Breast cancer cell line with low expression of HK2 and siRNA used by same

A breast cancer cell, low-expression technology, applied in the field of medical molecular biology and genetic engineering, can solve the problems of lack of targets for breast cancer treatment, limiting the effectiveness and extensiveness of breast cancer treatment methods, etc.

Active Publication Date: 2021-12-31
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Breast cancer exhibits great complexity and heterogeneity in terms of cell origin, histological morphology, disease grade, clinical manifestations, treatment response, and metastatic potential, which limits the effectiveness and extensiveness of existing breast cancer treatments
There is still a lack of clinical targets for breast cancer treatment

Method used

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  • Breast cancer cell line with low expression of HK2 and siRNA used by same
  • Breast cancer cell line with low expression of HK2 and siRNA used by same
  • Breast cancer cell line with low expression of HK2 and siRNA used by same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Design and Synthesis of SiRNA (HK2 siRNA) targeted by HK2 gene mRNA

[0065] The inventors of the inventors of the inventors designed HK2 siRNA according to human HK2 sequence in GenBank, and the basic principles of design are as follows: (1) Starting from the AUG start password of the transcript of human HK2, looking for "AA" two-line sequence, It also records the 19 base sequences of the 3 'end, as a potential siRNA target site; (2) Do not design siRNA, do not target the non-encoding area of ​​the 5' and 3 'ends, because these places have a rich regulatory protein binding area. And these UTR binding proteins or translation start complexes may affect the SiRNP nucleic acid ingenuclease complex binding mRNA to affect the effect of siRNA; (3) generally gc content in 10 nucleotides at 30% -70% In the range, a valid fragment is easier to find in a fragment of the GC content; (4) To avoid continuous repetition of the siRNA target sequence to form a secondary structure...

Embodiment 2

[0071] Example 2, 3 HK2 siRNA detection of HK2 interference effect in human breast cancer cells Zr-75-1

[0072] Human breast cancer cells Zr-75-1 is the product of the US ATCC cell library.

[0073] 1, transfection

[0074] (1) Treatment of human breast cancer cells Zr-75-1 in a culture dish (diameter of 6 cm) (6cm) containing 10% (v / v) neovasculum serum (diameter is 6 cm) (inoculated cell density in transfection) It is suitable for 70-80%.

[0075] (2) After completing step (1), SiRNA (NC siRNA, HK2 siRNA1, HK2 siRNA2 or HK2 siRNA3) was transfected with Lipofectamine 3000 (US Invitrogen) transfection (transfection method refer to Lipofectamine 3000 transfection reagent) Instructions), the cells were collected in 48 hours after transfection, and siRNA transfected human breast cancer cells Zr-75-1 were obtained.

[0076] 2, cDNA's acquisition

[0077] The total RNA transfected with Trizol (US INVITROGEN) was extracted from the total RNA transfected with human breast cancer cells...

Embodiment 3

[0104] Example 3, HK2 siRNA effect on Zr-75-1 proliferation of human breast cancer cells

[0105]The human breast cancer cells Zr-75-1 in step 1 obtained in Example 2 were inoculated to 96-well plates, and approximately 3,000 per well inoculated, and conventionally cultured to the adhesive wall; after the Cell County Kit-8 (Japan DOJINDO) Detected the OD value at 450 nm. After 5 days of continuous detection, the growth curve is drawn.

[0106] Growth curve figure 2 . The results showed that HK2 siRNA1, HK2 siRNA2 and HK2 SiRNA1, HK2 siRNA2 and HK2SiRNA3 can inhibit proliferation of human breast cancer cells Zr-75-1, and HK2 siRNA1 has the most obvious inhibitory effect on human breast cancer cells Zr-75-1 .

[0107] In combination of the above experimental results, HK2 siRNA1 was selected as a subsequent study of siRNA.

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Abstract

The invention discloses a breast cancer cell line with low expression of HK2 and siRNA used by the breast cancer cell line. The siRNA is oligo-nucleic acid siRNA1 (small interfering ribonucleic acid 1); the oligo-nucleic acid siRNA1 is composed of a single-stranded RNA molecule as shown in SEQ ID NO: 1 and a single-stranded RNA molecule as shown in SEQ ID NO: 2; and a 3' terminal of the oligo-nucleic acid siRNA1 is modified by two dT. Experiments prove that the oligo-nucleic acid siRNA1 can be used for inhibiting the growth of breast cancer tumors, inhibiting the proliferation of breast cancer cells, reducing the invasion ability of the breast cancer cells, reducing the glycolysis ability of the breast cancer cells and constructing a breast cancer cell model with low expression of the HK2. The cell line has an important application value.

Description

Technical field [0001] The invention belongs to the field of medical molecules biology and genetic engineering, and in particular to HK2 low expression of breast cancer cell lines and SiRNAs thereof. Background technique [0002] Breast cancer is one of the most common primary malignant tumors in female patients. Breast cancer exhibits great complexity and heterogeneity in terms of cell origin, histological form, disease grading, clinical manifestation, treatment reaction, and transfer potential, limiting the effectiveness and extensiveness of existing breast cancer treatment . At present, it is still lacking targets for breast cancer treatment. Therefore, the development mechanism of breast cancer is studied from the molecular level, and the tumor marker of the cancer pathway is determined and the signal path involved in the signal pathway, which is the prevention, screening and treatment of breast cancer, and is also a precise target. Treat a foundation to the treatment. [000...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/867C12N15/66C12N15/54C12N5/10A61K31/713A61P35/00A61P35/04
CPCC12N15/1137C12N15/86C12N9/1205C12N5/0693C12N5/0631A61P35/00A61P35/04C12N2310/14C12Y207/01001C12N2320/32C12N2740/15043C12N2800/107C12N2510/00C12N2310/531
Inventor 叶棋浓李玲张秀娟任鑫鑫罗菲
Owner ACADEMY OF MILITARY MEDICAL SCI
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