Molecular marker hsa-miR-374a of breast carcinoma and application thereof

A technology of hsa-mir-374a and molecular markers, applied in the field of molecular markers of breast cancer, can solve problems such as unseen research reports

Inactive Publication Date: 2010-01-27
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There is no research report on the relationship between hsa-miR-374a and breast cancer in the domestic and foreign literature searches of the prior art

Method used

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  • Molecular marker hsa-miR-374a of breast carcinoma and application thereof
  • Molecular marker hsa-miR-374a of breast carcinoma and application thereof
  • Molecular marker hsa-miR-374a of breast carcinoma and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 Detection of hsa-miR-374a expression level

[0018] With the consent of the Sun Yat-sen University Ethics Committee and the patient's family, fresh specimens of 13 cases of breast cancer tissue and 2 cases of normal breast tissue were collected, RNA was extracted by Trizol method, and hsa-miR-374a was detected by ABI 7500 fluorescent quantitative PCR instrument system There were three replicate wells for each sample. For the expression of hsa-miR-374a in each tissue, the RNA of one case of normal breast tissue was used as a standardized internal control. See the experimental results figure 1 In Figure A, figure 1 A is to detect the expression level of hsa-miR-374a in different breast cancer tissues and normal breast tissues; N1 and N2 represent 2 cases of normal breast tissues, and T1-T13 represent 13 cases of breast cancer tissues; it can be seen that hsa-miR-374a The expression of 374a in breast cancer tissue was significantly higher than that in normal b...

Embodiment 2

[0020] Example 2 Using in situ hybridization to detect the expression of hsa-miR-374a in paraffin-embedded tissue sections of breast cancer of different clinical grades

[0021] Randomly select paraffin-embedded tissue sections of breast cancer of different clinical grades, bake the slices in an oven at 45°C for 2hrs, dewax with xylene, wash xylene with 100%, 100%, 75%, 50%, 25% gradient alcohol and water, each for 5mins ;0.2M HCl 5mins; PBS 5mins; 40μg / ml proteinase K 25 ℃ 20mins; 0.2% glycine / PBS 5mins; PBS 5mins, 2 times; 4% formaldehyde 10mins; PBS 5mins, 2 times; PBS 5mins, 4 times; 5×SSC, 5mins, 2 times; pre-hybridization, 2hrs; incubation probe about 40hrs; 5×SSC, 5mins, 2 times; 50% formamide, 2×SSC 30mins, 2 times; 0.1% PBST, 5mins, 5 times; blocking solution (Roche) for 2hrs; anti-DIG antibody (Roche), 16hrs, 4°C; 0.1% PBST, 10mins, 4 times; staining solution, 10mins, 2 times; incubate NBT / BCIP, 12hrs ; PBST to terminate the reaction; 25%, 50%, 75%, 100% alcohol; dr...

Embodiment 3

[0022] Example 3 The effect of stable overexpression of hsa-miR-374a on the morphology of breast cancer epithelial cells MCF-7

[0023] To establish a cell line stably overexpressing hsa-miR-374a:

[0024] Calcium phosphate transfection to prepare retrovirus: 24 hours before transfection, pass 293FT to the required number of plates; on the day of transfection, pass the cells to be infected (MCF-7) to the required density; when transfecting, pass 20 μg ( 1 μg / μl) pMSCV-puro, 20 μg (1 μg / μl) packaging plasmid pIK, 380 μl 1×TE, 60 μl 2M CaCl 2 After mixing, 480 μl of 2×HEPES was slowly added dropwise, and after standing at room temperature for 30 minutes, the mixture was evenly added to the culture medium of 293FT passaged 24 hours ago, and 5 μl of chloroquine was added and mixed gently. Placed at 37°C, 5% CO 2 , constant temperature, constant humidity cell incubator for 6 hours after culture with 1 × solution A (1L solution A contains 7.149g HEPES, 1.802g D-glucose, 0.2236g KC...

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Abstract

The invention provides a new molecular marker hsa-miR-374a of breast carcinoma, that is, non-coding RNA gene hsa-miR-374a of micromolecule as a molecular marker of breast carcinoma. Expression of the molecular marker in tissues suffering from breast carcinoma is obviously higher than that of normal mammary tissues, and is associated with clinical classification of breast carcinoma, and expression of hsa-miR-374a in breast carcinoma cell line cultured in vitro is higher than that of normal mammary epithelial cells and immortalized normal mammary epithelial cells. The invention further provides application of the molecular marker of breast carcinoma to preparing an anti-breast cancer drug. The drug comprises effective amount of blocker capable of blocking expression of non-coding RNA gene hsa-miR-374a of micromolecule. The molecular marker provides new effective way for diagnosing and treating breast carcinoma. The invention also provides certain experience and foundation for further research of function of hsa-miR-374a and relation with other tumors.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a breast cancer molecular marker hsa-miR-374a and application thereof. Background technique [0002] Breast cancer is one of the most common malignant tumors in women. There are about 1.2 million new breast cancer cases and 500,000 deaths worldwide every year. It has the characteristics of high morbidity and mortality. One of the reasons for the high mortality rate of breast cancer is the spread and metastasis of breast cancer, and the prone sites of metastasis include lung, liver, brain, bone, and pleura. In recent years, the incidence of breast cancer in my country has been increasing year by year, seriously endangering women's lives and health. [0003] MicroRNA (microRNA) is a group of short-sequence RNAs widely present in eukaryotes that do not encode proteins, with a length of 20-24 bases, and are highly conserved, time-sequenced, and tissue-specific. It promotes ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11A61K48/00A61P35/00C12N15/113
Inventor 黎孟枫李隽蔡俊超黄勇波吴珏珩袁洁
Owner SUN YAT SEN UNIV
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