Functions and targets of let-7 micro rnas

A technology of let-7 and cells, applied in the field of molecular biology, can solve the problems of unknown details of regulatory pathways and networks, imprecise estimation of the number and identity of miRNA targets, etc.

Inactive Publication Date: 2010-03-17
ASURAGEN +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A limitation of this current approach is that details about the regulatory pathways and networks affected by any given miRNA remain largely unknown
As mentioned above, bioinformatics can only provide imprecise estimates of the number and identity of miRNA targets

Method used

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  • Functions and targets of let-7 micro rnas
  • Functions and targets of let-7 micro rnas
  • Functions and targets of let-7 micro rnas

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0241] B. Preparation of nucleic acid

[0242] Nucleic acids can be prepared by any technique known to those of ordinary skill in the art, such as chemical synthesis, enzymatic production or biological production. It is specifically considered that the miRNA probe of the present invention is chemically synthesized.

[0243] In some embodiments of the invention, miRNA is recovered or isolated from a biological sample. The miRNA can be recombinant, or it can be the cell's natural or endogenous miRNA (produced from the cell's genome). It is considered that biological samples can be processed to enhance the recovery of small RNA molecules such as miRNA. This method is described in US Patent Application Serial No. 10 / 667,126, which is specifically incorporated herein by reference. Generally, these methods involve lysing cells with a solution with guanidine salt and detergent.

[0244] Alternatively, nucleic acid synthesis can be performed according to standard methods. See, for examp...

Embodiment 1

[0285] Method for analyzing gene expression after miRNA transfection

[0286] The synthetic precursor miR miRNA (Ambion) was reverse-stained into quadruplicate samples of A549, HepG2 or HL-60 cells. A549 and HepG2 cells were transfected with siPORT NeoFX (Ambion) according to the manufacturer's recommendation using the following parameters: 200,000 cells per well in a 6-well plate, 5.0 μl NeoFX, 2.5 ml of miRNA at a final concentration of 30 nM. The cells were harvested 72 hours after transfection. HL-60 cells were transfected with the Gene Pulser Xcell System (Bio-Rad Laboratories, Inc.; Hercules, CA, USA) according to the manufacturer’s instructions and the following parameters: 500,000 cells in a volume of 150 μl per reaction, in a 2-mm electroporation cuvette (electroporation cuvette), a single square wave pulse of 25msec is used.

[0287] Total RNA was extracted with RNAqueous-4PCR (Ambion) according to the manufacturer's recommended protocol. The mRNA array analysis was p...

Embodiment 2

[0289] Gene expression analysis of A549, HEPG2 and HL-60 cells transfected with HSA-LET-7B

[0290] miRNAs are thought to affect gene expression mainly at the translation level. However, it has recently been reported that in some cases hsa-let-7 (Bagga et al., 2005) and other miRNAs (Lim et al., 2005) can reduce the mRNA level of direct targets, and this change can be used in microarray gene expression. Observed during analysis.

[0291] The experiments described herein can identify genes whose mRNA levels are affected by hsa-let-7 expression in human lung cancer (A549) cell lines, human liver cancer cell lines (HepG2), and human acute myeloid leukemia cell lines (HL-60). A549, HepG2 or HL-60 cells were transfected with the precursor miR hsa-let-7b (as a representative member of the hsa-let-7 miRNA family) as described in Example 1. The results of microarray gene expression analysis are shown in Tables 2, 3, and 4.

[0292] Table 2. Genes with altered mRNA expression levels in A5...

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Abstract

The present invention concerns methods and compositions for treating or assessing treatment of diseases related to mis-expression of genes or genetic pathways that can be modulated by let-7. Methods may include evaluating patients for genes or genetic pathways modulated by let-7, and / or using an expression profile to assess the condition of a patient or treating the patient with an appropriate miRNA.

Description

[0001] This application is related to US Patent Application Serial No. 11 / 141,707, filed May 31, 2005, and Serial No. 11 / 273,640, filed November 14, 2005, each of which is incorporated herein by reference in its entirety. technical field [0002] The present invention generally relates to the field of molecular biology. More specifically, the present invention relates to methods and compositions related to the diagnosis and treatment of diseases associated with biological pathways regulated directly or indirectly by the let-7 microRNA (miRNA) family. Background technique [0003] In 2001, several research groups isolated and characterized a large number of "microRNAs" (miRNAs) from C. elegans, Drosophila, and humans using cloning methods (Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001). Several hundred miRNAs have been identified in plants and animals including humans that appear to have no endogenous siRNAs. Thus, miRNAs, while similar to siRNAs, are ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/113
CPCC12N15/113C12N2330/10C12N2310/141A61P35/00
Inventor 查尔斯·D·约翰逊麦克·拜罗姆安德里斯·G·巴德弗兰克·J·斯莱克大卫·布朗德米特里·奥弗切兰柯凯文·克尔纳尔
Owner ASURAGEN
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