siRNA for inhibiting expression of FABP4 target gene and application of siRNA
A FABP4, gene expression technology, applied in the direction of DNA / RNA fragments, retroRNA virus, application, etc., can solve difficult gene silencing and other problems, achieve the effect of increasing HDL-C level and reducing plaque formation
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Embodiment 1
[0040] Example 1 Design and synthesis of siRNA targeting human, monkey and golden hamster FABP4
[0041] The FABP4 mRNA sequences of humans, golden hamsters and monkeys were obtained from Genebank by bioinformatics methods (RefSeqIDs are: NM_001442.2; XM_013110923.1; NM_001266067.1), and DNA man (V6) software was used to find mRNA conserved region sequences. The RNA structure analysis software was used to predict the secondary structure of the FABP4 mRNA sequence, and based on the siRNA design principles, the GC content, the secondary structure of the mRNA action site, and the base preference requirements of the siRNA were comprehensively analyzed. On the basis of siRNA online design software DSIR design, we further searched for suitable siRNA target sequences, and preliminarily screened out 4 pairs of siRNA sequences. Then use the BLAST software provided by NCBI GeneBank to select the transcript reference sequence database (Transcript Reference Sequences), and perform homolog...
Embodiment 2
[0058] Example 2 Screening of siRNA targeting FABP4 in humans, monkeys, and golden hamsters
[0059] 1. Resuscitate human mononuclear THP-1 cells, and culture them completely with RPMI-1640 containing 10% FBS. Based on a 37°C, 5% CO2 cell incubator, observe under a microscope that they are in a suspended state, and cultivate until the confluence reaches about 80%. Passaging at a ratio of 1:3. Add PMA to the passaged cell suspension to 100nM, inoculate in 6-well plate (10 5 cells / well), add 2mL RPMI-1640 complete medium to each well, culture at 37°C, 5% CO2 for 24h, and observe the cells in an adherent state under an inverted microscope, confirming that THP-1 cells have been induced to differentiate into macrophages.
[0060] 2. Use Lipofectamine 3000 to transfect 50nM siNC or siFABP4-144 or siFABP4-149 or siFABP4-150 or siFABP4-151 into the above induced differentiated human macrophages. After 72 hours of transfection, detect the expression of FABP4 protein in the cells by We...
Embodiment 3
[0061] Example 3 siFABP4-144 inhibits the expression and secretion of inflammatory factors downstream of human macrophage FABP4
[0062] After Lipofectamine 3000 transfected human macrophages with siFABP4-144 (5, 10, 30, 50, 100 nM) for 72 hours, Western blot and ELISA were used to detect the expression and secretion of FABP4 downstream inflammatory factor proteins in the cells, respectively. The results showed that siFABP4-144 dose-dependently inhibited the expression and secretion of FABP4 downstream inflammatory factors IL-6, IL-1β and TNF-α protein, while reducing the secretion of MCP-1 protein ( figure 2 A and B shown).
[0063] Human macrophages were transfected with 50 nM siFABP4-144, and after 24, 48, and 72 hours, Western blot and ELISA were used to detect the expression and secretion of inflammatory factor proteins downstream of FABP4 in the cells, respectively. The results showed that siFABP4-144 time-dependently inhibited the expression and secretion of FABP4 dow...
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