inflammation over the last 2 decades, the detailed mechanism of synovial hyperplasia and inflammation is not well understood. Recently, a rapid proliferation of synovial cells, overexpression of inflammatory genes, and impairment of apoptosis were noted to play a role in the persistence of abnormal cells involved in the disease process (2–4). Several studies have shown that an antiapoptotic protein, particularly in Bcl-2, is highly up-regulated in RA synovial fibroblasts as compared with osteoarthritis synovial fibroblasts and that the apoptotic process in RA synovial fibroblasts may be suppressed by the overexpression of Bcl-2 (5–7).
The microRNA (miRNA) are a family of noncoding RNAs that are believed to be important in many biologic processes through regulation of gene expression. Many miRNA are evolutionarily conserved across phyla. MicroRNA are single-stranded RNA molecules of ∼22 nucleotides in length. They play a crucial role in the regulation of gene expression by inhibiting protein translation or degradation of target messenger RNA (8). MicroRNA exhibit tissue-specific or developmental stage–specific patterns of expression and are associated with human diseases, such as cancer, leukemia, and viral infections (9, 10). Depending on the disease, the expression of miRNA is up-regulated in some conditions and down-regulated in others (11, 12). It is therefore possible that miRNA might be a novel therapeutic target for human diseases.
The results of several therapeutic trials examining the regulation of endogenous miRNA that are related to disease pathogenesis through in vivo administration of specific antisense oligoribonucleotides or double-stranded miRNA have been reported (13–15). Recently, miRNA-15a (miR-15a) was reported to down-regulate the expression of Bcl-2 by inhibiting protein translation, which leads to cell apoptosis (16). It was therefore hypothesized that the overexpression of miR-15a in the synovium plays a role in the treatment of RA by inducing cell apoptosis in the synovium due to Bcl-2 dysfunction.
The purpose of the present study was to investigate whether double-stranded miR-15a administered by intraarticular injection could be taken up by the cells in vivo, thus inducing cell apoptosis, in the synovium of arthritic mice.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
Recently, miRNA have attracted attention because there is evidence of miRNA functioning in human diseases; therefore, miRNA might be a novel therapeutic target for human diseases. For example, the expression of let-7 has been shown to be lower in cancerous lung tissue than in normal lung tissue, resulting in high levels of expression of the Ras gene (20). MicroRNA-146 is intensely expressed in RA synovial tissue (11). Therefore, regulation of the endogenous miRNA that are associated with human diseases could open the door to a new therapeutic strategy. Therapeutic trials aimed at silencing miRNA in vivo have been described (13, 14). Tazawa et al (15) reported that local injection of double-stranded miR-34a complexed with atelocollagen could suppress tumor growth in mice.
In the present study, we demonstrated the successful transfection of double-stranded miR-15a complexed with atelocollagen into synovial cells in the knee joints of arthritic mice. Atelocollagen has been reported to be a carrier biomaterial for gene delivery both in vitro and in vivo. Atelocollagen-mediated siRNA or miRNA delivery has been reported to be effective in gene silencing following local injection directly into tumors and to be effective in treating bone-metastatic tumors following intravenous injection because atelocollagen complexed with siRNA is resistant to nuclease and can be efficiently transduced into cells (15, 21). Schiffelers et al (22) reported that local electroporation of siRNA for tumor necrosis factor α in joint tissues could inhibit collagen-induced arthritis in mice by local interference with RNA. In the current study, we injected atelocollagen-mediated double-stranded miRNA into the knee joint because intraarticular injection is a method that is both simple and commonly used in the clinical setting. We found that synthetic double-stranded miRNA complexed with atelocollagen was efficiently taken up by cells and was functional in cells after intraarticular injection without electroporation. This technique may therefore serve as a useful tool for studying the function of miRNA through the overexpression of specific miRNA in arthritic disease.
In contrast, after injection of the double-stranded miR-15a–atelocollagen complex, miR-15a uptake by the liver was also noted, whereas the lung, spleen, kidney, and heart showed no significant uptake. The uptake of miR-15a in these internal organs in the experimental group was not as high as that in the knee joint. In addition, there was no difference in the expression of caspase 3 in the liver of mice in the experimental group and the control group, as determined by immunohistochemistry (data not shown). However, a part of the injected double-stranded miRNA dose may have moved from the joint cavity into the systemic circulation. These results must be addressed before clinical studies using systemic miRNA can be undertaken.
It has been reported that the function of miR-15a is in the down-regulation of Bcl-2 protein expression and the promotion of cell apoptosis without affecting the stability of messenger RNA (16). Bcl-2 is a central player in the genetic program of eukaryotic cells, favoring survival by inhibiting cell death (23). Bcl-2 is essential to the processes of apoptosis because it suppresses the initiation of the cell death process. In addition, overexpression of Bcl-2 protein has been reported in many types of human cancers, including leukemia, lymphoma, and carcinoma (24). The apoptotic process in RA synovial fibroblasts might be suppressed by the overexpression of Bcl-2. In the current study, miR-15a was down-regulated in arthritic mouse synovium and a high level of Bcl-2 expression was observed.
To determine whether an immune response to miRNA does or does not induce apoptosis, we used miR-124 (a neuron-specific miRNA) as the control miRNA. Cell apoptosis was not observed in arthritic synovium following the intraarticular injection of miR-124 (data not shown). Therefore, no innate immune response to miRNA, which then induced cell apoptosis, was detected.
This trial successfully induced cell apoptosis by the intraarticular injection of double-stranded miR-15a and inhibited the translation of Bcl-2 protein in arthritic synovium. The limitation of this study is that it was not possible to demonstrate a complete therapeutic effect, such as the remission of arthritis. Because the pathogenesis of arthritis is quite complicated, it would be impossible to resolve the condition simply by inducing cell apoptosis via inhibition of the Bcl-2 cascade. It would require a cocktail of several double-stranded miRNA or antisense oligoribonucleotides that would regulate inflammation, cell proliferation, etc. The current study showed the possibility of a new strategy for inducing the overexpression of miRNA in vivo by the intraarticular injection of double-stranded miRNA into the joint. Further study is needed, however, before clinical studies can be begun.
- Top of page
- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Nakasa had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Nagata, Nakasa, Ochi.
Acquisition of data. Nagata, Nakasa, Ishikawa, Shibuya, Yamasaki.
Analysis and interpretation of data. Nagata, Nakasa, Mochizuki, Miyaki, Adachi, Asahara.