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Application of mettl2 gene in the preparation of a kit for detecting the sensitivity of colorectal cancer to fluorouracil drug therapy

A technology for detection of reagents 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: 2022-07-01
SUN YAT SEN UNIV CANCER CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 the preparation of a kit for detecting the sensitivity of colorectal cancer to fluorouracil drug therapy
  • Application of mettl2 gene in the preparation of a kit for detecting the sensitivity of colorectal cancer to fluorouracil drug therapy
  • Application of mettl2 gene in the preparation of a kit for detecting the sensitivity of colorectal cancer to fluorouracil drug therapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Screening of 5-FU sensitive genes by genome-wide RNAi technology

[0034] This study utilized a genome-wide shRNA library LentiPlex TMPooledshRNA libraries (Sigma, USA) combined with next-generation sequencing analysis technology to construct a high-throughput screening system for 5-FU sensitivity-related genes in colorectal cancer (see the screening process for details). figure 1 ). The library is based on the lentiviral vector pLKO.1. The sequences of about 15,000 genes in the human genome are designed. On average, 3-5 shRNA sequences are designed for each gene. The total library is divided into 10 small pools (pool1-10). 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 0.5MOI (Multiplicity of infection), the purpose is to ensure that most cells are infected with no more than one virus particle, that is, each successfully infected cell only receive...

Embodiment 2

[0067] Example 2: Detection of METTL2 expression in HCT116 cells treated with 5-FU and 5-FU drug-resistant cell lines

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

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

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

[0071] (A) Total cell RNA was extracted by TRIzol method (performed according to the kit's operating instructions), and DNA concentration and purity were determined by nanodrop 2000;

[0072] (B) carrying out reverse transcription and synthesizing corresponding cDNA by above-mentioned RNA according to conventional methods;

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

[0074] T...

Embodiment 3

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

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

[0089] Table 4

[0090]

[0091] (2) The above lentivirus solution was routinely infected with HCT116 and SW620 cells, and screened 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) The METTL2 cDNA (purchased from Shandong Weizhen Biotechnology Co., Ltd.) was inserted into the XbaI and NheI sites of the pCDH-EF1-MCS-T2A-Puro (System Bioscience, USA) vector to co...

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Abstract

The invention belongs to the technical field of biomedicine, and discloses the application of METTL2 gene in the preparation of a kit for detecting the sensitivity of colorectal cancer to fluorouracil drug treatment. This application is based on the inventor's discovery that METTL2 protein expression can enhance the sensitivity of colorectal cancer cells to 5-FU, and that the high expression of METTL2 in the tumor tissue of colorectal cancer patients is positively correlated with the good prognosis of 5-FU chemotherapy. Therefore, clinicians can determine whether patients with colorectal cancer are suitable for fluorouracil treatment through the expression of METTL2 gene in patients with colorectal cancer, and formulate a personalized treatment plan.

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 fluorouracil drugs. Background technique [0002] The incidence and mortality of colorectal cancer (CRC) in the world 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-FU) was used in the clinic as the basic drug for colorectal cancer treatment in the 1950s, 5-FU and Capecitabine (oral drug) and other fluorouracil drugs (FU) are still Important components of first-line chemotherapy regimens (eg, CAPEOX, FOLFOX, FOLFIRI, XELOX, etc.) for patients with ...

Claims

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Application Information

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Patent Type & Authority Patents(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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