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Novel detection of miRNA for targeting interaction of tumor microenvironment and lung cancer stem cells and application

A technology for lung cancer and cancer, which is applied in the detection and application of miRNA, can solve the problem that the interaction needs to be further studied, and achieve the effect of inhibiting the formation and/or proliferation, improving the targeting, and inhibiting the interaction

Inactive Publication Date: 2018-10-02
BEIJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Therefore, the interaction between lung cancer tissue MSCs and lung cancer cells remains to be further studied

Method used

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  • Novel detection of miRNA for targeting interaction of tumor microenvironment and lung cancer stem cells and application
  • Novel detection of miRNA for targeting interaction of tumor microenvironment and lung cancer stem cells and application
  • Novel detection of miRNA for targeting interaction of tumor microenvironment and lung cancer stem cells and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] (1) 1. Surgery aseptic separation and resection of lung cancer tissue, immediately put in pre-cooled PBS containing double antibody, after the tissue is washed 5 times with PBS containing 10 times double antibody, use sterile bending shears in the ultra-clean table as soon as possible Cut the lung cancer tissue into tissue pieces about 1mm3 in size, and rinse them with complete culture solution 3 times until the supernatant is translucent, discard the supernatant, and add 5ml of PBS+10% FBS containing 0.1% type I collagenase at 37°C, 200 rpm, shake and digest for 1 hour; α-MEM+10% FBS culture solution terminates the digestion, centrifuge at 300g for 10 minutes, and remove the supernatant;

[0048] 2. Resuspend the cells in α-MEM (10% FBS) medium, and count the viable cells in trypan blue.

[0049] 3. Inoculate the cells in 25cm 2 Culture flask, 37℃, 5% CO 2 , Cultivate for 24 hours under saturated humidity conditions, change the medium to remove non-adherent suspension cells...

Embodiment 2

[0054] In this example, the inventor introduced the quantification process of miR-194 after the interaction between MSC derived from lung cancer tissue and lung cancer cells.

[0055] (1) Lung cancer cell lines H460 and H1299 were transfected with GFP lentiviral vector and were GFP positive. Lung cancer MSCs and lung cancer cells were co-cultured at 4:1 for 7 days. During this period, 90% of the cells were digested and passaged, due to tumor cell proliferation ability It is much higher than mesenchymal stem cells. Depending on the situation, a certain number of lung cancer MSCs should be supplemented to ensure the interaction between lung cancer MSCs and lung cancer cells. Seven days later, GFP-positive tumor cells were sorted by flow cytometry, resuspended in Trizol, and stored in a refrigerator at -70°C. Further determination of miRNA expression

[0056] 1. Extraction of total RNA

[0057] (1) After digesting the cells of the experimental group and the control group, wash them wi...

Embodiment 3

[0075] In this example, the inventor described in detail the experimental process of the lentivirus carrying miR-194 infecting lung cancer cells.

[0076] Preparation process of lentivirus carrying miR-194

[0077] (1) Culture 293T cells. The selection is in good growth condition, and it can be transfected when it reaches 80% confluence. It should not be used after passage more than 20 generations.

[0078] (2) Preparation of DNA-Lipofectamine 2000 complex. In a sterile 5ml tube, add 1.5ml serum-free Opti-MEM medium, followed by 4.2μg pLP1, 2μg pLP2, 2.8μg pLP / VSVG and 5μg luciferase and GFP double-labeled lentiviral expression plasmid, gently Mix well. In another sterile 5ml tube, put 42μl Lipofectamine TM 2000 was diluted in 1.5ml serum-free Opti-MEM medium, mixed gently, and placed at room temperature for 5 minutes. Attention, absorb Lipofectamine TM Mix gently before 2000. Mix the above 2 tubes of liquid and mix gently. Incubate at room temperature for 20 minutes to fo...

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Abstract

The invention provides novel detection of miRNA for targeting interaction of a tumor microenvironment and lung cancer stem cells and the application and further provides the application of a reagent in preparation of a medicine. The medicine is used for at least one of the following effects: preventing or treating cancers, inhibiting formation or proliferation of cancer stem cell spheres and inhibiting formation or proliferation of in-vivo cancers. The medicine is used for preventing or treating cancers, the reagent is used for over-expressing nucleic acids, and the nucleic acids have the following nucleotide sequences: 1) a nucleotide sequence shown as SEQ ID NO: 1; 2) identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% and at least 99% of the nucleotide sequence shown as SEQ ID NO: 1; or 3) mutation having one or more nucleotides compared with 1).

Description

Technical field [0001] The present invention relates to the field of biology. In particular, the present invention relates to the use of reagents in the preparation of drugs for the prevention or treatment of cancer. More specifically, the present invention relates to a novel target for the interaction of tumor microenvironment and lung cancer stem cells. Detection and application of miRNA. Background technique [0002] Lung cancer is one of the most malignant tumors in my country and even in the world. Its incidence and recurrence rate are extremely high, and it accounts for a high proportion of cancer-related deaths. In recent years, although the diagnosis and treatment of lung cancer have been greatly improved, the mortality rate is still high and showing a younger trend. With the rapid development of my country’s economy and urbanization, the deterioration of the air environment has also continued to increase, and the PM2.5 index of air pollution has also continued to rise. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886A61K31/7105A61K48/00A61P35/00
CPCA61P35/00A61K31/7105C12Q1/6886C12Q2600/158C12Q2600/136
Inventor 阎新龙岳文裴雪涛刘馨慧杨慧万令飞何丽娟
Owner BEIJING UNIV OF TECH
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