Small molecule RNA, preparation method thereof and application in pharmaceuticals for transcriptional activity of specificity up-regulated genes

A gene transcription, small molecule technology, applied in DNA preparation, DNA/RNA fragments, gene therapy, etc., can solve problems such as unclear function of microRNA

Active Publication Date: 2014-01-15
GUANGZHOU QIANYANG BIO-TECH PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, studies have found that many microRNAs in cells are not only distributed in the cytoplasm

Method used

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  • Small molecule RNA, preparation method thereof and application in pharmaceuticals for transcriptional activity of specificity up-regulated genes
  • Small molecule RNA, preparation method thereof and application in pharmaceuticals for transcriptional activity of specificity up-regulated genes
  • Small molecule RNA, preparation method thereof and application in pharmaceuticals for transcriptional activity of specificity up-regulated genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 The microRNA hsa-let-7i up-regulates the expression of interleukin 2 by targeting the TATA box

[0057] 1.1 Preparation method

[0058] Plasmids, microRNA mimics, interfering RNA

[0059] Replace the CMV promoter on the pMIR-Reporter vector (Promega) with restriction enzyme digestion with the -400 ~ +1bp segment near the transcription start site (TSS) of the human IL-2 promoter to construct the adult IL-2 promoter promoter Luciferase reporter carrier. Insert the precursor sequence of hsa-let-7i into the PLKO.1 vector (Sigma), and replace the puromycin gene on the vector with GFP to construct the PLKO-let-7i vector. Wild-type hsa-let-7i, mmu-let-7i, mutant hsa-let-7i, agomir-hsa-let-7i and corresponding negative controls were obtained from Shanghai Genepharma or Guangzhou Ribobio buy.

[0060] Antibodies and reagents

[0061] Anti-HA (MMS-101P) and anti-Pol II (8WG16) monoclonal antibodies were purchased from Covance, anti-TBP monoclonal antibodies (ChIP Ab+)...

Embodiment 2

[0093] Example 2 MicroRNAs hsa-miR-138, hsa-miR-92a, hsa-let-7c, and hsa-miR-181d specifically upregulate insulin, calcitonin, and calcitonin by targeting the TATA box sequence. Transcriptional activity of histone (H4A1) and c-myc genes.

[0094] 2.1 Preparation method

[0095] Computer prediction of microRNA and its target

[0096] According to the method in Example 1, the binding site of microRNA and gene promoter was predicted and screened.

[0097] Plasmids, microRNA mimics

[0098] According to the method of Example 1, a reporter vector for human insulin, CALCA, c-myc, H4-A1 and other promoters was constructed. Insert the precursor sequence of hsa-mir-181d into the PLKO.1 vector (Sigma). Wild-type hsa-miR-138, hsa-miR-92a, hsa-let-7c and the corresponding negative controls were purchased from Ribobio.

[0099] Dual luciferase report analysis

[0100] According to the method in Example 1, transfect the luciferase reporter vector and Renilla luciferase vector of the gene promote...

Embodiment 3

[0113] Example 3 Artificial synthesis of small interfering RNA targeting the gene TATA box specifically enhances the transcriptional activity of 78.9% of genes

[0114] 3.1 Preparation method

[0115] Targeting TATA box small interfering RNA sequence design

[0116] Three small interfering RNAs containing complementary sequences of TATA box were designed within the range of 20 bases each extending upstream and downstream with the TATA box sequence as the center.

[0117] Plasmids, small interfering RNA mimics

[0118] According to the method of Example 1, a reporter vector for adult interleukin 2, insulin, LHB, POMC, NPPA, IL6, HIV-1 virus, H4A1, APOE, CIRBP, BCL2L12, RHO, CALCA, GAPD and HBB promoters was constructed. The small interfering RNA and the corresponding negative control were synthesized by Ribobio.

[0119] Dual luciferase report analysis

[0120] According to the method in Example 1, transfect the luciferase reporter vector and Renilla luciferase vector of the gene promoter...

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Abstract

The invention discloses a method for specificity up-regulated gene expression through a targeting core promoter utilizing small molecule RNAs (including micro-RNAs and small interfering RNAs) and a series of regulation target genes. According to the invention, when a TATAbox sequence is contained in the promoter, a target site is in the range of expanding 20 basic groups from the upstream and downstream sides respectively by taking the TATAbox sequence as the center; when no TATAbox sequence is contained in the promoter, the target site is a 1-50 sequences on the upstream side of the genetic transcription initiation site. The micro-RNAs hsa-let-7i, hsa-miR-138, hsa-miR-92a, hsa-let-7c and hsa-miR-181d specifically regulate up the expression of interleukin-2, insulin, thyrocalcitonin, histone and c-myc gene respectively. Besides, aiming to the randomly-selected 19 gene promoters, the artificially-synthesized small interfering RNAs (siRNAs) can enhance the transcriptional activities of 78.9% of the genes. The invention aims to provide the method for specificity up-regulated gene expression utilizing micro-RNAs and has higher application values and broad application prospects in the biotechnology and the biomedicine field.

Description

technical field [0001] The present invention relates to the field of small molecule RNA, more specifically, relates to a kind of small molecule RNA and its preparation method and its application in the medicine for specifically up-regulating gene transcription activity. Background technique [0002] The precise regulation of gene expression is of great significance to the development of organisms, and the dysregulation of genes can cause many important diseases, such as cancer, immune diseases, neurological diseases, metabolic diseases (such as diabetes), etc. Therefore, the technology of precise regulation of gene expression has important research and clinical application value. The current method for regulating the expression of endogenous genes is mainly RNA interference (RNA interference) technology. RNA interference technology uses a double-stranded RNA molecule homologous to the target gene, and uses the RNA silencing pathway to inhibit the expression of the target ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/10A61K48/00
Inventor 张辉张意军
Owner GUANGZHOU QIANYANG BIO-TECH PHARM CO LTD
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