Kit applied to cancerogenic risk assessment of environmental pollution

A technology for risk assessment and kits, which can be used in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., and can solve problems such as environmental pollution

Pending Publication Date: 2022-01-04
BEIJING CHEST HOSPITAL CAPITAL MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since PAEs are not covalently bound to the polymer matrix, after slight physical and chemical processes such as heating, ionization, and acid treatment, PAEs can be released into the surrounding environment through evaporation or leaching from the plastic, causing environmental pollution

Method used

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  • Kit applied to cancerogenic risk assessment of environmental pollution
  • Kit applied to cancerogenic risk assessment of environmental pollution
  • Kit applied to cancerogenic risk assessment of environmental pollution

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1, Expression and Biological Function of MUC17 Gene in Lung Cancer Cells

[0057] 1. RT-PCR

[0058] 1.1 Cell RNA extraction and reverse transcription process

[0059] (1) The cell RNA extraction process is to select cells with good growth status, and use 1mL / 10 6 Add Trizol reagent (Invitrogen, USA, catalog number: 15596026) to each cell.

[0060] (2) Stand at room temperature for 10 minutes, fully crack and extract with chloroform, and add 0.2ml of chloroform for every 1mL of Trizol. Shake vigorously for 15 seconds, place at room temperature for 5 minutes, and centrifuge at 12000g for 15 minutes at 4°C.

[0061] (3) Transfer the colorless liquid in the upper layer after stratification to a new centrifuge tube, use pre-cooled isopropanol for precipitation, add 0.5mL isopropanol to every 1mL Trizol, and place on ice for 20min. Centrifuge at 12000g for 10min at 4°C and discard the supernatant.

[0062] (4) Wash the precipitate with pre-cooled 75% ethanol, ad...

Embodiment 2

[0113] Example 2, MUC17 gene methylation detection of cell lines after phthalates (PAEs) treatment

[0114] 1. Design and synthesis of primers for MUC17 gene methylation detection

[0115] Query and download the sequence of the gene MUC17 to be detected (GenBank Accession No.NC_000007.14 (Update Date8-Jul-2021)), and input the target sequence into EpiDesigner software for primer design. According to the software operation results, select and determine the appropriate primer design scheme. Synthetic primers, each 5OD.

[0116] The specific sequences of methylation detection primers are as follows:

[0117] Primer set 1 covers the DNA sequence (SEQ ID No.1) located at -3898bp to -3400bp at the transcription start site of MUC17 gene;

[0118] Primer set 1 is composed of F1 and R1, and its sequence is as follows:

[0119] Upstream primer F1: 5'-aggaagagag TTGTAGTTGGGAAGGAGTTTGTTT-3' (SEQ ID No.4),

[0120] Downstream primer R1: 5'-cagtaatacgactcactatagggagaaggctCCTCCACCTAATCTCC...

Embodiment 3

[0175] Example 3, DNA methylation inhibitors can reverse the expression silencing of MUC17 in respiratory epithelial cells and non-small cell lung cancer cells

[0176] 1. Cell RNA extraction and reverse transcription process

[0177] (1) DNA methylase inhibitor 5-Aza-2′-Deoxycytidine (5-Aza, 5-aza-2′-deoxycytidine, American Sigma-Aldrich Company, catalog number: 189825) dosing: when cells (A549 and BEAS-2B) When the growth density reaches 30%, add 5-Aza until its content in the culture system is 5 μM, replace the medium every 24 hours, and continue to add 5-Aza to its content in the culture system 5μM, a total of 96 hours of action.

[0178] (2) Select cells with good growth status, press 1mL / 10 6 Add Trizol reagent (Invitrogen, USA, catalog number: 15596026) to each cell. Place at room temperature for 10 minutes, fully lyse and extract with chloroform, adding 0.2ml of chloroform to every 1mL of Trizol.

[0179] (3) Shake vigorously for 15 seconds, place at room temperatu...

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Abstract

The invention discloses a kit applied to cancerogenic risk assessment of environmental pollution. The invention specifically discloses a specific methylation primer pair for detecting the methylation state or level of a CpG island in an MUC17 gene promoter region, as well as an application, kit and method of the specific methylation primer pair. The primer pair consists of a primer F3 (SEQ ID No. 8) and a primer R3 (SEQ ID No. 9). The kit for detecting the methylation of the DNA sequence in an MUC17 gene can specifically detect the DNA methylation level related to the MUC17 gene, and can be conveniently and quickly used in the aspects of environmental pollution carcinogenic risk assessment, lung cancer early screening, lung cancer risk prediction and the like, and the detection result can provide an evaluation method for early diagnosis of tumors.

Description

technical field [0001] The invention belongs to the technical field of molecular diagnosis, and in particular relates to a methylation marker kit used in environmental pollution carcinogenic risk assessment. Background technique [0002] Lung cancer is a high-incidence tumor in the world, ranking first in the incidence of malignant tumors in my country. In 2020, there will be 2.2 million new cases of lung cancer worldwide, and 1.8 million deaths (Sung H, Ferlay J, Siegel RL, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 2021, 71(3):209-249.). Although the survival rate of lung cancer patients has improved significantly in the past decade through early diagnosis and cancer treatment through biomarkers, the global incidence and mortality of lung cancer will still worsen with the increase of many risk factors . Among them, ambient air pollution and exposure to other respirable env...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6886C12Q1/6858C12Q2600/154C12Q2600/166C12Q2531/113C12Q2523/125C12Q2545/101C12Q2545/113
Inventor 林舒晔王海潮黄家强庞梦迪赵聪
Owner BEIJING CHEST HOSPITAL CAPITAL MEDICAL UNIV
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