SaRNA for activating PRDM5 expression in gastric cancer cells and application thereof
A technology for gastric cancer cells and gastric cancer, applied in the direction of DNA/RNA fragments, recombinant DNA technology, retroRNA virus, etc., can solve the problem of lack of homocysteine methyltransferase, etc., and achieve the effect of inhibiting proliferation and migration
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Embodiment 1
[0028] Example 1: Design of saRNA of tumor suppressor gene PRDM5.
[0029] Aiming at the tumor suppressor gene PRDM5, which is abnormally silenced in gastric cancer cells, starting from -1kb upstream of the transcription start site (TSS), and selecting a target site with a size of 21bp, the coding DNA of saRNA was synthesized. The sense nucleic acid strand and antisense nucleic acid strand of saRNA exist on the same nucleic acid strand, which is a hairpin single-stranded nucleic acid molecule, in which the complementary regions of the sense nucleic acid strand and the antisense nucleic acid strand form a double-stranded nucleic acid structure.
[0030] 1#PSC93384:
[0031] 5′-CcggACCCTGCGGTCTAGGAAATTTctcgagAAATTTCCTAGACCGCAGGGTtttttg-3′
[0032] 2#PSC93385:
[0033] 5′-CcggGCCCGGATCCGTTCCTGCCATctcgagATGGCAGGAACGGATCCGGGCtttttg-3′
[0034] In the following examples, two saRNA sequences of 1#PSC93384 and 2#PSC93385 were used for transfection.
Embodiment 2
[0035] Example 2: Construction of saRNA lentiviral vector
[0036] The linearized vector was obtained by digestion with restriction endonucleases. The primers were annealed to prepare the target fragments, and the PCR products of 1#PSC93384 and 2#PSC93385 sequences were purified and ligated into linear vectors respectively. The recombinant plasmid was transformed into Escherichia coli DH5α cells (Takara, Japan) for amplification, and after sequencing to verify the characteristics of the plasmid, the empty lentiviral plasmid or PRDM5 saRNA overexpression lentiviral plasmid, packaging plasmid and envelope plasmid were co-transfected into 293T Cells were harvested 48-72 hours after transfection (ie, unpurified cell supernatant), concentrated and purified to obtain a high-titer lentivirus preservation solution. Experimental results: The saRNA lentiviral vector was successfully constructed ( figure 1 ), carrier element information hU6-MCS-CBh-gcGFP-IRES-puromycin, observed in too...
Embodiment 3
[0037] Example 3: Immunohistochemical staining analysis of the expression of PRDM5 protein in gastric cancer
[0038] To analyze the expression of PRDM5 protein in gastric cancer, immunohistochemical analysis was performed on 162 cases of human gastric cancer tissues. The steps of immunohistochemical staining are as follows:
[0039] 1) Preparation of gastric cancer paraffin sections: Embed gastric cancer tissue in paraffin, section thickness about 5 μm, mount on glass slide, bake at 60°C for 2 hours;
[0040] 2) Dewaxing: xylene dewaxing twice, each 15 minutes;
[0041] 3) Hydration: Gradient hydration with anhydrous ethanol, 95% ethanol, 80% ethanol, and 70% ethanol for 5 minutes each, after washing with water, wash with PBS 3 times, 5 minutes each time;
[0042]4) Antigen heat repair: soak the slices in EDTA antigen repair solution with pH 9.0, it is better to add until the repair solution is not above the upper edge of the slice, heat and pressurize slowly until the air i...
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