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Inhibitor for targeted therapy of lung cancer, application of inhibitor as well as application of KAP1 as drug target in screening of anti-lung cancer drugs

An anti-lung cancer drug and targeted therapy technology, applied in the field of biomedicine, can solve problems such as strong toxic and side effects of drugs, poor treatment effect, and adverse reactions.

Inactive Publication Date: 2018-03-23
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the types of drug targets for lung cancer have been reported in the current prior art, some drugs screened by targets will produce adverse reactions after administration, such as strong drug side effects; and some drug targets have poor therapeutic effects. It is easy to delay the patient's condition and miss the best treatment period

Method used

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  • Inhibitor for targeted therapy of lung cancer, application of inhibitor as well as application of KAP1 as drug target in screening of anti-lung cancer drugs
  • Inhibitor for targeted therapy of lung cancer, application of inhibitor as well as application of KAP1 as drug target in screening of anti-lung cancer drugs
  • Inhibitor for targeted therapy of lung cancer, application of inhibitor as well as application of KAP1 as drug target in screening of anti-lung cancer drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Lung cancer tissues and adjacent normal tissues from 30 patients with non-small cell lung cancer in the Thoracic Surgery Department of the First People's Hospital of Yunnan Province were selected as test samples.

[0058] The tumor tissue and paracancerous tissue were put into different cryopreservation tubes, directly stored in liquid nitrogen, and stored in dry ice during transportation. To lyse cells, take a tissue block the size of a grain of rice, wash the blood stains of the tissue block with ice-cold PBS solution, remove excess liquid, and add 200 μL of RIPA lysate containing protease inhibitors. Use a tissue grinder to grind the tissue in a 1.5mL centrifuge tube. During the grinding process, do not leave the ice. Pause for 10 seconds every 5 seconds to prevent the tissue from overheating and destroying the integrity of the protein. It is completely normal that some connective tissue cannot be ground. After grinding, the solution was placed on ice for half an ho...

Embodiment 2

[0073] Plasmid construction

[0074] 1: Design and synthesis of gRNA

[0075] Design gDNA sequence

[0076] To design a pair of gDNA of about 20bp, we can design through the following online tools:

[0077] CRISPRDesign at MIT: http: / / crispr.mit.edu /

[0078] Design the gRNA sequence targeting the gene encoding KAP1, as shown in the following table:

[0079] Table 1 gRNA-KAP1 nucleic acid sequence

[0080]

[0081] Note: The lowercase part is the nucleic acid sequence connected to the plasmid, and the uppercase part is the gRNA sequence

[0082] 2: Cloning sgRNA into pSpCas9(BB) plasmid

[0083] (I) Each gRNA was dissolved to a final concentration of 100 μM, and the sgRNA was phosphorylated and annealed according to the following recipe.

[0084] Element

Dosage (ul)

sgRNA forward sequence (100uM)

1

sgRNA reverse sequence (100uM)

1

T4 ligation buffer 10X

1

T4PNK

1

water

6

Total

10

[...

Embodiment 3

[0111] Cell Proliferation:

[0112] 1. Observe the cells under a microscope. When the confluence of the A549-KAP1-KO cells prepared in Example 2 reaches 80% to 90%, discard the medium, add PBS solution to gently rinse, and discard the rinse solution.

[0113] 2. After adding a certain amount of trypsin, put the cells in 37°C, 5% CO 2 Put it in the cell culture incubator for about 3 minutes, then take it out and observe it under the microscope until the cells become round, then add 10% fetal bovine serum medium into the ultra-clean workbench to stop the digestion, and use a pipette to blow back and forth to break up the cells .

[0114] 3. Cell counting: take the hemocytometer, make sure it is clean, you can wipe it with alcohol, cover the cover glass on the hemocytometer, and cover the area with small squares on the hemocytometer. Use a micropipette to take 10 μL of the cell suspension and inject it from one end of the hemocytometer into the hemocytometer tank. Due to capill...

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Abstract

The invention provides an inhibitor gRNA for targeted therapy of lung cancer, application of the inhibitor as well as application of a KAP1 encoding gene as drug target in screening of anti-lung cancer drugs, belonging to the technical field of biological medicines. The inhibitor gRNA for the targeted therapy of the lung cancer can effectively inhibit the expression of the KAP1 encoding gene, thusremarkably inhibiting cell proliferation. The invention also discloses the application of the KAP1 encoding gene as the drug target in screening of the anti-lung cancer drugs. By detecting the expression level of KAP1 in clinical lung cancer tissues and adjacent normal lung tissues, the expression level of the KAP1 in the clinical lung cancer tissues is proved to be obviously higher than that inthe adjacent normal lung tissues. By inhibiting the expression level of the KAP1, the cell proliferation and cell migration are effectively inhibited, a theoretical basis is provided for clinical treatment of the lung cancer, and the new drug target is screened for application.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to an inhibitor of targeted therapy for lung cancer and its application, and the application of KAP1 as a drug target in screening anti-lung cancer drugs. Background technique [0002] At present, lung cancer is one of the most lethal tumors in the world, especially non-small cell lung cancer, which seriously threatens human health and life. Therefore, finding effective drugs and methods for treating lung cancer and completely conquering lung cancer is an important research topic in the medical field in my country and even in the world. [0003] In recent years, the research of oncology has developed very rapidly in my country and the world, and the continuous progress of science and technology has greatly promoted the treatment of tumors. With the advancement of tumor molecular biology research, the research on lung cancer-related genes has become increasingly in-d...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61K48/00A61P35/00
CPCA61K48/005C12N15/113C12N2310/10
Inventor 徐天瑞吴国金郭晓汐黄颖彭俊安输
Owner KUNMING UNIV OF SCI & TECH
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