Method for activating p21 gene expression
一种p21、基因的技术，应用在分子生物学领域，能够解决改变宿主基因组免疫反应等问题
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
preparation example Construction
[0056] As used herein, the term "synthesis" refers to the way of synthesizing oligonucleotides, including any way capable of synthesizing RNA, such as chemical synthesis, in vitro transcription, vector expression, and the like. The preparation method of the small activating nucleic acid molecule provided by the invention includes sequence design and sequence synthesis. The synthesis of the small activating nucleic acid molecular sequence can be done by chemical synthesis, or entrusted to a biotechnology company specialized in nucleic acid synthesis. Generally speaking, the chemical synthesis method includes the following four processes: (1) synthesis of oligoribonucleotides; (2) deprotection; (3) purification and separation; (4) desalting and annealing. For example, the specific steps of chemical synthesis of double-stranded RNA molecules such as saRNA in the present invention are as follows:
[0057] (1) Synthesis of oligoribonucleotides
[0058] Set to synthesize 1 micromo...
Embodiment 1
[0080] Example 1: Screening for functional small activating RNA (saRNA) targeting the p21 gene promoter region
[0081] In order to screen functional small activating RNAs that can activate the expression of the p21 gene, the 1 kb promoter sequence of the p21 gene was retrieved from the UCSC Genome database ( figure 1). A target site with a size of 19 bp was selected from -1 kb upstream of the transcription start site (TSS), and moved to the TSS site by moving 1 bp at a time to obtain a total of 982 target site sequences. The target sequence was filtered to exclude target sequences with GC content higher than 65% or lower than 35% and containing 5 or more consecutive identical nucleotides. After filtering, the remaining 439 target sequences entered the screening process as candidates. Based on these candidate sequences, corresponding double-stranded double-stranded RNA molecules are chemically synthesized. Wherein, the length of the sense and antisense strands of the double...
Embodiment 2
[0110] Example 2: saRNA induces p21 gene mRNA expression and inhibits cancer cell proliferation
[0111]To further evaluate the role of p21 saRNA in inducing p21 gene mRNA expression and inhibiting cancer cell proliferation, the saRNAs (RAG1-431, RAG1-553, RAG1-688) screened by QuantiGene 2.0 were transfected into the cancer cell line KU-7 (bladder cancer) , HCT116 (colon cancer) and HepG2 (hepatocellular carcinoma). The results showed that in all the above cell lines, saRNA could induce at least double the expression level of p21 gene mRNA and inhibit cell proliferation, revealing the efficacy of saRNA-mediated p21 induction. Specifically, RAG1-431, RAG1-553, and RAG1-688 were transfected into KU7 cells, respectively, which induced the expression of p21 mRNA by 14.0, 36.9, and 31.9 times, and produced survival rates of 71.7%, 60.7%, and 67.4% ( Figure 5 ). RAG1-431, RAG1-553, and RAG1-688 were transfected into HCT116 cells, respectively, to induce p21 mRNA expression by 2...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More