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Application of substance LF3 in regulation and control of expression of DCLK2 protein

A protein and substance technology, applied in the application field of expression, can solve the problems of cumbersome operation, non-specific effect reduction of other irrelevant genes, complex gene structure, etc., to prevent migration and invasion process, omit cell transfection procedures, avoid off-target effects

Pending Publication Date: 2021-06-01
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The gene structure of the DCLK family is relatively complex. Current studies have shown that its members DCLK1 and DCLK2 are positively expressed in tumor cells such as pancreatic cancer, cholangiocarcinoma, esophageal cancer, colorectal cancer, and breast cancer, and may be related to tumor metastasis and poor prognosis. , but its downstream working mechanism is currently unknown
[0003] Because it is a relatively new tumor marker, there is currently a lack of technology to target and regulate the expression of DCLK2 protein in the world. Previously, the shRNA method could only be used to regulate the expression level of DCLK2 in cells, but the operation is cumbersome and the cycle is long (1 to 2 weeks)), and Can have non-specific effects reducing expression of other unrelated genes

Method used

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  • Application of substance LF3 in regulation and control of expression of DCLK2 protein
  • Application of substance LF3 in regulation and control of expression of DCLK2 protein
  • Application of substance LF3 in regulation and control of expression of DCLK2 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0031] Example 1 Substance LF3 regulates the expression of DCLK2 protein

[0032] 1.1 RT-PCR experiment:

[0033] 1.1.1 Configure LF3 concentration of 10 μmol / L and 30 μmol / L treatment solution; get two kinds of breast cancer cells (MDA-MB-231 and MDA-MB-453); treat two kinds of breast cancer cells with two kinds of treatment solution respectively Process for 24 hours.

[0034] 1.1.2 Use TRIzol reagent (TaKaRa, #9109) to lyse the breast cancer cells in 1.1 above to extract total RNA.

[0035] 1.1.3 Use the cDNA synthesis kit (TaKaRa, RR036A) to reverse transcribe 10 μg of the total RNA extracted in step 1.2.1 into DNA. The real-time PCR kit (TaKaRa, RR820A) was used to perform fluorescence quantitative PCR experiments on the DCLK2 gene, and GAPDH was used as an internal reference gene. The instrument used for the PCR reaction was Applied Biosystems 7500 Real-time PCR System (Thermo Fisher).

[0036] The primer sequences used in PCR are listed below:

[0037] Primer sequen...

Embodiment 2

[0051] Example 2 Effect of LF3 on migration and invasion of breast cancer cells

[0052] 2.1 Scratch test:

[0053] Two breast cancer cells (MDA-MB-231 and MDA-MB-453) were divided into 1×10 6 / The density of each well is spread in a six-well plate; respectively cultured in medium with a final concentration of LF3 of 2 μmol / L and 10 μmol / L for 24 hours until the density is 80-90%; discard the medium and wash with PBS buffer Cells in the culture dish twice; use 10 μL plastic pipettetip to draw a scratch with consistent width in the culture well and take pictures, then observe the scratches continuously for several days, and take pictures and record them.

[0054] For best results at 48 hours, see image 3 and Figure 4 , is the cancer cell migration status of two breast cancer cells MDA-MB-231 and MDA-MB-453 treated with LF3 at a concentration of 2 μmol / L and 10 μmol / L at the 48th hour in the scratch test, where image 3 A and 4A are comparisons of migration photos at the ...

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Abstract

The invention relates to the field of biological medicine, in particular to application of a substance LF3 in regulation and control of expression of DCLK2 protein. The application scheme of the substance LF3 comprises a method for regulating and controlling the expression of the DCLK2 protein by the substance LF3, or application of the substance LF3 in preparation of drugs for regulating and controlling the expression of the DCLK2 protein and antitumor drugs. According to the application disclosed by the invention, the LF3 is proved to be a small-molecule inhibitor capable of inhibiting expression of the DCLK2 protein for the first time, migration and invasion processes of breast cancer cells are effectively prevented, a mode and a theoretical basis are provided for research on the DCLK2 protein and even a DCLK family, development of a target inhibitor of the DCLK2 is facilitated, and the LF3 is applied to treatment of diseases such as metastatic breast cancer and the like. Compared with a shRNA method based on plasmid expression, the scheme provided by the invention has the advantages that the effect is more quickly exerted, a tedious cell transfection procedure is omitted, and the off-target effect of the shRNA method is avoided.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to the application of substance LF3 in regulating the expression of DCLK2 protein. Background technique [0002] DCLK2 is a member of the DCLK protein family, its full name is doublecortin like kinase 2, the gene encodes a protein kinase. Its protein contains two N-terminal doublecortin domains, which are responsible for binding microtubules and regulating microtubule polymerization; and a C-terminal serine / threonine protein kinase domain with high homology to calmodulin-dependent protein kinase and a serine / proline-rich domain located between doublecortin and the protein kinase domain, which mediates its interaction with a variety of proteins. The gene structure of the DCLK family is relatively complex. Current studies have shown that its members DCLK1 and DCLK2 are positively expressed in tumor cells such as pancreatic cancer, cholangiocarcinoma, esophageal cancer, colorectal cancer, a...

Claims

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Application Information

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IPC IPC(8): A61K31/635A61P35/00A61P35/04
CPCA61K31/635A61P35/00A61P35/04
Inventor 石坚
Owner SOUTHERN MEDICAL UNIVERSITY
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