Micro-RNA Associated With Rheumatoid Arthritis
a technology of rheumatoid arthritis and microrna, which is applied in the field of microrna associated with rheumatoid arthritis, can solve the problems of difficult diagnosis of rheumatoid arthritis accurately, insufficient diagnosis method, and development of biological formulations (anti-tnf antibodies), and achieve the effect of suppressing inflammation and growth of ra synovial
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reference example 1
Expression Profile of miRNA in RA Synovial Cells
[0045]Synovial cells were obtained through the approval of an ethics committee from a patient with RA diagnosed in conformity with the standard of the American College of Rheumatology and a patient with osteoarthritis (OA) as a control who had given informed consent (RA synovial cells (RA-FLS) and OA synovial cells (OA-FLS)). Each of the cells were cultured and maintained in an RPMI medium containing 10% FCS and 4 mM L-glutamine. Small RNAs having 200 bases or less were extracted from the above-mentioned cells (mirVana™miRNA Bioarrays, manufactured by Ambion) to perform miRNA expression analysis. The small RNAs were extracted in accordance with a method known per se. The extracted 156 kinds of miRNAs were subjected to quantification by real-time RT-PCR (TaqMan® MicroRNA Assays manufactured by Applied Biosystems) and a ΔCt method.
[0046]The results confirmed that six kinds of miRNAs different by a factor of two or more in expression amou...
example 1
Expression of miRNA in RA-FLS
[0047]300 pmol of pre-miR-124a or control pre-miRNA (Ambion) were added to 1 mL of an OptiMEM medium (Invitrogen) together with 10 μL of RNAiMAX (Invitrogen). After that, the mixture was added to a plate having a size of 60 mm, and the temperature was kept at room temperature for 20 minutes. After that, 5 ml of an RA-FLS cell suspension (1×105 cells / ml) in an RPMI medium were seeded to the plate, and the mixture was cultured in a CO2 incubator for 24 to 96 hours. Quantification was performed by miR-124a-specific real-time RT-PCR (TaqMan® MicroRNA Assays, manufactured by Applied Biosystems) and a ΔCt method.
[0048]The results confirmed that the expression amount of miR-124a was high as compared to the expression amount of control miRNA in each time of culture as a result of the addition of an equal amount of pre-miRNA (FIG. 2).
experimental example 1
Influences of miR-124a on Growth of Various Cells
[0049]Influences of miR-124a on the growth of various cells were examined.
[0050]In the same manner as in Example 1, an examination was made on influences on the growth of cells in the case where the respective cells, OA-FLS (n=2), RA-FLS (n=4), E11 cells, and HeLa cells were transfected with 300 pmol of pre-miR-124a or control pre-miRNA (Ambion) and cultured in a CO2 incubator for 24 to 96 hours.
[0051]The measurement results of the degree of the growth of cells by an XTT method using a Cell Proliferation Kit II (manufactured by Roche Applied Science) confirmed that the growth of cells was clearly suppressed in RA-FLS, OA-FLS, E11, and HeLa cells, transfected with pre-miR-124a (FIG. 3).
[0052]Also in the case where the number of cells was measured by a trypan blue method, as illustrated in FIG. 4, it was confirmed that, in RA-FLS cells, the growth of cells was clearly suppressed in the cells transfected with pre-miR-124a.
[0053]Further, ...
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