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Method for mediating pyroptosis of lung adenocarcinoma NCI-H1299 cells by newly discovered miRNA-mRNA (micro ribonucleic acid-messenger ribonucleic acid) regulatory axis

A technology of NCI-H1299, regulating axis, applied in the field of biotechnology application, can solve the problem that the application of miR-1228-5p-PRKCDBP regulating axis cannot be mentioned, and achieve the effect of obvious clinical application value

Active Publication Date: 2021-06-29
SHANGHAI UNIV OF T C M
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] None of the existing studies have directly reported the application of miR-1228-5p and PRKCDBP in inhibiting lung adenocarcinoma NCI-H1299 through the mechanism of pyroptosis, and the application of the miR-1228-5p-PRKCDBP regulatory axis in anti-lung cancer has not been mentioned.

Method used

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  • Method for mediating pyroptosis of lung adenocarcinoma NCI-H1299 cells by newly discovered miRNA-mRNA (micro ribonucleic acid-messenger ribonucleic acid) regulatory axis
  • Method for mediating pyroptosis of lung adenocarcinoma NCI-H1299 cells by newly discovered miRNA-mRNA (micro ribonucleic acid-messenger ribonucleic acid) regulatory axis
  • Method for mediating pyroptosis of lung adenocarcinoma NCI-H1299 cells by newly discovered miRNA-mRNA (micro ribonucleic acid-messenger ribonucleic acid) regulatory axis

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] RT-qPCR primer design for miR-1228-5p.

[0047] Experimental method: According to the principle of RT-qPCR primer design, use Primer5.0 software to design real-time quantitative fluorescent PCR primers.

[0048] Experimental results: RT-qPCR primers for miR-1228-5p were obtained, and the primer sequences are shown in SEQ ID NO: 4-6.

Embodiment 2

[0050] The miR-1228-5p inhibitors used for miR-1228-5p knockdown were constructed.

[0051] Experimental method: According to the miR-1228-5p sequence, miR-1228-5p / NC inhibitors were designed and chemically synthesized. The inhibition efficiency of the obtained inhibitors was detected by NCI-H1299 cell transfection and RT-qPCR.

[0052] Experimental results: miR-1228-5p / NC inhibitor was obtained, the sequence of which is shown in SEQ ID NO: 2-3. After the obtained inhibitors were transfected, the miR-1228-5p gene was significantly decreased compared with the normal cells and the empty plasmid transfection group. figure 1 As shown, the expression level of miR-1228-5p was 0.14-0.25 of the blank cells, and miR-1228-5p inhibitors were successfully constructed.

Embodiment 3

[0054] After miR-1228-5p inhibitors transfected NCI-H1299 cells, Annexin V / PI flow cytometry cell death detection.

[0055] Experimental method: Normal cultured NCI-H1299 cells were mixed with 5×10 5 pcs / ml density, 2ml / well seeded into 6-well plate. After 24 H adherent culture, miR-1228-5p / NC inhibitors were transfected into cells using Lipofectamine 2000 and Opti MEM. After 24 H intervention, the cells were collected, and the cell death was detected by using the FITC-Annexin V flow cytometry kit.

[0056] Experimental results: if figure 2 As shown, the death number of lung adenocarcinoma NCI-H1299 cells transfected with miR-1228-5p inhibitors was significantly higher than that of normal culture and empty plasmid transfection, indicating that miR-1228-5p inhibitors have a significant effect on lung adenocarcinoma NCI-H1299 cells has a significant inhibitory effect.

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Abstract

The invention relates to an action mechanism and an implementation method for mediating pyroptosis of lung adenocarcinoma NCI-H1299 cells by a newly discovered miRNA-mRNA (micro ribonucleic acid-messenger ribonucleic acid) regulatory axis, namely a miR-1228-5p-PRKCDBP regulatory axis, and belongs to the field of biotechnology application. The invention provides the application of miR-1228-5p and PRKCDBP in the field of pyroptosis of the lung adenocarcinoma NCI-H1299 cells for the first time, provides construction of a miR-1228-5p knockdown vector and a PRKCDBP overexpression vector, and defines the action mechanism of mediating pyroptosis of the lung adenocarcinoma NCI-H1299 cells by the miR-1228-5p knock-down vector and the PRKCDBP overexpression vector. Meanwhile, the interaction relationship between the miR-1228-5p and the PRKCDBP gene is proved in vitro, and a basis for the existence of the miR-1228-5p-PRKCDBP regulatory axis is provided. The invention provides the new mechanism of mediating the pyroptosis of the lung adenocarcinoma NCI-H1299 cells by knocking down the miR-1228-5p, overexpressing the PRKCDBP and the miR-1228-5p-PRKCDBP regulatory axis, so that the development and the preparation of the anti-lung adenocarcinoma medicines and the gene editing therapy according to the miR-1228-5p-PRKCDBP regulatory axis have obvious clinical application values.

Description

technical field [0001] The invention relates to a method for newly discovering that a miRNA-mRNA regulation axis mediates pyroptosis of lung adenocarcinoma NCI-H1299 cells, and belongs to the field of biotechnology applications. Background technique [0002] Lung cancer is still the tumor with the highest mortality rate in the world. With the development of science and technology, although the treatment methods for lung cancer are constantly improving, the clinical effect is still limited. Modern medicine believes that the regulation of genes plays a decisive role in the occurrence, development and outcome of diseases. The idea of ​​gene therapy is expected to break through the shackles of traditional treatments in the future and become an important means to cure diseases. [0003] NCI-H1299 cells are a typical p53 gene-deficient lung adenocarcinoma cells. The p53 gene is an important tumor suppressor gene, and mutations of this gene occur in more than 50% of all malignant...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/7088A61P35/00C12N15/85C12Q1/02
CPCA61K31/7088A61P35/00C12N15/85G01N33/5011G01N2500/10
Inventor 蒋日磊许家佗刘平李倩陈筱雷
Owner SHANGHAI UNIV OF T C M