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Screening method of lung cancer specific transcription regulation and control element

A transcriptional regulatory element and screening method technology, applied in the field of molecular biology, can solve the problems of low resolution and difficult to determine the core region of the screening sequence, etc., and achieve the effects of enhanced expression, simple operation, and high expression

Inactive Publication Date: 2016-04-20
HUAQIAO UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the resolution of ChIP-chip / seq is low at present, and the sequencing signal peaks are widely distributed in the genome, and it is difficult to determine the core region of the screening sequence according to the signal peaks

Method used

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  • Screening method of lung cancer specific transcription regulation and control element
  • Screening method of lung cancer specific transcription regulation and control element
  • Screening method of lung cancer specific transcription regulation and control element

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Prediction analysis of DKK1 enhancer regulatory elements in the genome

[0029] The Encyclopedia of DNA Elements project (The Encyclopedia of DNA Elements project, ENCODE) is a large-scale international cooperation project funded by the National Human Genome Research Institute, which aims to analyze all functional DNA elements in the genome; the second phase of the project will extend the entire scope to the entire human genome , involving the identification and sequencing of RNA species, the determination of coding and non-coding genes, chromatin openness, histone modification, transcription factor binding sites, DNA methylation and other 177 related analyzes covering 235 different cell lines . The research results related to the ENCOND project are published free of charge in the UCSC (University of California Santa Cruz) human genome browser, that is, the UCSC database.

[0030] According to the relevant information released by the ENCODE project team on th...

Embodiment 2

[0045] Example 2 Construction of Luciferase Recombinant Plasmid Driven by Lung Cancer Specific DKK1 Gene Regulatory Candidate Elements

[0046] The DKK1 promoter has good lung cancer specificity, so a proximal promoter fragment with high activity was selected and constructed into the reporter gene plasmid pGL3-baisc plasmid by genetic engineering. Meanwhile, SV40 was used as an early promoter as a positive control. The predicted TRE is placed in the firefly luciferase (Luciferase) reporter gene plasmid, and for the detection of promoter activity, the predicted TRE element is placed upstream of the reporter gene; while the enhancer verification requires the screening fragment to be placed in a relatively active Before a weak promoter element, an insulator element needs to be placed between the enhancer and the promoter element.

[0047] (1) Construction of luciferase recombinant plasmid driven by DKK1 gene proximal promoter fragment

[0048] The firefly luciferase reporter ge...

Embodiment 3

[0059] Example 3 Functional Verification of DKK1 Gene Candidate Regulatory Elements

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Abstract

The invention provides a screening method of a lung cancer specific transcription regulation and control element. The method comprises the following steps: acquiring epigenetic information in an area about 27kb long surrounding a DKK1 gene locus in a non-small cell lung cancer A549 cell by virtue of a UCSC database, predicting and obtaining 12 candidate enhancer transcription regulation and control element fragments, and respectively inserting the 12 enhancer transcription regulation and control element fragments to the upstream of a near-end promoter of DKK1 to successfully establish luciferase recombinant plasmids; and respectively transfecting the luciferase recombinant plasmids to the non-small cell lung cancer A549 cell, a non-small cell lung cancer H460 cell, a small cell lung cancer H446 cell, an esophagus cancer Eca-109 cell, a normal liver cell Chang liver and a human embryonic kidney cell HEK293, detecting the activity of relevant candidate elements by virtue of a dual-luciferase reporter gene detection system, and screening the regulation and control element having relatively high enhancing expression function on the DKK1 promoter. The screening method has the characteristics of simple operation.

Description

【Technical field】 [0001] The invention belongs to the field of molecular biology, relates to gene expression regulation, and specifically relates to a screening method for lung cancer-specific transcription regulation elements. 【Background technique】 [0002] DKK1 is an antagonist of the canonical wnt signaling pathway, it can bind to Lrp5 / 6 receptor protein, and produce competitive inhibitory effect on the wnt signaling pathway. In the detection of the serum concentration of DKK1, compared with other malignant tumors, benign lung diseases and healthy controls, the serum DKK1 concentration of lung cancer patients is significantly higher, which can be used as a serum marker for the diagnosis of lung cancer, but the study found that the DKK1 promoter is in The activity in lung cancer was not very high. The space- and time-specific expression regulation of eukaryotic genes is an extremely complicated process. Some DNA sequences involved in the regulation of gene transcription ...

Claims

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Application Information

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IPC IPC(8): C12Q1/66
CPCC12Q1/66
Inventor 许瑞安李文茂高燕吴雅清蔡双凤崔秀灵
Owner HUAQIAO UNIVERSITY
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