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Small molecule noncoding RNA gene hsa-mir-101 and antineoplastic use thereof

A tumor and tumor cell technology, applied in anti-tumor drugs, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of unseen hsa-miR-101 and primary liver cancer research reports, etc., to reduce proliferation and incidence. Tumor ability, the effect of promoting sensitivity

Inactive Publication Date: 2008-12-31
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] After searching the domestic and foreign literature of the prior art, so far there is no research report on the relationship between hsa-miR-101 and primary liver cancer

Method used

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  • Small molecule noncoding RNA gene hsa-mir-101 and antineoplastic use thereof
  • Small molecule noncoding RNA gene hsa-mir-101 and antineoplastic use thereof
  • Small molecule noncoding RNA gene hsa-mir-101 and antineoplastic use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Example 1: Detection of the expression level of hsa-miR-101 in liver cancer tissue specimens

[0015] Extract total RNA from liver cancer and non-cancerous liver tissues, mix the RNA sample with RNA loading buffer in a ratio of 3:1, denature at 80°C for 10 minutes, and then place it on ice for 5 minutes; Under 10W electric power, use 15% denaturing polyacrylamide gel for about 40min electrophoresis separation; after electrophoresis is completed, cut the corners of the gel and mark, assemble the gel and membrane in order in the semi-dry transfer tank, constant flow 8mA / cm 2 Transfer the membrane for 1 hour; remove the Hybond N+ nylon membrane and cut the corner to make a mark, 70,000μJ / cm 2 UV cross-link and dry bake at 80°C for 2h; after wetting the nylon membrane with 2×SSC, spread it on the inner wall of the hybridization tube, and remove the air bubbles between the membrane and the tube wall, add the pre-hybridization solution, and put it into the hybridization box. Rota...

Embodiment 2

[0017] Example 2: Overexpression of hsa-miR-101

[0018] Introducing the small RNA of hsa-miR-101 into cells is achieved by using Invitrogen's transfection reagent Lipofectamine RNAi MAX to reverse. Take a 24-well plate as an example. Other well plates can be scaled up according to the size of the wells. Or shrink: trypsinize the cells and resuspend them in DMEM medium containing 10% FBS without dual antibody to make the cell density 3×10 4 Cells / ml; dilute small RNA in 100μl opti-MEM, tap the tube wall to mix; add 1μl Lipofectamine RNAi MAX to each tube, tap the tube wall to mix, and leave it at room temperature for 15 minutes; add RNAiMAX / siRNA diluent Then add 500μl of cell diluent to the 24-well plate (to make the cell density approximately 30-50% confluent after 24h of transfection), mix well and incubate in an incubator for a certain period of time before performing related functional tests.

Embodiment 3

[0019] Example 3: Detection of cell apoptosis

[0020] After processing the cells as needed, aspirate the culture supernatant, fix with 4% paraformaldehyde at room temperature for 15 minutes, stain with 1μg / ml (final concentration) DAPI staining solution at room temperature for 5 minutes, and observe the nucleus morphology under a Lycra fluorescence microscope. Cells with chromatin constriction and DNA fragmentation are considered to be apoptotic cells, and the apoptosis rate is calculated according to the following method:

[0021] Apoptosis rate (%) = number of apoptotic cells / total number of cells (total number of cells ≥500)

[0022] We tested the effect of hsa-miR-101 on tumor cell apoptosis under the conditions of serum starvation and chemotherapeutic drug treatment, and found that the experimental group overexpressing hsa-miR-101 in HepG2 cells (ATCC in the United States) died after 48 hours of serum starvation. The death rate is ~2 times that of the negative control (NC) g...

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Abstract

The invention discloses a small-molecular RNA gene hsa-miR-101 relevant to a primary hepatocellular carcinoma as well as an application thereof. The invention also provides a method for analyzing the correlation between the small-molecular RNA gene and the tumor formation and development, as well as purposes of the small-molecular RNA gene hsa-miR-101 at the aspect of primary liver cancer treatment. The invention aims to provide the small-molecular RNA gene hsa-miR-101 acting as the application of tumor therapeutic drugs, and has great practical significance and broad applying prospect in the medical and biopharmaceutical fields.

Description

Technical field [0001] The present invention relates to a small molecule non-coding RNA gene hsa-miR-101 related to primary liver cancer, and the use of the gene in assisting diagnosis and treatment of primary liver cancer. The invention belongs to the field of biotechnology. technical background [0002] Primary hepatocellular carcinoma is currently one of the most common malignant tumors in the world. my country is the main place where liver cancer occurs. The incidence of liver cancer is as high as 100,000 molecules. In recent years, the incidence of liver cancer has increased significantly, which has seriously endangered human health and life safety. [0003] MicroRNA (microRNA) is a type of small non-coding ribonucleic acid that is widely present in various biological species, and its length is about 20-22 bases. It does not code for proteins or polypeptides, but through specific binding with the 3'UTR (3' untranslated region) of the mRNA (messenger RNA) of the target gene t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11A61K31/7088A61P35/00C12N15/113
Inventor 庄诗美苏杭杨建荣杨金娥程家森
Owner SUN YAT SEN UNIV
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