Reduction of pyridoxine 5'-phosphate oxidase (PNPO) for treating ovarian cancer
An inhibitor and protein technology, applied in the field of molecular biology, can solve problems such as no PNPO, and achieve high knockout efficiency
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Embodiment 1
[0047] Example 1 Synthesis of PNPO-siRNA and verification of PNPO knockout effect
[0048] 1. Design of PNPO-siRNA
[0049] Find a suitable target sequence, design and screen to obtain PNPO-siRNA against PNPO mRNA:
[0050]Sense strand (Sense): 5'-GACUGGCUCUAUGAGAGAC-dTdT-3'(SEQ ID NO.1),
[0051] Antisense strand (Antisense): 5'-GUCUCUCAUAGAGCCAGUC-dTdT-3' (SEQ ID NO.2).
[0052] 2. Validation of PNPO-siRNA silencing effect
[0053] Ovarian cancer cells OVCAR-3 were plated for 24 hours and given PNPO-siRNA, specifically using 10 μl of Roche X-tremeGENE siRNA transfection reagent + 2 μg of PNPO-siRNA transfection, and the control group was transfected with 10 μl of Roche X-tremeGENE siRNA transfection reagent + NC- siRNA 2 μg. Protein and mRNA were extracted 24 hours after transfection. The knockout efficiency was verified by qPCR and Western-blot.
[0054] The NC-siRNA sequence is as follows:
[0055] Sense: 5'-GACGUUGGACAUCGGAUCA-dTdT (SEQ ID NO.5),
[0056] Antisens...
Embodiment 2
[0061] Example 2 Synthesis of PNPO-shRNA and verification of PNPO knockout effect
[0062] 1. Construction of lentiviral interference vector
[0063] (1) PNPO-shRNA sequence information
[0064] Upstream strand (Top strand): 5'-gatccGACTGGCTCTATGAGAGACTTCAAGAGAGTCTCTCATAGAGCCAGTCTTTTTTg-3' (SEQ ID NO.3),
[0065] Downstream strand (Bottom strand): 5'-aattcAAAAAGACTGGCTCTATGAGAGACTCTCTTGAAGTCTCTCATAGAGCCAGTCg-3' (SEQ ID NO. 4).
[0066] (2) Construction of RNAi lentiviral recombinant plasmid
[0067] 1) Annealing of shRNA;
[0068] 2) Lentiviral vector (pHY-LV-KD5.1, purchased from Han Yin Biotechnology (Shanghai) Co., Ltd., see the vector map Figure 7 ) digestion and recovery: the pHY-LV-KD5.1 vector was linearized by restriction enzymes BamH I (GGATCC) and EcoR I (GAATTC);
[0069] 3) The connection of the shRNA carrier and the PNPO-shRNA DNA double-stranded sequence;
[0070] 4) transfer the ligation product into the prepared bacterial competent cells for transformati...
Embodiment 3
[0079] Example 3 Knockout of PNPO inhibits the proliferation, migration and invasion of ovarian cancer cells
[0080] 1. Knockout of PNPO in ovarian cancer cell lines SK-OV-3 and OVCAR-3 can inhibit the proliferation of ovarian cancer cells
[0081] As in Example 2, the virus was added at MOI=15 (SK-OV-3) or MOI=20 (OVCAR-3) to establish a stably transfected cell line (PNPO-shRNA). The same amount of lentiviral particles (NC) was idling in the control group. After the knockout efficiency was verified by qPCR and Western-blot, the proliferation efficiency was detected by WST method. Specifically, the absorbance of OD450nm at 0h, 12h, 24h, 48h, and 72h was measured with a microplate reader, and 10 μl of WST-1, put it back into the incubator and continue culturing for 2 hours.
[0082] Result: For the experimental results of WST method, see image 3 A and image 3 b. The results showed that the proliferation of ovarian cancer cells was inhibited after knocking out PNPO.
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