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MM (multiple myeloma)-resistant small-interference RNA (ribonucleic acid) and application thereof

A multiple myeloma, ribonucleic acid technology, applied in the fields of application, antineoplastic drugs, medical preparations containing active ingredients, etc., can solve the problem of lack of effective therapeutic molecular targets

Inactive Publication Date: 2015-04-29
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The technical problem to be solved by the present invention is to provide an effective solution to the current lack of effective therapeutic molecular targets in the treatment of multiple myeloma, that is, an anti-multiple myeloma small interfering ribonucleic acid target Polynucleotide, the application of the target polynucleotide in the preparation or screening of anti-multiple myeloma drugs

Method used

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  • MM (multiple myeloma)-resistant small-interference RNA (ribonucleic acid) and application thereof
  • MM (multiple myeloma)-resistant small-interference RNA (ribonucleic acid) and application thereof
  • MM (multiple myeloma)-resistant small-interference RNA (ribonucleic acid) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Design and synthesis of siRNA against SUMO-1 gene

[0053] Design and synthesize SUMO-1 small interfering RNA (siRNA), wherein the target sequence in the SUMO-1 gene of the siRNA is as shown in Table 1:

[0054]siRNA target sequence (CDNA) of table 1 SUMO-1 gene

[0055] serial number

serial number

sequence

siRNA-1

SEQ ID NO:1

CTGGGAATGGAGGAAGAAG

siRNA-2

SEQ ID NO:2

CAATGAATTCACTCAGGTT

siRNA-3

SEQ ID NO:3

GGACAGGATAGCAGTGAGA

[0056] According to the sequence in the above table, the siRNA sequence was synthesized by Ribo (Guangzhou) Company, and ddH 2 O dilute the synthesized siRNA nucleotide molecules to a concentration of 100 μM for later use. The sequence information and structural information of the resulting siRNA are as follows:

[0057] Fragment sequence of siRNA-1: sense strand 5'CUGGGAAUGGAGGAAGAAG dTdT 3' (as shown in SEQ ID NO:4 in the sequence listing);

[0058] Antisense st...

Embodiment 2

[0063] Example 2: Using siRNA to transfect myeloma cells

[0064] Myeloma cells NCI-H929 (the cells were purchased from ADCC) and RPMI-8226 (the cells were purchased from ADCC) in the logarithmic growth phase were centrifuged for 5 min, the supernatant was discarded, and the cells were resuspended with PBS, and then Centrifuge for 5min. Use the ELX800 microplate reader to adjust the cell concentration to 30×10 4 cells / L, inoculated into a six-well plate overnight. The cationic liposome was lipo2000, and the siRNA-3 obtained in Example 1 was dissolved in water, and the final concentration of the obtained aqueous solution was adjusted to 100 nM. The cationic liposome and siRNA solutions were diluted with optimum respectively, and left at room temperature for 5 minutes for later use. Mix the cationic liposome solution and the siRNA solution so that the liposome fully encapsulates the siRNA, and let it stand at room temperature for later use. After 20 min, add the mixture to t...

Embodiment 3

[0083] Example 3: Effect of SUMO-1 Gene Expression Inhibition on Apoptotic Rates of Different Multiple Myeloma Cells

[0084] Annexin V-FITC-PI kit (purchased from KGI, Shanghai) and flow cytometry (model BD FACS calibur 4, USA) were used for detection.

[0085] Flow cytometry: Suspended cells were collected by centrifugation (2000rpm for 5min); cells were washed twice with PBS (2000rpm for 5min) to collect 1-5×10 5 Cells; add 500 μL Binding Buffer to suspend cells; add 5 μL Annexin V-FITC and mix well, then add 5 μL Propidium Iodide, mix well; room temperature, dark, react for 5-15 minutes; flow cytometry detection.

[0086] MTS method: divide cells into 10 × 10 3Inoculate each well in a 96-well plate, add 20 μl MTS (purchased from Promega) after 48 hours of treatment, 37 ° C, 5% CO 2 After incubating in the cell culture box for 2 hours, use a Flex Station 3 (purchased from Molecular Devices) microplate reader to detect (490 / 690nm).

[0087] From the target polynucleotide ...

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Abstract

The invention relates to the field of biotechnology and medicine, and discloses an MM (multiple myeloma)-resistant small-interference RNA (ribonucleic acid) and an application thereof. The nucleotide sequence of a target polynucleotide is shown in any one of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 in a sequence table. Excessive SUMO (small ubiquitin-related modifier) modification is related to poor prognosis of MM, deep research on effects caused by SUMO modification Wnt / beta-catenin on MM cell proliferation and apoptosis as well as molecular mechanisms is beneficial to discovery of the source of the progress of the myeloma, and prognosis and regulatory mechanisms of the MM are further clarified; new therapeutic targets are developed for the key of recurrence of the MM. The provided target DNA has very important practical significance in realizing safer and more effective anti-myeloma treatment and curing the MM finally.

Description

technical field [0001] The invention relates to the fields of biotechnology and medicine, in particular to a small interference ribonucleic acid against multiple myeloma and its application. Background technique [0002] Multiple myeloma (Multiple myeloma, MM) is the second highest incidence of malignant tumors in the blood system. It is still incurable and seriously threatens the life and health of the people. Conventional chemotherapy and even new drugs have little effect on maintaining self-renewing myeloma stem cells while reducing tumor burden, which is considered to be the key to the ultimate progression and recurrence of MM. Therefore, further research on the source of MM recurrence is urgently needed to develop new, safer and more effective targeted therapies to truly improve the survival and prognosis of MM patients. [0003] Wnt gene is a proto-oncogene, which is the key to the occurrence of various tumors and an important regulatory signal in the development of t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/113A61K31/713A61P35/00
Inventor 周莉莉黄禾菁侯建
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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