Small interference RNA molecule (SiRNA) capable of effectively killing and wounding tumour cell pointed at PLK1 mRNA, its mixture and use

A ribonucleic acid, tumor cell technology, applied in the field of nucleic acid, can solve the problem of no treatment method

Inactive Publication Date: 2005-08-10
杭州新瑞佳生物医药技术开发有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Malignant tumor is a major disease that endangers human life and health, but so far there is still no effective treatment for it

Method used

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  • Small interference RNA molecule (SiRNA) capable of effectively killing and wounding tumour cell pointed at PLK1 mRNA, its mixture and use
  • Small interference RNA molecule (SiRNA) capable of effectively killing and wounding tumour cell pointed at PLK1 mRNA, its mixture and use
  • Small interference RNA molecule (SiRNA) capable of effectively killing and wounding tumour cell pointed at PLK1 mRNA, its mixture and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Synthesis of siRNA

[0049] The synthesis of siRNA can be entrusted to commercial companies that openly carry out synthesis business, such as Dharmacon in the United States, specifically through www.dharmacon.com It was known that all siRNA molecules were deprotected at the 2-position, desalted, purified, and annealed to form double strands, and then dissolved in DEPC-treated distilled water.

[0050] The preparation method of the small interfering RNA molecule (SiRNA) provided by the present invention can also adopt the existing solid-phase chemical synthesis method. This method can be found in: Wincott F, DiRenzo A, Shaffer C, Grimm S, Tracz D, Workman C, Sweedler D, Gonzalez C, Scaringe S and Usman N. Synthesis, degradation, analysis and purification of RNA and ribozymes. Nucleic Acids Res. 1995 , 23: 2677-84.

[0051] The small interfering RNA molecule (SiRNA) of No. 2 in the previous table is an example: the whole chemical synthesis can be roughly divi...

Embodiment 2

[0060] Example 2 Cell culture and transfection of siRNA

[0061] 1. Cell culture. SW480 is a colon cancer cell line isolated from colon cancer tissue (Leibovitz A, Stinson JC, McCombs WB 3rd, McCoy CE, Mazur KC, Mabry ND. Classification of human colorectal adenocarcinoma cell lines. Cancer Res. 1976; 36 (12): 4562-9.). The cells were cultured in vitro with DMEM medium containing 10% fetal bovine serum. Bcap-37, D6 and SMMU-7721 are tumor cells established in China, and their culture conditions are RPMI 1640 medium containing 10% fetal bovine serum.

[0062] 2. SiRNA transfection: SiRNA was transferred into tumor cells by means of Oligofectamine from Invitrogen (www.invitrogen.com), and the specific steps were carried out according to the instructions. The brief description is as follows, 5×10 4 Tumor cells were inoculated overnight on 24-well culture plates, transferred into siRNA the next day, and then cultured for 72 hours to detect the level of PLK1 mRNA in the cells. ...

Embodiment 3

[0065] Example 3 Inhibitory effect of siRNA on tumor cell PLK1 mRNA

[0066] 1. Selection of SiRNA: In the present invention, a total of 10 SiRNA molecules targeting the PLK1 mRNA coding region were selected, namely the 10 SiRNA molecules listed in the above table. The attack site is between 243-1599 of the start codon, and the G / C in the 19 complementary double-stranded nucleotide sequences in each siRNA molecule is between 37%-63%.

[0067] 2. Effects of 10 SiRNAs on PLK1 mRNA expression in SW480 cells:

[0068] A. The effect of 10 SiRNAs with a concentration of 50nM on PLK1 mRNA in SW480 cells:

[0069] After 50nM of these 10 SiRNAs acted on SW480 cells for 72 hours, when observing their effects on PLK1 mRNA levels, it was found that No. 1, No. 4 and No. 9 SiRNA molecules had the most obvious inhibitory effects on PLK1, reaching 91%, 94% and 89%. In addition, the 2nd, 3rd, 5th, 7th, 8th also had greater than 50% inhibitory effect on PLK1 mRNA, and the 6th, 10th A also ha...

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Abstract

A small-interference RNA (SiRNA) molecular directed to PLKI mRNA for killing tumor cells, its mixture and its application are disclosed. It is a double-stranded RNA molecular. Its nucleoside sequence is also disclosed.

Description

technical field [0001] The invention relates to the field of nucleic acid technology, in particular to a small interfering ribonucleic acid molecule (SiRNA) targeting PLK1 mRNA for effectively killing tumor cells, a mixture thereof and applications thereof. Background technique [0002] Malignant tumor is a major disease that endangers human life and health, but so far there is still no effective treatment for it. Scientists have long been trying to develop new drugs that can effectively treat malignant tumors. Contents of the invention [0003] The technical problem to be solved by the present invention is to provide a small interfering ribonucleic acid molecule (SiRNA) targeting PLK1 mRNA that can effectively kill tumor cells as an active ingredient of an antitumor drug. For this reason, the present invention adopts the following technical solutions: it is a double-stranded RNA molecule and its nucleotide sequence has at...

Claims

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Application Information

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IPC IPC(8): A61K31/7088A61P35/00C07H21/00
Inventor 丁佳逸
Owner 杭州新瑞佳生物医药技术开发有限公司
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