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A kind of siRNA of silencing type IV collagen expression and its application

A collagen and silencing technology, applied in the field of RNA silencing, can solve the problems of systematic research on tumor infiltration and migration without tumor's own secretion of ECM, inhibition of tumor infiltration and migration, and lack of research reports.

Active Publication Date: 2021-09-14
THE SECOND AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The research on the effect of ECM on tumor invasion and migration in the tumor microenvironment mostly focuses on the research on the ECM produced by fibroblasts. There are no reports on the regulation of ECM, but there is no systematic study on the effect of tumor self-secretion of ECM on tumor invasion and migration, and there is no strategy targeting the tumor microenvironment or tumor cells themselves to inhibit the expression of tumor self-ECM to inhibit tumor invasion and migration; tumor microenvironment and tumor self-secretion The relationship between ECM, the mechanism of regulating the secretion of ECM by tumor cells, and the specific role of ECM secreted by tumor cells in tumor migration have not been reported; no research reported

Method used

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  • A kind of siRNA of silencing type IV collagen expression and its application
  • A kind of siRNA of silencing type IV collagen expression and its application
  • A kind of siRNA of silencing type IV collagen expression and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Cell Culture and Treatment

[0039] Purpose of the experiment: To prepare cell samples for subsequent experiments.

[0040] Experimental materials: Liver cancer cell line SMMC-7721 was purchased from Subcomb Biotechnology Co., Ltd. (Shanghai, China); liver cancer cell line HepG2 and breast cancer cell line MDA-MB-231 were purchased from the American Germplasm Collection Center (ATCC, VA , United States) obtained. All cells were cultured in Dulbecco's modified Eagle medium (DMEM, Gibco, NY, USA) containing 10% fetal bovine serum (FBS, Gibco, NY, USA).

[0041] Experimental procedure: All cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C, containing 5% carbon dioxide concentration, under humid conditions.

Embodiment 2

[0042] siRNA preparation and transfection of embodiment 2 liver cancer cells

[0043] Purpose of the experiment: To prepare liver cancer cells knocked down by CoL4α1, PITPNM3, and Snail genes for subsequent experiments.

[0044] Experimental materials: HepG2 mediated by liposome-mediated liver cancer cell transfection, SMMC-7721, serum-free medium Opti-mem I (Invitrogen, CA, USA), Lipofectamine2000 reagent (Invitrogen, CA, USA), siRNA produced by Ribobio Synthesized by Science and Technology (Guangzhou, China), the base sequence is shown in Table 1 below.

[0045] Table 1 Oligonucleotide sequences of siRNAs

[0046]

[0047]

[0048] Experimental steps:

[0049] 1) Inoculate cells into a 6-well plate one day before transfection, so that the cell density is about 60% on the day of transfection, and culture the cells in 10% FBS DMEM medium without antibiotics. Remove the medium before adding siRNA, and add 400ul serum-free DMEM to each well to remove the influence of se...

Embodiment 3

[0053] Example 3 CCL18 Promotes the Expression of Type IV Collagen in Tumor Cells

[0054] Purpose of the experiment: To detect the ability of the cells obtained in Examples 1 and 2 to express collagen IV under the action of CCL18, and to verify the inhibitory effect of siRNA on the expression of collagen IV in liver cancer cells mediated by CCL18.

[0055] Experimental materials: recombinant human CCL18 cytokine (PeproTech, NJ, USA), DMEM medium (DMEM, Gibco, NY, USA), fetal bovine serum (FBS, Gibco, NY, USA), rabbit anti-human Collagen IV polyclonal antibody (Cell Signaling technology, MA, U.S.), rabbit anti-human GAPDH polyclonal antibody (Cell Signaling technology, MA, U.S.), PVDF membrane (Millipore, MA, U.S.), protein marker (Thermo, MA, U.S.), skimmed milk powder (Erie, China), phenylmethylsulfonyl fluoride (PMSF, Shanghai, Shanghai, China), phosphatase inhibitor cocktail (China, Shanghai, China), RIPA cell lysate (China, Shanghai, China), ECL luminescent liquid (Thermo...

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Abstract

The invention discloses the application of siRNA capable of silencing the expression of type IV collagen in the preparation of antitumor drugs. The present invention utilizes the strategy of RNA interference to silence the expression of type IV collagen of liver cancer cells, which can inhibit the role of CCL18 derived from tumor-associated macrophages in promoting the invasion and migration of liver cancer, thereby achieving the purpose of anti-tumor.

Description

technical field [0001] The invention relates to the technical field of RNA silencing, in particular to an siRNA for silencing the expression of type IV collagen and its application. Background technique [0002] The distant metastasis of tumor is realized through the blood circulation or lymph circulation. Previous studies believed that tumor cells migrated through the blood circulation or lymph circulation in the form of single cells. Recent studies believed that tumor cells migrated in the form of a cell cluster. Migrating cells The clumps contain tumor cells, fibroblasts, immune cells and extracellular matrix (ECM). In essence, tumor cells migrate with the "soil" they depend on for survival, which can avoid anoikis to the greatest extent. In the migrating cell mass, the extracellular matrix secreted by the tumor cells themselves plays a key role. [0003] In the tumor microenvironment, CCL18 (a chemokine) derived from tumor-associated macrophages (TAM) binds to the funct...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K31/713A61P35/00
Inventor 陈静琦张慧曾波航朱必胜
Owner THE SECOND AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV
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