MicroRNA used for inducing leukemia cell differentiation

A molecular, tumor cell technology, applied in the field of double-stranded RNA molecules and their applications

Inactive Publication Date: 2012-08-08
SINOGENOMAX
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] At the same time, the possibility of designing siRNAs against known gene sequences is limited by the limited number of genes known to be disease targets

Method used

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  • MicroRNA used for inducing leukemia cell differentiation
  • MicroRNA used for inducing leukemia cell differentiation
  • MicroRNA used for inducing leukemia cell differentiation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Screening of 12 positive siRNAs

[0049] K562 cells were cultured in IMDM medium (purchased from Thermo-Fisher, catalog number SH30228.01B) supplemented with 10% fetal bovine serum (purchased from Sigma, catalog number SH30228.01B) in a cell culture incubator (purchased from Sanyo) at 37°C with 5% carbon dioxide (Unless otherwise indicated, the K562 cell culture method is the same in the following examples), will contain 3x10 7 Random siRNA library mixed plasmids with different siRNA coding sequences were transfected into 6.7x10 748 hours after transfection, add G418 (purchased from Merck, product number 345810; final concentration 800ug / ml) to the culture medium to kill untransfected cells; after 14 days, adjust the cell concentration to 1×10 7 cells / ml, according to every 10 6 Ratio of adding 1ug of antibody to each cell Use anti-CD235 antibody (purchased from R&D, product number MAB1228) to label the cells at 4°C for half an hour, then add Pan Mouse Ig...

Embodiment 2

[0051] Example 2: 12 siRNAs increase the expression of CD235 in K562 cells to varying degrees

[0052] In addition to 12 randomly synthesized siRNAs, a negative control group (Con) was set up: only the transfection reagent HiPerFect Transfection Reagent was added during transfection, and no siRNA group was added.

[0053] K562 cells were cultured according to the method of Example 1, and when the cells reached (at a confluence of about 60%), these siRNA duplexes were respectively transiently transfected into K562 cells separately with HiPerFect Transfection Reagent (purchased from Qiagen, Cat#301704), The concentration of siRNA was 25nM during each transfection, and the second transfection was carried out 60 hours after the first transfection, and the cells were collected 48 hours and 72 hours after the second transfection, and were used to detect the indicators of erythroid differentiation, thereby The role of these siRNAs in inducing erythroid differentiation was identified....

Embodiment 3

[0056] Example 3: clone-67siRNA increases CD235 expression in K562 cells

[0057] The culture method and transfection method of CD235 cells were the same as in Example 2, except for the clone-67siRNA obtained in Example 2, all identification experiments were set up with two negative control groups (unless otherwise specified, the negative control groups in the following examples were Same as this): transfection only added transfection reagent HiPerFect Transfection Reagent, no siRNA group (Con group); transfection does not act on any gene negative control Allstars negative control siRNA (purchased from Qiagen, Cat#1027280) group (Allstars group).

[0058] Detection of CD235 expression in K562 cells transiently transfected with clone-67siRNA, Con and Allstars: Cells in each group were collected 72 hours after two transfections, and FITC-labeled anti-CD235 antibody (purchased from BD Pharmingen, catalog number 559943) was used at 4°C After staining for 1 hour, the cells were wa...

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Abstract

The invention provides microRNA used for inducing leukemia cell differentiation and application thereof in the induction of erythroid differentiation of tumor cell lines K562. The microRNA not only has the advantages of cheapness, no toxic or side effect, long duration, high transfection efficiency and the like, but also has the characteristics of stronger action and effectiveness of various indexes for tumor cell differentiation compared with a conventional human leukemia cell erythroid differentiation siRNA inducer.

Description

technical field [0001] The invention relates to a double-stranded RNA molecule and its application, in particular to a double-stranded RNA molecule capable of inducing the erythroid differentiation of leukemia cells and its application. Background technique [0002] According to the theory of tumor stem cells (for example, see C.T.Jordan, M.L.Guzman, M.Noble, Cancer Stem Cells, N.Engl.J.Med.355 (2006) 1253-1261) and the research progress of abnormal tumor cell differentiation disorders, it has been proposed in recent years A new idea of ​​tumor treatment: tumor induction differentiation therapy, that is, the use of differentiation inducers to make tumor cells differentiate into normal cells, reduce or reverse the malignant phenotype of tumor cells, so as to treat tumors (for example, see M. Leszczyniecka et al., Differentiation therapy of human cancer: basic science and clinical applications, Pharmacol. Therapeut. 90(2001) 105-156). Among them, retinoic acid and arsenic age...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/11C12N15/63A61K48/00A61P35/02
Inventor 陈梅红范翠青朱宁熊元卢雅彬
Owner SINOGENOMAX
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