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Application of D374Y mutant of PCSK9 protein in hepatoma cell migration restraining

A technology for inhibiting liver cancer and cell migration, which can be applied in the biological field and can solve problems such as the impact of PCSK9 on the migration of liver cancer cells that has not been seen

Active Publication Date: 2015-10-21
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no relevant report on the effect of PCSK9 on the migration of liver cancer cells in the current research reports

Method used

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  • Application of D374Y mutant of PCSK9 protein in hepatoma cell migration restraining
  • Application of D374Y mutant of PCSK9 protein in hepatoma cell migration restraining
  • Application of D374Y mutant of PCSK9 protein in hepatoma cell migration restraining

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1, the construction of human PCSK9 gene wild type, D374Y mutant overexpression vector

[0055] The present invention relates to human PCSK9 gene wild type (Genebank: NM_174936, 2079bp) and D374Y mutant sequence (G at 1120bp is mutated to T, and the aspartic acid codon GAC is mutated to tyrosine codon TAC). The nucleotide sequence of the wild-type PCSK9 gene is specifically shown in sequence 4 in the sequence listing, encoding the wild-type PCSK9 protein shown in sequence 3 in the sequence listing. The nucleotide sequence of the D374Y mutant sequence of the PCSK9 gene is specifically shown in sequence 2 in the sequence listing, which encodes the D374Y mutant of the PCSK9 protein shown in sequence 1 in the sequence listing.

[0056] In this example, the wild type PCSK9 gene (sequence 4) and the D374Y mutant sequence (sequence 2) of the PCSK9 gene were respectively inserted between the multiple cloning sites NotI and NsiI of the lentiviral vector LV6 to construc...

Embodiment 2

[0058] Example 2, Normal translation and secretion of human PCSK9 gene overexpression vector in HepG2 cells

[0059] In order to verify the working efficiency of the PCSK9 gene overexpression vector, the recombinant expression vectors LV6-PCSK9 and LV6-D374Y constructed in Example 1 were respectively transfected into HepG2 cells, and positive cell pools with puromycin resistance were obtained by screening.

[0060] 1. Western blot was used to detect the expression of PCSK9 protein in cells

[0061] The specific detection method is as follows:

[0062] 1) Extract total protein from HepG2 cells transfected with recombinant expression vectors LV6-PCSK9 and LV6-D374Y respectively, and perform protein quantification. 10 μg of each protein was heat-denatured at 95°C for 5 min in loading buffer (CST), separated by 10% SDS-PAGE electrophoresis, and then transferred to NC membrane (Millipore).

[0063] 2) After the membrane transfer is completed, wash with 20 mL TBS on a decolorizing...

Embodiment 3

[0077] Example 3, the overexpression of the D374Y mutant of the human PCSK9 gene does not affect the proliferation of HepG2 cells

[0078] The unregulated overexpression of genes may affect the normal proliferation ability of cells, and the high expression of D374Y mutant of PCSK9 may have potential effects on the proliferation of HepG2 cells. In order to verify this hypothesis, the inventor of the present invention has carried out following experiment:

[0079] 1. CCK8 method was used to detect the proliferation ability of cells

[0080] The present invention uses CCK8 and immunofluorescence methods to detect the proliferation ability of PCSK9 overexpression cells (LV6-D374Y group in Example 2) and blank control cells (LV6-NC group in Example 2). Among them, the CCK8 experiment was performed according to the instructions of the CCK8 cell proliferation detection kit of Dongren Chemical Technology (Shanghai) Co., Ltd. The specific operation is as follows:

[0081]Firstly, th...

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Abstract

The invention discloses an application of a D374Y mutant of PCSK9 protein in hepatoma cell migration restraining. The provided application is concretely any one of the following applications that a1: a product which restrains hepatoma cell migration is prepared and a2: the hepatoma cell migration is restrained of protein shown in a first sequence of a sequence table. An experiment proves that overexpression of the D374Y mutant of the PCSK9 protein has no obvious effect on the multiplication capacity of HepG2 cells but can obviously restrain the migration capacity of the cells; restraining of the D374Y mutant of the PCSK9 protein for hepatoma cells is acted through regulating and controlling of an ErK signal channel. The application has significant meaning on treatment of hepatomas.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to the application of a D374Y mutant of PCSK9 protein in inhibiting the migration of liver cancer cells. Background technique [0002] Proprotein convertase subtilisin-like / kexin type 9 (PCSK9) is the third major ADH (autosomal dominant hypercholesterolemia) gene located after LDLR and APOB. The gene was first cloned by Seidah et al. and defined as apoptosis-regulated convertase 1 (apoptosis-regulated convertase 1, Narc-1), pointing out its potential role in liver regeneration and neuronal differentiation. PCSK9 protein is mainly expressed in cells with regeneration and differentiation capabilities. The function of PCSK9 in liver cells is mainly manifested in the post-translational regulation of (low-density lipoprotein receptor, LDLR): PCSK9 secreted protein can bind to LDLR on the liver cell membrane and mediate the intracellular lysozyme of PCSK9-LDLR complex Therefore, the high expre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/64C12N15/57C12N15/867C12N5/10A61P35/00
CPCC12N9/6454C12Y304/21061
Inventor 牟玉莲李奎黄雷刘岚高倩魏景亮刘洋吴添文夏颖
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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