Application of siRNA suppressing human Ezrin gene expression in the research on esophagus carcinoma proliferation

A gene expression and cancer cell technology, applied in the field of siRNA, can solve the problem that the mechanism of Ezrin function and its expression change has not been well elucidated

Inactive Publication Date: 2007-03-21
SHANTOU UNIV MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the function of Ezrin in esophageal cancer and other tumors and the mechanism of its expression change have not been well elucidated so far.

Method used

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  • Application of siRNA suppressing human Ezrin gene expression in the research on esophagus carcinoma proliferation
  • Application of siRNA suppressing human Ezrin gene expression in the research on esophagus carcinoma proliferation
  • Application of siRNA suppressing human Ezrin gene expression in the research on esophagus carcinoma proliferation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Design, synthesis and annealing of siRNA targeting Ezrin gene.

[0046] Two sets of siRNA sequences were designed according to the design software provided by Dharmacon on the Internet, as shown in Table 1:

[0047] siRNA Target Sequence

Numbering

Region

start

5'TCCACTATGTGGATAATAA3' (SEQ ID NO: 3)

PSE1

ORF

274

5'ACTTTGGCCTCCACTATGT3' (SEQ ID NO: 4)

PSE2

ORF

265

[0048] The design principles are as follows:

[0049] (1) Starting from the AUG start codon of the mRNA, look for 5'AA(N19)TT,

[0050] Where N is any base, and the sequence length is 19bp (as shown in Table 1);

[0051] (2) Three or more G or C cannot appear consecutively on the target sequence;

[0052] (3) Avoid the interference sequence at the beginning and end of the target gene;

[0053] (4) Select sequences with a GC content between 30% and 52%;

[0054] (5) targeting the ORF region;

[0055] (6) Homology comparison o...

Embodiment 2

[0058] Construction of embodiment 2 expression vectors PSE1 and PSE2

[0059] 1. Linearization and recovery of the carrier: Add the following reagents in sequence to the centrifuge tube (total volume 50 μl): 37.5 μl of sterile water, 5 μl of pSUPER.neo vector, 5 μl of 10× buffer B, 0.5 μl of acetylated BSA (10 mg / ml) μl and HindIII (20u / μl) 2μl. After mixing, add 2 μl of BglII (20u / μl) to the mixture after incubating in a 37°C water bath for 1 hour, and incubate in a 37°C water bath for another 1 hour. Take 5 μl of electrophoresis for 1% agarose gel electrophoresis, and use the undigested plasmid as a control to identify whether the vector has become linear. Recover the linear pSUPER.neo vector fragment according to the instructions of the Quick PCR purification kit.

[0060] Add 5 times the volume of PB buffer to the enzyme digestion mixture, then transfer the mixture to the QIAquick spin column, centrifuge at 12000×g for 1min; discard the effluent, add 750μl PE buffer to t...

Embodiment 3

[0063] Embodiment 3 Analysis of stable transfection and interference effect

[0064] The identified recombinants were transfected into human esophageal cancer cell line EC109 cells, and an empty vector was also transfected as a control. In order to obtain cells stably expressing the plasmid, we first used a higher concentration of G418 (400μg / ml) for drug screening. EC109 cells transfected with blank plasmid PSCs and cells transfected with the above siRNAs (referred to as PSE1 and PSE2, respectively) survived due to the neo gene on the plasmid vector, which has G418 resistance; in contrast, untransfected The control EC109 cells with the above plasmids were killed by G418 toxicity. In order to prevent the loss of the expression plasmid during continuous subculture, the cells are still cultured in a growth environment with a relatively low drug concentration (200 μg / ml). If the plasmid is lost, the cells will still be killed due to the toxic effect of G418 . Under the above c...

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Abstract

The present invention relates to siRNA field, and discloses the application of siRNA suppressing human Ezrin gene expression in the research on esophagus carcinoma cell proliferation. The present invention designs and synthesizes two pairs of siRNA's targeting human Ezrin gene, and constitutes the expression vector for them to express inside mammal cell successfully. These siRNA's can interfere the expression of Ezrin gene effectively and specifically, and esophagus carcinoma cell strain with lower Ezrin expression is established. The present invention lays foundation for the deep research on the function of Ezrin in esophagus carcinoma and other Ezrin related tumors. The present invention researches the relationship between Ezrin and the generation, development and cleaving proliferation of esophagus carcinoma, and lays foundation for revealing the pathogenesis of esophagus carcinoma and treating esophagus carcinoma.

Description

technical field [0001] The invention relates to the field of siRNA, in particular to the application of siRNA inhibiting the expression of human Ezrin gene in the study of proliferation of esophageal cancer cells. Background technique [0002] RNAi is a process of post-transcriptional silencing of homologous target genes mediated by double-stranded RNA. RNAi can induce the degradation of endogenous target gene mRNA by artificially introducing double-stranded RNA with the same sequence as the endogenous target gene, thereby reducing gene expression. express purpose. As an emerging gene blocking technology, RNAi technology has obvious advantages, such as high specificity, high interference efficiency and simple operation. Therefore, it is widely used in gene function research, antiviral infection, tumor treatment and other hot fields. [0003] Esophageal cancer is a common malignant tumor of the digestive system, which seriously affects human health. The 5-year survival rat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00C12Q1/68C12N15/11C12N15/63C12N15/113
Inventor 许丽艳谢剑君李恩民
Owner SHANTOU UNIV MEDICAL COLLEGE
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