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Lowering Pyridoxine Phosphate Oxidase in 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

Active Publication Date: 2018-02-27
JINSHAN HOSPITAL FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are no related studies on PNPO in ovarian cancer, and there are no related reports that PNPO inhibits tumor cell proliferation, migration, and invasion.

Method used

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  • Lowering Pyridoxine Phosphate Oxidase in Ovarian Cancer
  • Lowering Pyridoxine Phosphate Oxidase in Ovarian Cancer
  • Lowering Pyridoxine Phosphate Oxidase in Ovarian Cancer

Examples

Experimental program
Comparison scheme
Effect test

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] Ovary 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] Antisense: ...

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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PUM

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Abstract

The present invention relates to treatment of ovarian cancer by reducing pyridoxine phosphate oxidase (PNPO). The present invention has confirmed through experiments that reducing PNPO can inhibit the proliferation, migration and invasion of ovarian cancer cell lines, and lead to cell cycle G2 / M phase arrest, suggesting that PNPO is a potential new target for the treatment of ovarian cancer, which can target Patients with high expression of PNPO in ovarian cancer can be treated by reducing the expression of PNPO to achieve the purpose of treatment, and at the same time, it is beneficial to further study the mechanism of occurrence and development of ovarian cancer.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to the treatment of ovarian cancer, and more specifically to the treatment of ovarian cancer by reducing pyridoxine phosphate oxidase. Background technique [0002] Pyridoxine phosphate oxidase (PNPO) is an oxidase that catalyzes the synthesis of pyridoxal phosphate (PLP, the active form of vitamin B6) from pyridoxine phosphate (PNP) and pyridoxamine phosphate (PMP). PLP is a coenzyme of various enzymes, and participates in more than 140 kinds of catalytic reactions, such as the metabolism of amino acids such as homocysteine ​​and the synthesis of neurotransmitters such as catecholamines, etc. [0003] PNPO enzyme inactivity was detected in tumors of liver or nerve origin (1998-Emily O.Ngo); in breast cancer T47D cells (tamoxifen-sensitive, ER-positive), the expression of PNPO can be inhibited by estradiol and tamoxifen Regulation of PNPO (2006Mariam H); multiple case rep...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61K31/7088C12N15/867A61P35/00
Inventor 许国雄张凌云
Owner JINSHAN HOSPITAL FUDAN UNIV
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