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Application of miRNA-214 inhibitor for inhibition of regulatory T cells

A miRNA-214, inhibitor technology, applied in the field of tumor therapy

Active Publication Date: 2015-07-01
JIANGSU MICROMEDMARK BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cell proliferation and apoptosis are often abnormal in tumors, so it is speculated that the abnormal deletion, mutation or overexpression of miRNA will lead to the occurrence of human diseases.

Method used

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  • Application of miRNA-214 inhibitor for inhibition of regulatory T cells
  • Application of miRNA-214 inhibitor for inhibition of regulatory T cells
  • Application of miRNA-214 inhibitor for inhibition of regulatory T cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Example 1 Construction of tumor transplantation model mice

[0100] First, mouse sarcoma S-180 cells (from the Cell Bank of the Chinese Academy of Sciences) and mouse Lewis lung cancer cells (LLC) (from the Cell Bank of the Chinese Academy of Sciences) were selected to construct tumor transplantation model mice; Plasma and cell microparticles (MVs); Real time-PCR method was used to detect the expression level of miR-214 in the plasma and cell microparticles of mice with different tumor transplantation models.

[0101] 1.1 Construction of mouse sarcoma S-180 cell transplantation model mice and Lewis lung cancer cell (LLC) transplantation model mice

[0102] C57BL / 6J mice were selected and divided into three groups:

[0103] i. Control group: C57BL / 6J mice, inject 100uL normal saline subcutaneously;

[0104] ii. Sarcoma S-180 cell transplantation model mice: C57BL / 6J mice, subcutaneously injected with sarcoma S-180 cells (100uL, 10 6 cells / only) to establish a tumor mo...

Embodiment 2

[0118] Example 2 LLC MVs promote Treg proliferation and tumor growth

[0119] The first group of experiments: first collect LLC MVs, and then inject LLC MVs intravenously into C57BL / 6J mice. On the 14th day, take the whole blood of the mice, T cells in the spleen, and collect blood and spleen samples; then use the Real-time PCR method To detect the expression level of miR-214 in plasma, the eBiosience Treg flow detection kit (provided by eBiosience) was used to detect CD4 in the whole blood and spleen of each group of mice on a flow cytometer. + CD25 high Foxp3 + cell content. The schematic diagram of the experimental design process is shown in Figure 2A .

[0120] The second set of experiments: first collect LLC MVs, then inject LLC MVs intravenously into C57BL / 6J mice, transplant s-180 tumors to the mice on the 14th day, collect blood and tumor tissue samples on the 24th day; then use Real-time PCR The method was used to detect the expression level of miR-214 in plasma...

Embodiment 3

[0142] Example 3 Effect of miR-214-deleted cell microparticles on Treg cells

[0143] Methods: Antisense oligonucleotide (antisense oligonucleotide) was used to knock down miR-214 in LLC cells, and the MVs in the culture medium were collected to obtain miR-214-depleted cell microparticles derived from LLC cells; then Miltenyi CD4+ T cell magnetic bead sorting kit (Miltenyi Biotechnology Co., Ltd., Germany) sorted and cultured CD4+ T cells in mouse spleen; the eBiosience Treg flow detection kit (eBiosience Co., Ltd.) was used to detect on the flow cytometer mouse CD4 in each group + CD25 high Foxp3 + cell content.

[0144] 3.1 Culture of mouse Lewis lung cancer cell line (LLC cells) (from the Cell Bank of the Chinese Academy of Sciences)

[0145] Under the culture conditions of complete medium, high glucose DMEN, 10% FBS, 4mmol / L glutamine, and double antibodies, the LLC mouse Lewis lung cancer cell line (from the Cell Bank of the Chinese Academy of Sciences) was cultivated...

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Abstract

Provided is a use of miRNA-214 inhibitor in inhibiting regulatory T cells (Treg cells). The microRNA-214 (miRNA-214) can promote Treg cells, and can assist tumours in immune escape. The experiments demonstrate that the inhibition of miRNA-214 can inhibit the growth of Treg cells, thus inhibiting the growth of tumours. Therefore, miRNA-214 can be used for developing an anti-tumour drug or a drug inhibiting immune response hyperactivity.

Description

technical field [0001] The present invention relates to the field of tumor treatment, in particular to the application of miRNA-214 inhibitors for suppressing regulatory T cells (Treg) and tumor immune escape, thereby preventing or treating tumors Background technique [0002] microRNA is derived from the long-chain RNA initial transcription product (Pri-miRNA) with a length of about 1000bp, and the Pri-miRNA molecule is cleaved by Drosha enzyme in the nucleus to form a miRNA precursor with a stem-loop structure of about 60-80nt in length. Premise After the miRNA is transported to the cytoplasm, it is further cleaved into a double-stranded miRNA of about 18-26 nt. After the double-stranded miRNA is unwrapped, the mature miRNA enters into the RNA-induced gene silencing complex (RNA-induced silencing complex, RISC), complete or incomplete pairing with the complementary mRNA, and degrades the target mRNA or represses its expression. [0003] Although the proportion of microRNA...

Claims

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Application Information

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IPC IPC(8): A61K48/00C12Q1/68C12Q1/04A61P35/00
CPCA61K48/00C12N15/113A61P35/00C12N2310/113C12Q1/6876C12Q2600/106C12Q2600/158
Inventor 张辰宇曾科张峻峰殷媛蔡星
Owner JIANGSU MICROMEDMARK BIOTECH
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