New application of FAR1 gene

A FAR1, gene expression technology, applied in the field of medicine, can solve problems such as unclear molecular mechanism

Pending Publication Date: 2022-06-07
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few studies on the function of FAR1 protein in the process of cancer development, and the molecular mechanism involved is still unclear. direction

Method used

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  • New application of FAR1 gene
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  • New application of FAR1 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Construction of FAR knockout plasmid

[0024] CRISPR / Cas9 is an adaptive immune defense formed by bacteria and archaea during the long-term evolution to defend against invading viruses and foreign DNA. Microorganisms integrate the invading DNA sequences into their own genomes, and when they are invaded again, they cut the DNA or RNA of viruses, etc., so as to resist the invasion of viruses. In recent years, CRISPR / Cas9 has been humanized for gene editing, which provides new solutions for the treatment of diseases by performing specific DNA modifications on targeted genes. CRISPR (ClusteredRegularly Interspaced Short Palindromic Repeat) requires two parts when editing genes: Cas9 nuclease and gRNA, of which Cas9 nuclease has been integrated into commercial vectors, while gRNA is an important component of the CRISPR knockout system part. There is an RNA sequence of about 20 nt in the CRISPR sequence that targets the target gene. Cas9 and gRNA combine to form ...

Embodiment 2

[0041] Example 2: Construction of FAR1 gene knockout stable cell line

[0042] 1. Select A549 cells with the highest substitution number and in good condition and inoculate them in a 6-well plate to ensure that the cell density is about 75% after 24 hours. Use RPMI-1640 medium containing only 10% fetal bovine serum at 37°C, 5% CO 2 cultured in a cell incubator;

[0043] 2. Before transfecting the plasmid, wash the cells with sterile 1×PBS, add Optim medium, and transfect 1.2 μg plasmid per well. In addition, transfect the PX459 plasmid with unlinked sgRNA as a negative control (while maintaining the total amount of transfected plasmid). Under the premise of the mass of 1.2 μg, the two groups of sgRNAs were mixed together and transfected into A549 cells), and after 6 h of transfection, the medium was changed to a complete medium containing serum but no antibiotics to continue the culture;

[0044] 3. After 24 hours of transfection, the cells were transferred into a 100mm cell...

Embodiment 3

[0073] Example 3: Non-targeted lipid metabolomics detection experiment

[0074] 1) Prepare cells: A549 lung adenocarcinoma cells, AKF cells, and normal alveolar epithelial cells HPAEpic in good condition with a cell density of about 95% (guaranteed that the amount of cells per dish is ≥ 10) 7 1 cells), and washed twice with 1×PBS, collected the cells into a 2 mL centrifuge tube with a cell scraper, centrifuged to remove the supernatant, and stored the cell pellet in liquid nitrogen;

[0075] 2) Take 100mg of sample and put it in a 5mL centrifuge tube, add 1.5mL of chloroform-methanol-water mixture (2:2:1, -20°C), and add 5 steel balls;

[0076] 3) Put the sample into the high-throughput tissue grinder and process it at 60Hz for 1.5min;

[0077] 4) Let the sample stand on ice for 30min, add 0.38mL of pure water, vortex for 30s, and place on ice for 10min;

[0078] 5) Centrifuge at 12000r for 5min at room temperature, and take 600μL of the lower layer in a new 2mL centrifuge t...

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Abstract

The invention discloses a new application of an FAR1 gene, namely an application of screening a drug for treating non-small cell lung cancer aiming at inhibiting the expression of the FAR1 gene, and the new application is characterized in that an FAR1 gene knockout plasmid is constructed through a CRISPR/Cas9 technology, and an A549-KO-FAR1 stable cell line is further constructed; lipid changes among normal alveolar epithelial cells, wild type A549 and A549-KO-FAR1 cell strains are compared and analyzed through lipid metabonomics, it is found that lipid metabolism in the A549 is disordered, after the FAR1 gene is knocked out, part of PC and TG can recover to the level of the normal alveolar cells, FAR1 protein inhibits proliferation and migration of non-small cell lung cancer by adjusting lipid metabolism of cancer cells, and the non-small cell lung cancer can be effectively inhibited. Therefore, the purpose of treating the non-small cell lung cancer can be achieved, the FAR1 gene can be used as a target spot for treating the non-small cell lung cancer, and a wide prospect is provided for development of non-small cell lung cancer treatment drugs targeting the FAR1 gene in the future.

Description

technical field [0001] The invention belongs to the technical field of medicine, and specifically relates to a new use of the FAR1 gene. Background technique [0002] Non-small cell lung cancer (NSCLC) accounts for more than 80% of all lung cancers and is closely related to smoking habits and environmental pollution. Lung cancer is the number one killer of cancer diseases, killing more than one million patients worldwide each year. The incidence and mortality of lung cancer are closely related to each other. Despite the continuous improvement and innovation of diagnostic techniques and treatment methods, the overall 5-year survival rate of lung cancer is still very low, and the global situation is not optimistic. The pathogenesis of tumors is complex and changeable, and tumor-targeted therapy has always played a key role in the diagnosis and treatment of NSCLC. Therefore, in-depth exploration of the signaling network of lung cancer cells and the study of reliable therapeut...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/53C12N5/10C12Q1/02
CPCC12N15/85C12N9/0008C12Y102/01C12N5/0688C12N5/0693G01N33/5044C12N2800/107C12N2503/02G01N2500/10
Inventor 王敏郝佩琪徐天瑞安输苏慧玲
Owner KUNMING UNIV OF SCI & TECH
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