Application of long-chain non-coding RNA in preparation of tumor angiogenesis inhibitor

A long-chain non-coding, tumor vascular technology, applied in the field of genetic medicine, can solve the problems of unclear long-chain non-coding RNA related mechanisms, cell cycle arrest, etc.

Inactive Publication Date: 2019-08-16
SUZHOU UNIV
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  • Application Information

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Problems solved by technology

In the prior art, when conducting basic and clinical research on leukemia, some long non-coding RNAs were obtained through gene chip identification technology, which are closely related to AML. Malignant biological behaviors have a negative regulatory effect, which can inhibit the growth of tumor cells and arrest the cell cycle in the G2 / M phase; however, the relevant mechanisms of these long non-coding RNAs are still unclear

Method used

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  • Application of long-chain non-coding RNA in preparation of tumor angiogenesis inhibitor
  • Application of long-chain non-coding RNA in preparation of tumor angiogenesis inhibitor
  • Application of long-chain non-coding RNA in preparation of tumor angiogenesis inhibitor

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1 Angiogenesis Experiment

[0026] The cloned long-chain non-coding RNA (sequence is SEQ ID NO: 1, prepared according to the existing method) was loaded on a conventional lentiviral vector, and directly infected K562 cells to construct a long-chain non-coding RNA high-expression stable cell line (LLEST). Virus-infected cells were used as controls (non-highly expressed leukemia K562 stable cell lines); the expression results of the two cell lines are shown in the appendix figure 1 . The specific methods of cloning, loading and infection are conventional techniques.

[0027] Angiogenesis Experimental Procedures

[0028] (1) Pre-cool the 96-well plate, spare cell supernatant, 200ul tip, and pipette in a 4°C refrigerator or on ice for 30 minutes, take out the Matrigel Matrigel from the -20°C refrigerator, and place it at 4°C Refrigerator until it thaws;

[0029] (2) Laying gel: Take 50 μl / well Matrigel matrigel and add it to a 96-well plate, and do it on ice. Av...

Embodiment 2

[0034] Example 2 Detection of PDGFA gene level

[0035] According to the method of Example 1, leukemia K562 stable cell lines with high expression and non-high expression of long-chain non-coding RNA were constructed, the cells were collected, RNA was extracted by Trizol one-step method, and the PDGFA gene level was detected by qRT-PCR method. Compared with the blank group, the PDGFA gene level decreased in the long non-coding RNA high expression group, see appendix image 3 . The existing commercially available Oblimersen drug (which is a single-stranded DNA molecule consisting of 18 bases and complementary to Bcl-2 mRNA, which can inhibit the expression of Bcl-2 family proteins by inhibiting Bcl-2 mRNA) was used for comparative experiments. According to the above method, it was found that compared with the blank group, the PDGFA gene expression level was similar (the difference was less than 3%), and it had almost no inhibitory effect on tumor angiogenesis. At the same tim...

Embodiment 3

[0036] Example 3 Detection of PDGFA protein level

[0037] According to the method of Example 1, leukemia K562 stable cell lines with high expression and non-high expression of long-chain non-coding RNA were constructed, and the cell supernatant was collected after routine culture, and the PDGFA protein level was detected by ELISA method. In the leukemia cell line K562, compared with the control group, the expression level of PDGFA decreased in the long-chain non-coding RNA high expression group, see appendix Figure 4 .

[0038] The invention packs long-chain non-coding RNA into a lentiviral vector to construct an angiogenesis inhibitor, detect angiogenesis, and detect tumor cell PDGFA gene and protein levels. By inhibiting its expression with gene medicine, it can inhibit the formation of tumor blood vessels, reverse the source of oxygen and nutrients for tumor cells, thereby inhibiting the growth of tumors and improving the anti-tumor efficacy.

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Abstract

The invention discloses application of long-chain non-coding RNA in the preparation of a tumor angiogenesis inhibitor. The influence and mechanism of a long-chain non-coding RNA gene on tumor angiogenesis are disclosed for the first time, tumor angiogenesis can be inhibited by influencing PDGFA level, and patients can be benefited during leukemia treatment.

Description

technical field [0001] The invention belongs to gene medicine technology, in particular to an angiogenesis inhibitor based on long chain non-coding RNA. Background technique [0002] Tumor angiogenesis is closely related to tumor growth and metastasis. Tumor growth can be divided into avascular phase and vascular phase. In the avascular phase, tumors mainly rely on the diffusion of surrounding tissues to obtain nutrients and excrete metabolites. The tumor diameter does not exceed 1~ At the vascular stage, new capillaries appear in the tumor and gain the ability to grow further, and the tumor grows rapidly and metastasizes. Many studies have found that the high expression of bcl-2 gene is related to the inhibition of tumor angiogenesis, and mt-p53 gene may promote tumor angiogenesis by down-regulating bcl-2. [0003] Long non-coding RNA (Long non-coding RNA, LncRNA) accounts for about 41% of non-coding RNA (non-coding RNAs, ncRNAs), which does not have the function of coding...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/7105A61P35/00
CPCA61K31/7105A61P35/00
Inventor 祁小飞
Owner SUZHOU UNIV
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