Method for transcriptional gene expression regulation and application

A technology of transcription level and gene expression, which is applied in the direction of introducing foreign genetic material, DNA/RNA fragments, recombinant DNA technology, etc. by using vectors, and can solve problems that do not involve gene upregulation or activation at the transcription level, and achieve powerful gene regulation functions, Effective effect, anticancer effect

Inactive Publication Date: 2012-03-14
JINSHAN HOSPITAL FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the current RNA interference technology degrades the mRNA of the target gene or suppresses the translation of the mRNA of the target gene at the post-transcriptional level (that is, at the mRNA level) to exert the corresponding gene silencing effect, but does not include gene silencing at the transcriptional level, and even Not involved in the field of small RNA-mediated gene upregulation or activation at the transcriptional level

Method used

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  • Method for transcriptional gene expression regulation and application
  • Method for transcriptional gene expression regulation and application
  • Method for transcriptional gene expression regulation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Design of small double-stranded RNA

[0047] Through literature search, determine the transcription start point and core promoter region of E-cadherin gene (eg http: / / www.ncbi.nlm.nih.gov / Gene ID: 999). Log in to the website of the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ), and download the E-cadherin gene sequence. Within the E-cadherin promoter region (1000 bp above the transcription start point), avoid high CpG regions, and select relatively complex base combinations of C, T, A and G (mainly to avoid the simple structure of the target sequence, The 19-base sequence with high repeatability) is used as the target of the small double-stranded RNA, so that the dsRNA sequence is designed according to the selected target, and the designed dsRNA is used with the blast database of the National Center for Biotechnology Information website and the whole genome For matching, select a dsRNA sequence with better specificity as the scr...

Embodiment 2

[0053] Example 2 Effects of small double-stranded RNAs on cell growth, metastasis, and invasion

[0054] Transfection of double-reporter-validated dsRNA into prostate cancer cell lines (PC-3 and DU-145) and liver cancer cell lines (97L, 97H, and LM3) to evaluate the screened cells using cell proliferation assays, scratch assays, and invasion assays Changes in biological behaviors such as growth, metastasis, and invasion of tumor cells after the action of dsRNA.

[0055] The synthetic small double-stranded RNA was transfected into prostate cancer cell lines (PC-3 and DU-145) and liver cancer cell lines (97L, 97H and LM3) using liposome-based transfection reagents, and harvested 3 days after transfection Cells, total RNA and total protein were extracted, and then the expression of the target gene was detected by fluorescent quantitative PCR and Western blotting. If the expression of the target gene is up-regulated or down-regulated after dsRNA intervention, the dsRNA will be us...

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Abstract

The invention specifically relates to a method for transcriptional gene expression regulation by using small double-stranded RNAs, which belongs to the fields of biomedicine and molecular biology. The method comprises the following steps: screening and determining an effect site of a target gene on a genomic DNA sequence; designing specific siRNA or shRNA directed to the effect site; transfectingspecific siRNA or shRNA of a target sequence into nucleus. According to the invention, small double-stranded RNAs, including siRNA, shRNA, chemically or biochemically modified siRNA, shRNA built in related plasmids, etc., are utilized to realize transcriptional gene down-regulation or siliencing, or transcriptional gene up-regulation or activation. The small double-stranded RNAs have a powerful gene regulation function in transcription, particularly in treatment and research of malignant tumors, transcriptional down-regulated cancer genes or up-regulated cancer suppressor genes can perform very effective antitumor action.

Description

technical field [0001] The invention belongs to the field of biomedicine and molecular biology, especially the technical field of gene regulation in molecular biology. In particular, it relates to a method for regulating gene expression at the transcriptional level by using small double-stranded RNA. Background technique [0002] At present, gene regulation technology, especially RNA interference technology, has been widely used in the field of biomedical research. Small double-stranded RNA (double-stranded RNA, dsRNA) can efficiently and specifically block the expression of specific genes in the body, promote the degradation of target gene mRNA, and induce cells to show a phenotype of specific gene deletion, which is called RNA interference ( RNA interference, RNAi), or Post-transcriptional Gene Silencing (PTGS). At present, these technologies have been widely used in the field of biomedical research, and have become a simple and effective genetic tool to replace gene kno...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/63
Inventor 吴志明陈刚
Owner JINSHAN HOSPITAL FUDAN UNIV
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