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Application of METTL2 gene in preparation of kit for detecting treatment sensitivity of colorectal cancer fluorouracil drugs

A technology of fluorouracil and colorectal cancer, applied in the field of biomedicine, can solve the problem of low efficiency and achieve the effect of optimizing the structure of medical resources

Active Publication Date: 2020-08-11
SUN YAT SEN UNIV CANCER CENT
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

However, the effective rate of 5-FU as a single drug is only 10-15%, and the effective rate of combined leucovorin (Leucovorin, LV) can be increased from 11% to 25%, but the overall effective rate is still low, and the sensitivity of tumors to FU has become One of the most important factors restricting the efficacy and prognosis of patients with chemotherapy

Method used

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  • Application of METTL2 gene in preparation of kit for detecting treatment sensitivity of colorectal cancer fluorouracil drugs
  • Application of METTL2 gene in preparation of kit for detecting treatment sensitivity of colorectal cancer fluorouracil drugs
  • Application of METTL2 gene in preparation of kit for detecting treatment sensitivity of colorectal cancer fluorouracil drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Genome-wide RNAi technology screening for 5-FU sensitive genes

[0034] This study utilized a genome-wide shRNA library LentiPlex TMPooledshRNA libraries (Sigma, USA) combined with next-generation sequencing analysis technology to construct a screening system for high-throughput screening of genes related to 5-FU sensitivity in colorectal cancer (for details of the screening process, see figure 1 ). Based on the lentiviral vector pLKO.1, the library has designed sequences for about 15,000 genes in the human genome, with an average of 3-5 shRNA sequences designed for each gene. The total library is divided into 10 small pools (pool1-10), and each small The library contains targeted sequences for approximately 1500 genes. In the experiment, we used the RNAi library to infect HCT116 cells and HT-29 cells at 0.5 MOI (Multiplicity of infection), the purpose is to ensure that most cells are infected with no more than one virus particle, that is, each cell succe...

Embodiment 2

[0067] Example 2: Relationship between METTL2 and 5-FU sensitivity in colorectal cancer

[0068] 1. Expression changes of METTL2 before and after treatment with 5-FU in colorectal cancer cells

[0069](1) Colorectal cancer cell line HCT116 cells were treated with 5-FU at different concentrations (0, 0.5 μM, 1 μM, 2 μM, 4 μM, 8 μM) for 24 h; the cells were collected to extract RNA and protein, and the mRNA and protein levels of METTL2 were determined.

[0070] (2) mRNA level detection (RT-qPCR method):

[0071] (A) Extract total cellular RNA by TRIzol method (executed according to the kit operation guide), and measure DNA concentration and purity in nanodrop 2000;

[0072] (B) performing reverse transcription of the above RNA according to conventional methods to synthesize the corresponding cDNA;

[0073] (C) Quantitative PCR (qPCR) method to detect the relative content of mRNA (with GAPDH as internal control):

[0074] The qPCR primers for METTL2 can be any pair in the foll...

Embodiment 3

[0087] Example 3: Construction of colorectal cancer cell lines with stable knockdown or overexpression of METTL2 and its effect on proliferation ability.

[0088] (1) According to the operating instructions of the pLKO.1 vector (Sigma-Aldrich, USA), design and synthesize shRNA targeting the target sequence in the following table (Table 4), insert into the Age I and EcoR I sites of the pLKO.1 plasmid, and package in 293T cells To obtain the lentivirus expressing the corresponding shRNA.

[0089] Table 4

[0090]

[0091] (2) Routinely infect HCT116 and SW620 cells with the above-mentioned lentivirus solution, and screen with puromycin to obtain stable knockdown METTL2 cell lines (HCT116 / sh1 and HCT116 / sh2, SW620 / sh1 and SW620 / sh2) or negative control cell lines ( HCT116 / shNC, SW620 / shNC).

[0092] (3) Insert METTL2 cDNA (purchased from Shandong Weizhen Biotechnology Co., Ltd.) into the XbaI and NheI sites of the pCDH-EF1-MCS-T2A-Puro (System Bioscience, USA) vector to cons...

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Abstract

The invention belongs to the technical field of biological medicines, and discloses application of an METTL2 gene in preparation of a kit for detecting the treatment sensitivity of colorectal cancer fluorouracil drugs. The application is based on the invention. The inventor discovers that METTL2 protein expression can enhance the sensitivity of colorectal cancer cells to 5-FU, and tumor tissue high expression METTL2 of a colorectal cancer patient is positively correlated with good prognosis of 5-FU chemotherapy. Therefore, a clinician can determine whether the colorectal cancer patient is suitable for fluorouracil drug treatment or not according to the expression condition of the METTL2 gene of the colorectal cancer patient, and formulate a personalized treatment scheme.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and particularly relates to the application of METTL2 gene in the preparation of a kit for detecting the treatment sensitivity of colorectal cancer to fluorouracil drugs. Background technique [0002] The morbidity and mortality of colorectal cancer (Colorectal Cancer, CRC) ranks third among all types of tumors in the world. In recent decades, with the development of my country's economy and science and technology, people's living standards have been greatly improved, and with the popularization of Western-style diet patterns, the incidence of colorectal cancer has been increasing year by year. Since 5-fluorouracil (5-fluorouracil, 5-FU) was used clinically as a basic drug for the treatment of colorectal cancer in the 1950s, fluorouracil drugs (FU) such as 5-FU and Capecitabine (oral medicine) are still used today. An important component of first-line chemotherapy regimens (eg, CAPEOX, FOLFO...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886G01N33/574
CPCC12Q1/6886G01N33/57484G01N33/57419G01N33/57446C12Q2600/106C12Q2600/118C12Q2600/158G01N2800/52
Inventor 刘然义彭绮华岳欣
Owner SUN YAT SEN UNIV CANCER CENT
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