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Antitumor application of has-miR-520e

A technology of has-mir-520e and uses, which is applied in the field of treating primary hepatocellular carcinoma to achieve the effect of reducing proliferation

Inactive Publication Date: 2012-02-22
TIANJIN TOPTECH BIO SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Has-mir-520e is located at 19q13.42, there is no report on the relationship between has-mir-520e and the diagnosis and treatment of liver cancer

Method used

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  • Antitumor application of has-miR-520e
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  • Antitumor application of has-miR-520e

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Real-time quantitative PCR analysis of has-miR-520e expression:

[0020] 1. Cell line:

[0021]

[0022] Sample preparation: Surgical resection specimens from 11 cases of primary hepatocellular carcinoma (HCC) were collected, frozen at -80°C, and transported in liquid nitrogen. According to clinicopathological analysis, all belonged to stage II and III, and the specimens were provided by Tianjin First Central Hospital.

[0023]

[0024] HBsAg: positive (+) or negative (-);

[0025] 2. Extraction of total RNA: Trizol reagent was used to extract the total RNA of cells and tissues, and Trizol reagent was purchased from Invitrogen

[0026] 1) Add 1ml Trizol to the cell culture bottle, let it react at room temperature for 15min, repeatedly suck and mix with the tip of the pipette, transfer to RNase-free 1.5ml Ep tube; or take 0.1-0.2g of fresh animal tissue and put it in a tissue homogenizer Add pre-cooled 1ml Trizol to a glass homogenizer, and homogenize...

Embodiment 2

[0062] Example 2: The effect of has-miR-520e on the proliferation of liver cancer cells

[0063] The cell proliferation ability was detected by MTT method, two HCC cell lines (HepG2, H7402) and two normal liver cell lines (Chang liver, L-O2) were selected, and the liver cancer cells were transfected with miR-520e mimics (miR-520 mimics) And normal hepatocytes transfected with miR-520e inhibitor (miR-520e inhibitor) as the experimental group, random sequence (Negative Control, NC and Inhibitor Negative Control, Inhibitor Control) as the control group. miRNA was designed and synthesized by Guangzhou Ruibo Biotechnology Company.

[0064] 1) Cell inoculation: the above-mentioned cells in the logarithmic growth phase were made into a single cell suspension with RPMI1640 or DMEM culture medium containing 10% fetal bovine serum, and seeded into a 96-well cell culture plate with 4000-5000 cells per well. The volume of each well is 100ul.

[0065] 2) Cell culture and transfection: Af...

Embodiment 3

[0072] Example 3: The influence of has-miR-520e on the malignant proliferation of liver cancer cells detected by plate colony formation

[0073] 1) Inoculation of cells: after transfection, liver cancer cells were inoculated in a flat-bottomed 6-well culture plate, 1000 cells / well, the culture plate was shaken to make the cells evenly distributed, and cultured in a 37°C, 5% CO2 incubator.

[0074] 2) Cell culture: Stop the culture when visible clones appear in the culture plate, discard the culture medium, and wash twice with PBS.

[0075] 3) Cell fixation: fix with 2ml of pure methanol for 15min.

[0076] 4) Cell staining: discard the fixative, add an appropriate amount of methylene blue staining solution for staining for 10-30 minutes, slowly wash away the staining solution with running water, and dry at room temperature.

[0077] 5) Observation: Microscopic observation, counting the number of clones greater than 50 cells.

[0078] 6) Calculation of clone formation rate: n...

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Abstract

The invention discloses has-miR-520e associated with primary hepatocellular carcinoma and its application in preparation of medicaments for treating tumors. The invention finds that has-miR-520e has low expression in hepatoma cells, and the expression level in tumor tissues of hepatoma patients is much lower than the expression level in adjacent tissues; and furthermore, has-miR-520e can effectively inhibit the proliferation of hepatoma cells and inhibit the growth of tumors. The has-miR-520e is a sequence as shown in SEQIDNO:1. The invention also provides an application of has-miR-520e in the preparation of medicaments for treating tumors. The invention has great practical significance and extensive potential application prospects in fields of medicine and biological pharmacy.

Description

Technical field: [0001] The present invention relates to a has-miR-520e related to primary liver cancer and the use of the miRNA in treating primary liver cell carcinoma. Background technique: [0002] Primary liver cancer (primary hepaticcaminoma, PHC) is one of the most common and most malignant tumors in the world. my country is one of the areas with high incidence of liver cancer, with an incidence rate of about 30.3 / 100,000. About 140,000 people die from PHC every year, accounting for more than 50% of the death toll of PHC in the world, which seriously threatens people's health and life. Liver cancer is known as the "king of cancers" because it is not easy to detect early, develops rapidly, easily metastasizes, and is difficult to treat. At present, the treatment methods for liver cancer include surgical treatment, hepatic artery embolization chemotherapy, radiotherapy, intratumoral ethanol injection, biological therapy, and traditional Chinese medicine treatment. [...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K48/00A61P35/00
Inventor 张晓东叶丽虹张帅
Owner TIANJIN TOPTECH BIO SCI & TECH
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