Application of human YTHDF1 (Youth-Tech-Health Domain Family Member 1) gene

A gene and gene expression technology, applied in the field of new uses of genes, can solve the problems of lung cancer cell occurrence, incomplete development, survival rate of less than 15%, and unsatisfactory clinical efficacy.

Active Publication Date: 2019-02-15
KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current treatment for lung cancer includes surgical resection, combined chemotherapy based on platinum (such as cisplatin or carboplatin), radiotherapy, and targeted drug therapy, etc., but the clinical efficacy is still unsatisfactory, and the 5-year survival rate is not more than 15%. The main reason is that the occurrence and development of lung cancer cells are not fully understood

Method used

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  • Application of human YTHDF1 (Youth-Tech-Health Domain Family Member 1) gene
  • Application of human YTHDF1 (Youth-Tech-Health Domain Family Member 1) gene
  • Application of human YTHDF1 (Youth-Tech-Health Domain Family Member 1) gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: RT-PCR assay detects the expression of YTHDF1 in lung cancer cell lines

[0054] 1. Extraction of total cellular RNA

[0055] (1) When normal lung epithelial cells and lung cancer cells grow to 80% coverage, remove the supernatant, wash away the serum with PBS, add 1mL TRizol, and place it horizontally for 5 minutes to ensure that TRizol is evenly distributed on the cell surface and lyse the cells. Let the cells fall off the culture dish, transfer all the liquid to the centrifuge tube, and repeatedly pipette until there is no obvious large precipitate in the lysate; stand at room temperature for 5 minutes;

[0056] (2) Centrifuge at 12,000g for 5min in a centrifuge at 4°C, and transfer the supernatant to a new 1.5mL centrifuge tube;

[0057] (3) Add 200 μL of chloroform, vortex, and centrifuge at 12,000 g for 15 min in a centrifuge at 4°C, after which the liquid is divided into three layers;

[0058] (4) Absorb the upper aqueous phase (be careful not to tou...

Embodiment 2

[0076] Example 2: Western blot detection of YTHDF1 protein expression

[0077] 1. WB (Western Blotting) detection

[0078] 1.1 Extraction of total cell protein

[0079] After the cells were treated according to the specific experiment, the supernatant medium was discarded, and washed once with PBS; the corresponding cell lysate was added according to the amount of the cell pellet, and the freeze-thaw lysed 3 times, during which the pipetting was continued; 4°C, 12000rpm centrifugation After 10 min, the supernatant was discarded and the precipitate was used for subsequent experiments.

[0080] 1.2 Protein concentration detection and denaturation treatment

[0081] The protein concentration detection kit is Biyuntian BCA protein concentration detection kit (enhanced type), item number: P0010S, the method is as follows:

[0082] Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of the standard into the standard wells of the 96-well plate, and add the standard diluent to make up to 20 μL, s...

Embodiment 3

[0087] Embodiment 3: clinical experiment

[0088] 1. Immunohistochemical experiment

[0089] Lung cancer tissue chips and lung cancer pathological tissue slices were obtained in cooperation with the Department of Pathology, Xiangya Hospital, Central South University. Tumor samples from nude mice were fixed in formalin and then sliced ​​in wax. The samples were baked at 80°C for 2 hours. Soak in xylene for dewaxing, gradient alcohol dehydration, immerse in 0.3% hydrogen peroxide (methanol and water preparation) solution for 15 minutes to remove tissue peroxidase, rinse with Tris buffer saline (TBS), and microwave at 95°C for 20 minutes for antigen retrieval. Recover at room temperature for 1 hour; then wash three times with TBS, incubate with 5% goat serum for 30 minutes to remove non-specific binding, primary antibody anti-YTHDF1 (Proteintech, 11515-1-AP, 1:200), anti-cleaved-caspase 3 (CC3 :CST, 9661S, 1:1000), anti-Ki67 (MAXIM Biotechnologies, MAB-0005, ready to use), anti-...

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Abstract

The invention discloses novel application of a human YTHDF1 (Youth-Tech-Health Domain Family Member 1) gene, that is, a human YTHDF1 gene expression amount detection reagent is applied to preparationof lung cancer clinical diagnosis reagents, a human YTHDF1 gene is applied to preparation of lung cancer medicines, compared with normal pulmonary epithelial cells, the YTHDF1 gene is highly expressed in lung cancer cells, RNA (Ribonucleic Acid) designed according to the YTHDF1 gene is capable of interfering slow viruses, inhibiting expression of YTHDF1, remarkably inhibiting proliferation of thelung cancer cells, retarding tumor cell division at a G0 / G1 phase and promoting cell apoptosis, inhibiting formation of nude mouse transplanted tumour, and furthermore the purpose of treating lung cancer can be achieved; YTHDF1 gene knockout mouse tumor formation capability detection shows that cell proliferation is remarkably inhibited, and cell apoptosis is remarkably increased; the results show that the YTHDF1 gene can be used as a target for lung cancer treatment, and wide prospects are provided for development of lung cancer treatment medicines based on YTHDF1 genes in future.

Description

technical field [0001] The present invention relates to a new application of gene, especially the new application of human YTHDF1 gene. Background technique [0002] Acquired modifications include methylation, acetylation, and ubiquitination. RNA modification is a type of acquired modification. So far, more than 100 chemical modifications have been discovered on different intracellular RNAs such as tRNA, rRNA, mRNA, and long non-coding RNA. N6-methyladenosine, which was discovered in the 1970s, is the most common modification type of intracellular mRNA and long non-coding RNA, and it exists widely in viruses, yeast, and even mammals. Recent studies have shown that abnormal m6A modification can lead to abnormal brain development and other diseases, including cancer. RNA-binding proteins (RNA-binding proteins) are key factors in post-transcriptional processing that regulate RNA splicing, stability, localization, and translation, and many RBPs are aberrantly expressed in dif...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886A61K31/713A61P35/00
CPCA61K31/713A61P35/00C12Q1/6886C12Q2600/158
Inventor 陈勇彬杨翠萍石玉林吴梦鸽江丽萍申秋硕
Owner KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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