Method for screening microRNA having transcribing and post-transcriptional gene silencing functions

A technology of gene silencing and transcription level, applied in biochemical equipment and methods, DNA/RNA fragments, screening process, etc., can solve the problems of inability to completely block gene expression, inapplicability, side effects, etc., and achieve good gene silencing effect , significant inhibitory effect, good adaptability and specificity

Active Publication Date: 2014-02-12
SHANGHAI INST OF MATERIA MEDICA CHINESE ACAD OF SCI
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Problems solved by technology

[0004] At present, siRNA-mediated gene silencing technology has been widely used in the scientific research of gene function, and is the most commonly used gene silencing technology in current research. Both are of great significance, but RNAi technology has its limitations: (1) In mammalian cells, RNAi cannot completely block the expression of genes, especially high-expression genes; (2) RNAi is not applicable to all For example, dsRNA has no obvious inhibitory effect on some genes that function in nerve cells; (3) the off-target effect of RNAi is obvious; (4) SiRNA can activate interferon-responsive genes in the body and cause side effects

Method used

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  • Method for screening microRNA having transcribing and post-transcriptional gene silencing functions
  • Method for screening microRNA having transcribing and post-transcriptional gene silencing functions
  • Method for screening microRNA having transcribing and post-transcriptional gene silencing functions

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preparation Embodiment 1m

[0077] Preparation Example 1 Preparation of microRNA 552 (miR-552)

[0078] Using cytochrome P450 enzyme subtype 2E1 (Cytochrome P450 2E1, CYP2E1) as the target gene, microRNA 552 (miR-552) with dual inhibitory ability was screened through the following steps.

[0079] Step 1): Use the miRBase database to predict the microRNA binding site of the 5' flanking region (where the promoter may exist) 1kb before the CYP2E1 transcription start site, and obtain a series of microRNAs that may bind to the promoter region of the target gene ( See figure 2 );

[0080] Step 2): Use the TargetScan database to predict the microRNA binding site of the 3'untranslated region of CYP2E1, and obtain a series of microRNAs that may bind to the 3'untranslated region of CYP2E1 (see image 3 );

[0081] Step 3): Use chemically synthesized microRNA mimics (mimics) to detect the effect of the microRNA obtained in step 1) and step 2) on the protein expression of the target gene, and screen to obtain mi...

preparation Embodiment 2m

[0088] Preparation Example 2 Preparation of microRNA 1254 (miR-1254)

[0089] According to the aforementioned method, we selected the target gene type I heme oxidase (Haemoxygenase 1, HMOX1), and used the same computer prediction and experimental methods to obtain microRNA 1254 (miR-1254) with both transcriptional and post-transcriptional gene silencing capabilities ( See Figure 8 ), this data illustrates the effectiveness of the method of the present invention.

[0090] Experimental Example

Embodiment 1

[0091] Example 1 The inhibitory effect of miR-552 on the expression levels of CYP2E1 mRNA and protein was detected in human liver cancer cells PLC / PRF / 5.

[0092] 25nM, 50nM, 100nM, 200nM miR-552 were transfected into human liver cancer cells PLC / PRF / 5, and the protein expression of CYP2E1 in the cells was detected by Western blot, and CYP2E1 was detected by reverse transcription-real-time quantitative PCR Changes in mRNA expression. Such as Figure 4 and 5 As shown, miR-552 can dose-dependently cause a decrease in the expression of CYP2E1 protein ( Figure 5 ), CYP2E1 mRNA level decreased slightly, no dose-related ( Figure 5 ).

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Abstract

The invention provides a method for screening microRNA having transcribing and post-transcriptional gene silencing functions. The microRNA obtained by screening through the method can be combined with 5' flanking region specific sites to reduce the target gene mRNA expression (having a transcription inhibition capability), and can also be combined with 3' non-translational region specific sites to reduce the target gene protein expression effect (having a post-transcriptional / translational inhibition capability).

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, relates to a method for screening microRNAs with dual-level gene silencing functions in transcription and post-transcription. mechanism (dual regulation). Background technique [0002] Gene silencing is a broad concept, which is the process of epigenetic regulation of gene expression. It usually refers to the phenomenon that a specific gene in an organism is not expressed or expressed at a lower than normal level, but it is not caused by changes in the genetic material itself. This phenomenon exists widely in the biological world. It is an endogenous gene expression regulation mechanism in organisms, and it is also one of the important techniques for inhibiting gene expression in biological research. Reducing the expression level of a gene and then observing the resulting phenomenon is a very effective means to study the function of this gene. Using nucleic acids as drug targ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/10C12Q1/68
CPCC12N2320/11C12N15/111C12N2310/141C12N15/113
Inventor 任进王以政苗玲玲宫丽崑戚新明李成刚邢国振
Owner SHANGHAI INST OF MATERIA MEDICA CHINESE ACAD OF SCI
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