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A kind of inhibitor of p4hb gene expression and its application

A gene expression and inhibitor technology, applied in gene therapy, genetic engineering, medical preparations containing active ingredients, etc., can solve the problem of not being found

Active Publication Date: 2019-07-12
SHANGHAI SEVENTH PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the occurrence and development of liver cancer is a relatively complicated process, and there are thousands of abnormally expressed genes. At present, no gene that plays a key role in liver cancer tissue has been found, and the occurrence or progression of liver cancer can be curbed by inhibiting this gene.

Method used

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  • A kind of inhibitor of p4hb gene expression and its application
  • A kind of inhibitor of p4hb gene expression and its application
  • A kind of inhibitor of p4hb gene expression and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0058] Human liver cancer cells Huh-7, HepG2, PLC5, Hep3B, SK-Hep-1 cells and normal liver cell line L02 were cultured with DMEM full medium containing 100 U / ml double antibody and 10% fetal bovine serum for 48 hours. The culture environment is: 37°C, 5% CO 2 , 100% humidity; change the medium once every 2 days, use a microscope to observe the number of cell proliferation every day, when the cells grow in logarithmic phase, use the Western blot method to detect the expression of P4HB in each cell line: the specific steps are as follows:

[0059] ① Cell preparation

[0060] After 24 hours of cell transfection, the expression of protein in each group was detected by Western blot method;

[0061] ② Related liquid preparation

[0062] A.2M Tris-HCl (pH 8.8) 100m1

[0063] Weigh 24.2g Tris base; add 50mlddH2O; slowly add concentrated hydrochloric acid to pH 8.8 (about 4m1); let the solution cool to room temperature, the pH will rise, add ddH 2 0 to 100ml.

[0064] B.1M Tris-HC...

Embodiment 2

[0146] Human liver cancer cells Huh-7, HepG2, PLC5, Hep3B, SK-Hep-1 cells and normal liver cell line L02 were collected, RNA was extracted by kit method, and the concentration of extracted RNA was adjusted for PCR detection. The PCR forward primer of P4HB gene is: 5'-GGAATGGAGACACGGCTTC-3'; the PCR reverse primer of P4HB is: 5'-TTCAGCCAGTTCACGATGTC-3'. The reaction procedure of the PCR is shown in Table 3.

[0147] Table 3 PCR reaction program

[0148]

[0149] After qRT-PCR, the expression of P4HB gene in human liver cancer cells Huh-7, HepG2, PLC5, Hep3B, SK-Hep-1 cells and normal liver cell line L02 was obtained as follows: figure 2 shown.

[0150] Depend on figure 2 It can be seen that based on the expression of P4HB gene in normal tissue, the expression of P4HB mRNA in liver cancer cells Huh-7, HepG2, PLC5, Hep3B and SK-Hep-1 cells was significantly higher than that in normal tissue. expression volume.

Embodiment 3

[0152] Combine P4HB siRNA with vector lipofectamine TM The connection of 2000 is a physical connection, through lipofectame TM The positive charge of 2000 physically encapsulates the negatively charged siRNA within the lipofectamnie. After the siRNA and lipofectamine are mixed, the physical connection is realized.

[0153] HepG2 and Huh-7 cells were transfected with P4HB inhibitor siRNA molecule, control siRNA, P4HB vector and empty vector for 24 hours, respectively. The treated HepG2 and Huh-7 cells were obtained. Specifically, the commercial vector lipofectaminTM2000 was used for transfection, and the steps were as follows:

[0154] First, centrifuge the Ep tube containing 1OD (3.0nmol) siRNA dry powder on a desktop centrifuge for 4 seconds, then slowly open the tube cap, add 150ul DEPC-treated water to dissolve, cover the cap, shake and dissolve, so that the final concentration of siRNA is 20uM. In order to avoid repeated freezing and thawing, divide it into 0.1ml EP t...

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Abstract

The invention provides an inhibitor for P4HB gene expression. The inhibitor is a siRNA molecule of a nucleotide sequence shown by SEQ ID NO. 1. According to the inhibitor provided by the invention, the nucleotide sequence of the siRNA molecule has complementary combination with mRNA generated by the P4HB gene expression, so that the P4HB gene at a target site is prevented from being expressed as protein, the P4HB gene is kept silent, and a purpose of inhibiting the P4HB gene expression can be achieved. The invention also provides application of the inhibitor in preparing a drug for preventing or treating liver cancer. The inhibitor provided by the invention has complementary combination with mRNA generated by the P4HB gene expression, so that the P4HB gene is kept silent, the epithelial-myofibroblast transdifferentiation (EMT) can play an opposite effect. Meanwhile, the silence of P4HB can inhibit in vivo tumor formation of the liver cancer cells.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to an inhibitor of P4HB gene expression and application thereof. Background technique [0002] In the field of traditional medicine, organic compounds and antibodies are specific drugs for the treatment of various diseases. In recent years, with the application of molecular biology in the field of medicine and the introduction of new treatment concepts, the function of controlling specific gene expression in organisms Sexual nucleic acid has attracted much attention as a new type of medicine or diagnostic drug. [0003] At present, the functional nucleic acid as a new type of medicine or diagnostic drug mainly includes RNAi (RNA interference: RNA interference). The main mechanism of action of the drug is the gene silencing of RNAi. Functional nucleic acid aptamer, or antisense nucleic acid that binds to target mRNA to inhibit its translation, thereby specifically pr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113A61K48/00A61K31/7088A61P35/00
CPCC12N15/113C12N2310/14
Inventor 夏伟叶颖王国玉庄菊花
Owner SHANGHAI SEVENTH PEOPLES HOSPITAL
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