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Human GPD2 gene inhibitor and application thereof

Active Publication Date: 2019-12-13
WUXI NO 3 PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are very few studies on the relationship between GPD2 and tumors. Some studies have found that there is a significant difference in the expression of GPD2 in the GP4 matrix after Gleason score GP3 in prostate cancer; other studies have found that there are significant differences in the immunostaining of GPD2 in breast cancer and adjacent tumors. The staining was significantly higher than that of the adjacent cancer; Interfering with the expression of GPD2 can inhibit the proliferation of breast cancer cell MDA-MB-231

Method used

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  • Human GPD2 gene inhibitor and application thereof
  • Human GPD2 gene inhibitor and application thereof
  • Human GPD2 gene inhibitor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Construction of lentiviral vector for suppressing human GPD2 gene

[0053] 1. RNA interference target design and double-stranded DNA oligo preparation

[0054] (1) Screening targets for human GPD2 gene inhibitors

[0055] The GPD2 (NM_000408) gene information was retrieved from Genbank; according to the RNA interference sequence design principle, an effective shRNA interference target for the GPD2 gene was selected, specifically TGTATTAGAGAGTATCAAT (SEQ ID NO: 1).

[0056] (2) DNA oligo sequence synthesis

[0057] Design the shRNA interference sequence based on the selected target sequence, and add appropriate restriction endonuclease sites at both ends to complete the vector construction. In addition, a TTTTT termination signal was added to the 3' end of the positive strand, and a complementary sequence to the termination signal was added to the 5' end of the anti-strand. After the design is completed, send it to Jierui Company to synthesize single-stranded DNA olig...

Embodiment 2

[0108] Real-time fluorescent quantitative RT-PCR method to detect the silencing efficiency of GPD2 gene

[0109] The breast cancer MDA-MB-231 cells in the logarithmic growth phase were trypsinized to make a cell suspension (the number of cells was about 5×10 4 / mL) were seeded in 6-well plates and cultured until the cell confluency reached about 30%. According to the multiplicity of infection (MOI, RKO: 10) value, an appropriate amount of the virus prepared in Example 1 was added, the culture medium was replaced after 24 hours of cultivation, and the cells were collected after the infection time reached 4 days. Total RNA was extracted according to the instruction manual of Invitrogen's Trizol. According to the M-MLV operating manual of Promega Company, the RNA was reverse-transcribed to obtain cDNA (see the table below for the reverse transcription reaction system, react at 42°C for 1 h), and then bathed in a water bath at 70°C for 10 minutes to inactivate the reverse transcr...

Embodiment 3

[0117] Detection of proliferation ability of tumor cells infected with lentivirus by MTT assay

[0118] Breast cancer MDA-MB-231 cells were trypsinized and seeded in a 12-well plate with a cell density of 10-15%. The next day, replace with fresh medium containing 5 μg / ml polybrene. The lentivirus of Example 1 was added to the culture plate according to the multiplicity of infection (MOI, RKO: 10) value, and fresh medium was replaced after 12-24 hours of infection. After 72 hours of infection, the fluorescence was observed under a fluorescence microscope, and the infection efficiency reached 90%.

[0119] Trypsinize the virus-infected cells in the logarithmic growth phase, resuspend the complete medium into a cell suspension; inoculate in a 96-well plate, 100 μL per well; store at 37°C, 5% CO 2 Cultivate in an incubator; add 10 μL of 5 mg / mL MTT 4 hours before the end of the culture; discard the culture solution and add 100 mL of DMSO to terminate the reaction; detect the OD ...

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Abstract

The invention discloses a human GPD2 gene inhibitor and an application thereof. Specifically, a human GPD2 gene is taken as a target, a proper target gene sequence is selected, and a nucleic acid molecule, a recombinant vector, a slow virus and other inhibitors for effectively inhibiting expression of the GPD2 gene and effectively inhibiting proliferation of human tumor cells, especially breast cancer tumor cells, are designed by a RNA interfering mode; an application of the human GPD2 gene in screening tumor treatment drugs, preparing tumor treatment drugs or preparing tumor diagnosis drugs is provided, and tumor treatment ways is enriched.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a human GPD2 gene inhibitor and application thereof. Background technique [0002] RNA interference (RNA interference, RNAi) refers to the specific degradation of intracellular mRNA mediated by endogenous or exogenous double-stranded RNA, resulting in the silencing of the expression of target genes and the loss of corresponding functional phenotypes. It can efficiently and specifically block the expression of a specific gene in the body, leading to its degradation, thereby causing the silencing of a specific gene in the organism, and causing the cells to show the absence of a certain gene phenotype. It is a commonly used research gene emerging in recent years. Functional, laboratory techniques for finding cures for disease. At present, RNAi-specific inhibition of gene expression has been applied in genetic diseases, viral infectious diseases, and gene therapy of c...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/867A61K31/7105A61K48/00A61P35/00
CPCA61K31/7105A61P35/00C12N15/113C12N15/86C12N2310/10C12N2740/15043
Inventor 王北陈丹丹袁凤来过小强
Owner WUXI NO 3 PEOPLES HOSPITAL
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