siRNA (small interfering ribonucleic acid) for inhibiting ER81 gene expression and application of siRNA in breast cancer cells

A breast cancer cell and gene expression technology, which is applied in the application field of inhibiting the expression level of ER81 gene in breast cancer cells, can solve the problems of insufficient understanding of the interaction, etc., and achieve an obvious inhibitory effect

Inactive Publication Date: 2014-07-16
杨举伦
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the human body is an organic and complex interconnected network system; and the occurrence of human tumors is a multi-step, multi-stage, multi-gene involved systemic process; tumor cell proliferation, apoptosis, infiltr

Method used

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  • siRNA (small interfering ribonucleic acid) for inhibiting ER81 gene expression and application of siRNA in breast cancer cells
  • siRNA (small interfering ribonucleic acid) for inhibiting ER81 gene expression and application of siRNA in breast cancer cells
  • siRNA (small interfering ribonucleic acid) for inhibiting ER81 gene expression and application of siRNA in breast cancer cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Construction of recombinant siRNA expression vector targeting human ER81 gene

[0049] 1. Synthesis of siDNA gene fragment targeting human ER81 gene

[0050] The human ER81 mRNA sequence was searched in the gene database GenBank on the NCBI website. There are 7 transcription mutants in the ER81 gene, and the gene accession numbers are: NM_004956, NM_001163147, NM_001163148, NM_001163149, NM_001163150, NM_001163151, NM_001163152. Find the common sequence of 7 transcriptional mutants by sequence alignment and use their common sequence in Genscript siRNA Target Finder (https: / / www.genscript.com / ssl-bin / app / rnai ) to find several siRNA target sequences with the highest scores. Finally, the designed siRNA target sequences were compared in GenBank by BLAST to remove sequences that may hybridize with other irrelevant genes. Finally, the target sequences at the upper, middle and lower positions of the mRNA were selected to obtain the following sequences: ETV1A: G...

Embodiment 2

[0073] Example 2: Inhibitory effect of recombinant siRNA expression vector on ER81 gene in breast cancer cell line MDA-MB-231 and its influence on cell biological characteristics

[0074] 1. The recombinant siRNA expression vector was stably transfected into MDA-MB-231 cells

[0075] Dilute 0.8 μg of each recombinant siRNA expression vector (pGenesil-ETV1A, pGenesil-ETV1B, pGenesil-ETV1C, pGenesil-ETV1C, pGenesil-NC) and the original siRNA expression vector pGenesil-1, mix gently. Dilute 2 μl of Lipofectamine with 298 μl of OPTI-MEM low serum medium TM 2000 (purchased from Invitrogen), incubated at room temperature for 5 minutes. Diluted expression vector DNA and diluted Lipofectamine TM 2000 equal volumes were mixed and incubated at room temperature for 20 minutes after mixing. Take out the laid 24-well culture plate, discard the original culture medium, then add 600 μl of transfection mixture dropwise to each well, mix well and put the culture plate back into the incub...

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Abstract

The invention discloses siRNA (small interfering ribonucleic acid) for specifically inhibiting human ER81 gene expression, and a preparation method and screening method of siRNA. An ER81 gene is used as a target point sequence, and aiming at the homologous sequence of seven transcription mutant coding regions of the gene, an interference target point is selected from each of the upstream side, the midstream side and the downstream side so as to construct three recombinant siRNA expression vectors. The siRNA recombinant expression vectors is transferred into a breast cancer cell line MDA-MB-231 and an siRNA segment which can be used for obviously inhibiting the ER81 gene expression is finally screened out by using a DNA (deoxyribonucleic acid)-mediated RNA interfering technology. Experimental results show that screened siRNA can be used for specifically and efficiently inhibiting the ER81 gene expression so as to inhibit the proliferation of the breast cancer cells and promote cell apoptosis. Screened siRNA disclosed by the invention provides a new way for preparing related medicaments for treating breast cancers and shows good application prospects.

Description

technical field [0001] The invention belongs to the field of medical molecular biology, and in particular relates to the construction and screening of an siRNA expression vector targeting human ER81 gene and its application in inhibiting the expression level of ER81 gene in breast cancer cells. In particular, it relates to the use of siRNA to specifically inhibit the expression of ER81 gene through the RNA interference pathway and its use in killing breast cancer cells and inducing cell apoptosis. Background technique [0002] Breast cancer is one of the common malignant tumors in women. About 1.3 million women in the world develop breast cancer every year, and 500,000 women die of breast cancer. Its incidence rate is always on the rise, especially in the past 20 years. The occurrence of breast cancer involves the imbalance of apoptosis and proliferation, the disorder of cell differentiation, the dysfunction of oncogenes and tumor suppressor genes, and the abnormalities of ...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61K48/00A61P35/00
Inventor 杨举伦冯强黎贵芸
Owner 杨举伦
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