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Method and reagent for inhibiting invasiveness of cancer cells

A technology of inhibitors and cells, applied in pharmaceutical formulations, medical preparations containing active ingredients, antineoplastic drugs, etc.

Inactive Publication Date: 2012-09-12
山东益康药业股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the role of miR-320 in tumorigenesis, migration, and invasion has not been reported in this field at present.

Method used

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  • Method and reagent for inhibiting invasiveness of cancer cells
  • Method and reagent for inhibiting invasiveness of cancer cells
  • Method and reagent for inhibiting invasiveness of cancer cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0142] Example 1, Pfn1 3'UTR contains the target site of conserved miR-320a

[0143] To study the role of miR-320a in tumorigenesis, the inventors first used a recently reported biochemical approach (see Karginov FV et al. A biochemical approach to identifying microRNA targets. Proceedings of the National Academy of Sciences of the United States of America 2007; 104(49): 19291-6) identified the target gene of miR-320a. The main principle of this method is to co-immunoprecipitate the miRNA and its target gene mRNA in RISC with the antibody against Ago2, the core component of RISC, The species of target genes of the corresponding miRNAs are then identified. As a result, the present inventors identified Pfn1 as a candidate target gene of miR-320a.

[0144] Then, the inventors used miRanda (http: / / www.microrna.org / microrna / home.do), TargetScan (http: / / www.targetscan.org / ) and PicTar (http: / / www.pictar.org / ) and other miRNA target gene prediction software also predicted that Pfn...

Embodiment 2

[0146] Example 2, Pfn1 protein expression level and miR-320a expression level are negatively correlated

[0147] First, the inventors detected the relationship between miR-320a and Pfn1 expression in some cell lines, such as figure 2 a-d. In lung cancer cell lines A549 and 95-D, the expression level of Pfn1 mRNA in 95-D cells was 5-fold higher than that in A549, while the level of Pfn1 protein in 95-D cells was lower than that in A549, which indicated that the protein level of Pfn1 in 95-D The translation process may be blocked. In comparison, the expression level of miR-320a in 95-D cells was 6-fold higher than that in A549 cells. Similar results were also observed in prostate cells PC-3 and DU-145. All in all, a negative correlation between the protein expression level of Pfn1 and the expression level of miR-320a can be observed in these cells tested.

[0148] These results above suggest that miR-320a negatively regulates the protein expression of Pfn1 at the post-trans...

Embodiment 3

[0149] Example 3, MiR-320a can act on the 3'UTR of Pfn1 mRNA to inhibit the expression of Pfn1

[0150] In order to further confirm that miR-320a can directly target and regulate Pfn1, the inventors then detected whether miR-320a can bind to the 3'UTR of Pfn1 mRNA and prevent protein translation. For this reason, the inventor has constructed the following reporter gene carrier: the 3'UTR of the full-length Pfn1 of people is cloned into the 3'UTR region of the luciferase reporter gene (Wt UTR, image 3 a), while also constructing a control vector, that is, a vector with 3 nucleotide mutations in the seed region of the target site of miR-320a (Mut UTR, image 3 a) To remove the binding of miR-320a to this site.

[0151] The above-mentioned vectors were co-transfected with miR-320a or control miRNA into 293T cells, or on this basis, antisense nucleic acid of miR-320a (anti-miR-320a) or control antisense nucleic acid (Controlanti-miR) was transfected to To inhibit the function of ...

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Abstract

The invention relates to a method and a reagent for inhibiting invasiveness of cancer cells, discloses the application of a miT-320a inhibitor which is used for preparing a composition for inhibiting tumor cells, and the application of a Pfnl inhibitor which is used for preparing the composition for inhibiting tumor cells, and further discloses a method for screening potential substances for inhibiting tumors. The invention discloses the correlation between miR-320a and the migration and invasion of tumor cells for the first time, and Pfnl participates in and mediates the process, therefore, a new target is provided for the prevention and treatment of tumors.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention relates to a method and a reagent for inhibiting the invasiveness of cancer cells. Background technique [0002] Tumor metastasis is an important cause of death in cancer patients. Tumor metastasis goes through a complex process: first, primary tumor cells invade adjacent tissues and enter lymphatic vessels or blood vessels, that is, enter blood vessel infiltration (Intravasation); then migrate through blood vessels into distal capillaries; Infiltration (Extravasation), and finally through proliferation from micrometastases (Micrometastases) to macroscopic metastases. Factors associated with tumor metastasis have been identified, such as growth factors, cytokines, chemokines, precursors of angiogenic factors, extracellular matrix remodeling molecules, and certain transcription factors. But so far, the understanding of the molecular and cellular mechanisms of tum...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K45/00A61K48/00A61P35/00
Inventor 金由辛史毅赵波涛
Owner 山东益康药业股份有限公司
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